Baculovirus Expression Vector System Manual

Instruction Manual6th Edition, May 1999

Instruction Manual6th Edition, May 1999

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Baculovirus ExpressionVector System Manual6th Edition May 1999

Instruction Manual

For information or to place an order, please call:1-800-848-MABS (6227)For Technical Assistance call:1-800-TALK-TEC (825-5832)10975 Torreyana Road • San Diego, CA 92121 • USATel: (619)812-8800 • Fax: (619) 812-8888URL: http://www.pharmingen.com

General Methods

6xHis and GST Purification

Direct Cloning

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Table of Contents

Baculovirus Memorandum of Agreement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vi

Opening Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii

1. The Baculovirus Expression Vector System . . . . . . . . . . . . . . . . . . . . . . . . 1

2. Advantages of using the Baculovirus Expression Vector System . . . . . . . 3

3. AcNPV Baculovirus DNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7AcNPV C6 Wild-type Baculovirus DNA . . . . . . . . . . . . . . . . . . . . . . . . . 8BaculoGold™ Linearized Baculovirus DNA . . . . . . . . . . . . . . . . . . . . . . 8Linearized AcRP23.lacZ Baculovirus DNA . . . . . . . . . . . . . . . . . . . . . . . 9Linearized AcUW1.lacZ Baculovirus DNA . . . . . . . . . . . . . . . . . . . . . . . 10

4. General Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 114.1 Selecting an Appropriate Baculovirus Transfer Vector . . . . . . . . . . . . . . 114.2 Optimizing Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144.3 Cloning your Gene into a Baculovirus Transfer Vector . . . . . . . . . . . . . 14

Preparing Vector and Insert . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14Ligating Vector and Insert . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16Propagating Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Purifying Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

4.4 Insect Cell Lines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18General Handling Techniques . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Monolayer Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Freezing and Thawing Insect Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22

4.5 Producing and Maintaining AcNPV-derived Baculoviruses . . . . . . . . . . 22Generating Recombinant Baculoviruses by Co-Transfection . . . . . . . . . 23End-point Dilution Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Plaque Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26Plaque Pickup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Amplifying Virus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Storing Virus Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Isolating AcNPV Particles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30Isolating AcNPV DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

4.6 Expressing Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31Monolayer Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Suspension Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

4.7 Purifying Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Non-secreted Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Cell Lysate Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Secreted Recombinant Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

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5. Purification Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39

5.1 6xHis Expression and Purification Kit . . . . . . . . . . . . . . . . . . . . . . . . . . 39

Batch Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42

5.2 GST Expression and Purification Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . 43

Batch Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44

Column Purification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

Dialyzing GST-Fusion Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

5.3 Checking Purity and Recovery of Recombinant Protein . . . . . . . . . . . . 46

5.4 Cleaving Fusion Proteins using Site-specific Proteases . . . . . . . . . . . . . . 46

Thrombin Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46

Factor Xa Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47

5.5 Generating 32P-Labeled GST or 6xHis Fusion Proteins . . . . . . . . . . . . . . 47

6. Generating Recombinant Baculovirus by Direct Cloning . . . . . . . . . . . . 49

7. Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

7.1 Cloning Inserts into Baculovirus Transfer Vectors . . . . . . . . . . . . . . . . . 53

7.2 Insect Cell Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53

7.3 Co-transfection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54

7.4 Plaque Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

7.5 Virus Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

7.6 Recombinant Protein Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56

7.7 6xHis Expression and Purification System . . . . . . . . . . . . . . . . . . . . . . 57

7.8 GST Expression and Purification System . . . . . . . . . . . . . . . . . . . . . . . . 58

7.9 Thrombin Cleavage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59

8. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61

Appendix A: BaculoGold™ Starter Package and Transfection Kit . . . . . . . . . . 65

Appendix B: 6xHis Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67

Appendix C: GST Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

Appendix D: vEHuni and vECuni Baculovirus Reagent Sets . . . . . . . . . . . . . . 75

Appendix E: Baculovirus Transfer Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

I. Polyhedrin Locus-based Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

Fusion Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

BioColors™ Baculovirus Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

Multiple Promoter Transfer Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . 96

II. p10 Locus-based Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 100

Multiple Promoter Transfer Vectors . . . . . . . . . . . . . . . . . . . . . . . . . . . 101

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103

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Figures1. The Baculovirus life cycle in vivo and in vitro . . . . . . . . . . . . . . . . . . . . . . . . 2

2. Design of AcNPV BaculoGold™ DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3. Design of AcRP23.lacZ DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

4. Design of AcUW1.lacZ DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

5. Experimental scheme using BEVS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

6. Monolayer and suspension Sf cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

7. Comparison of uninfected and infected Sf9 cell monolayers . . . . . . . . . . . . 22

8. 12-well End-point Dilution Assay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25

9. Western blot analysis of Retinoblastoma protein (Rb) in plaques . . . . . . . . . 27

10. Examples of recombinant protein expression levels in

Baculovirus-infected Sf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

11. Characterization of native and Baculovirus-expressed

Retinoblastoma protein (Rb) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

12. Functional activity of Baculovirus-expressed recombinant protein . . . . . . . . 35

13. Expression, purification and cleavage of fusion proteins . . . . . . . . . . . . . . . 40

14. Strategy for directly cloning EcoRI fragments into the AcMNPV genome . . . . 50

15. Baculovirus vectors for direct cloning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51

16. BioColors™ in Sf9 cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

17. Separation of Baculovirus-expression GFP and BFP using

fluorescence-activated cell sorting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

Tables1. Comparison of BEVS and bacterial expression systems . . . . . . . . . . . . . . . . . 3

2. Analysis of recombination frequencies by plaque assays . . . . . . . . . . . . . . . . . 7

3. Vector selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

4. Recommended cell numbers and approximate densities for various assays . . . 18

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BACULOVIRUS MEMORANDUM OF AGREEMENTNON-EXCLUSIVE RIGHTS TO USE BACULOVIRUS EXPRESSIONVECTOR SYSTEM TECHNOLOGY FOR RESEARCH PURPOSES

I. BACKGROUND

The Texas Agricultural Experiment Station (TAES) claims rights to technology devel-oped by Dr. Max D. Summers of the Department of Entomology relating to a recombi-nant Baculovirus expression vector system (BEVS) and the use of such vectors in insectcell culture media for expression of cloned genetic material. TAES is making the systemand its components available for noncommercial research purposes. This Baculovirusexpression vector system and related subject matter are claimed in two United StatesPatents, Numbers 4,745,051 and 4,879,236. Commercial rights to BEVS or productsthereof are subject to a non-exclusive license, terms of which will be made availableupon written request. Information and materials received from TAES relating to BEVSmust be taken with the understanding that it is subject to a restrictive license forresearch purposes only.

II. TERMS AND CONDITIONS OF AGREEMENT

(1) All information and material received under this Agreement shall be used for researchpurposes only.

(2) Access and distribution of the vectors and information must be limited to Recipient andto those personnel who report to Recipient, hereinafter referred to as "Recipient."

(3) Recipient agrees to supply TAES preprints of any publications resulting from the use ofthe BEVS material promptly upon receipt of notice of acceptance from the publishingjournal. Preprints should be sent to the attention of the Coordinator of Research Devel-opment for Industrial Relations, Texas Agricultural Experiment Station, Texas A&M Uni-versity, College Station, Texas 77843-2162.

(4) Recipient and those who report to Recipients are aware of the proprietary interestinvolved herein and commit to honoring the terms and conditions of this Agreement.

(5) Recipient accepts the biological material with the knowledge that it is experimentalbiological material and that is provided by TAES without warranty of any sort,expressed or implied. Recipient agrees to comply with all applicable governmental reg-ulations for the handling thereof. Recipients shall hold TAES harmless for any damageswhich may be alleged to result in connection with the use and possession of therequested materials as provided in this Agreement, subject to any relevant state or fed-eral government limitations.

(6) This Agreement and Recipient’s right to use biological material become effective uponbreaking the seal of the package containing biological material and automatically termi-nates if Recipient fails to comply with any provisions of this Agreement.

(7) TAES retains ownership and all rights to biological material not expressly granted andnothing in this Agreement constitutes a waiver of TAES' rights under U.S. Federal, State,or Patent law.

NOTE: THESE RESTRICTIONS DO NOT APPLY TO INFORMATION OR TECHNOLOGYWHICH RECIPIENT CAN SHOW ARE IN THE PUBLIC DOMAIN OR FOR WHICHHE/SHE HAD PREVIOUSLY RECEIVED OR DEVELOPED IN GOOD FAITH THROUGHCHANNELS INDEPENDENT OF THE TEXAS AGRICULTURAL EXPERIMENT STATION.

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Opening RemarksAll reagents and materials listed in this manual are for research use only.

Safety RequirementsThese research products have not been approved for human or animal diagnostic ortherapeutic use. We suggest that all purchasers follow the NIH guidelines that havebeen developed for recombinant DNA experiments. All PharMingen products shouldbe handled only by qualified persons trained in laboratory safety procedures. Theabsence of a product warning is not to be construed as an indication that the prod-uct is safe. All possible hazards of many biological products may not be known at thistime. Always use good laboratory procedures when handling any of these products.

WarrantyInformation presented in this manual is accurate to the best of our knowledge. It isnot, however, guaranteed as such. It is the user’s responsibility to investigate and ver-ify the suitability of the supplied materials and procedures for a particular purpose.PharMingen expressly disclaims all warranties of merchantability and fitness for aparticular purpose with respect to the use or suitability of the reagents and materi-als. PharMingen shall in no event be responsible for damages of any nature, directlyor indirectly resulting from the use of the products of these kits.

DisclaimerThis manual is a practical guide for researchers to become familiar with the Bac-ulovirus expression technology as a tool to overexpress foreign genes. It is notintended as a replacement to a textbook about Baculoviruses but rather to serve asan introduction to Baculovirus nomenclature and cite key references to guide theinterested reader to additional literature. The information disclosed herein is not tobe construed as a recommendation to use the above product in violation of anypatents. PharMingen will not be held responsible for patent infringement or otherviolations that may occur with the use of our products. For commercial use of the6xHis/Ni-NTA system, licenses may be granted by Hoffmann-La Roche Ltd. (Basel,Switzerland). Please contact QIAGEN Inc., 9600 De Soto Avenue, Chatsworth, CA91311 for further information. All Baculovirus and related products sold by PharMin-gen are for research use only. The Polymerase Chain Reaction (PCR) is a processpatented by Hoffmann-La Roche, Inc. Triton is a trademark of Union Carbide Chemi-cals and Plastics Co.

Technical Assistance and Ordering InformationAt your request, we will furnish technical assistance and information about our prod-ucts. Call 800-TALK-TEC (825-5832) to talk to a Technical Service Specialist. Our spe-cialists have the education and experience necessary to answer your technical ques-tions regarding the reagents and materials listed in this manual. All technical assistanceis provided gratis and you assume sole responsibility for results you obtain by relyingon that assistance. We make no warranties of any kind with respect to technical assis-tance or information we provide. Call 800-848-MABS (848-6227) to place an order.

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AbbreviationsAcNPV Autographa californica nuclear polyhedrosis virusAmp Ampicillinβ-gal β-galactosidaseBEVS Baculovirus expression vector systemBFP Blue fluorescent proteinBSA Bovine serum albuminBV BaculovirusCIAP Calf intestinal alkaline phosphataseCsCl Cesium chlorideDTE DithioerythritolDTT DithiothreitolECV Extracellular virusEDTA Ethylenediamine tetraacetic acidEtBr Ethidium bromideFACS Fluorescent activated cell sortingFBS Fetal bovine serumGFP Green fluorescent proteinGST Glutathione S-transferaseh Hourkb KilobaseskD KilodaltonLB Luria-bertani (broth)MCS Multiple cloning sitemin MinuteMOI Multiplicity of infection (plaque-forming units/cell number)NaPi Sodium phosphate NaPPi Sodium pyrophosphateORF Open reading frameOV Occluded virus particlesPAGE Polyacrylamide gel electrophoresisPBS Phosphate buffered salinepfu Plaque-forming unit(s) = virusPi Inorganic phosphatepi Post infectionPMSF Phenylmethylsulfonyl fluorideRb Retinoblastoma proteinRT Room temperatureSf Spodoptera frugiperda Sj Schistosoma japonicumSDS Sodium dodecyl sulfateTBE Tris borate/EDTATE Tris/EDTAU Unitv/v Volume: volume ratiowt WildtypeX-gal 5-bromo-4-chloro-3-indolyl-β-D-galactopyranosideYP Yellow proteinyr Year

1

The 6xHis vectors were developed by PharMingen and produced in collaboration with QIAGEN

1The Baculovirus Expression Vector System

The Baculovirus Expression Vector System (BEVS) is one of the most powerful and ver-satile eukaryotic expression systems available.1,2 The BEVS is a helper-independentviral system which has been used to express heterologous genes from many differentsources, including fungi, plants, bacteria and viruses, in insect cells. The BaculovirusDNA used in PharMingen’s BEVS is the Autographa californica nuclear polyhedrosisvirus (AcNPV). In this system several Baculovirus genes nonessential in the tissue cul-ture life cycle (polyhedrin, p10, basic) may be replaced by heterologous genes. Sincethe Baculovirus genome is generally too large to easily insert foreign genes, heterologousgenes are cloned into transfer vectors.† Co-transfection of the transfer vector andAcNPV DNA into Spodoptera frugiperda (Sf) cells allows recombination between homol-ogous sites, transferring the heterologous gene from the vector to the AcNPV DNA.AcNPV infection of Sf cells results in the shut-off of host gene expression allowing fora high rate of recombinant mRNA and protein production. Recombinant proteins canbe produced at levels ranging between 0.1% and 50% of the total insect cell protein.Factors influencing foreign gene expression are discussed, although it is difficult to pre-cisely predict how efficiently different genes will be expressed.

Baculoviruses (family Baculoviridae) belong to a diverse group of large double-stranded DNA viruses that infect many different species of insects as their naturalhosts.3 Baculovirus strains are highly species-specific and are not known to propagatein any non-invertebrate host. The Baculovirus genome is replicated and transcribed inthe nuclei of infected host cells where the large Baculovirus DNA (between 80 and200 kb) is packaged into rod-shaped nucleocapsids.4 Since the size of these nucleocap-sids is flexible, recombinant Baculovirus particles can accommodate large amounts offoreign DNA. AcNPV is the most extensively studied Baculovirus strain. Its entiregenome has been mapped and fully sequenced.5-7 Infectious AcNPV particles enter sus-ceptible insect cells by facilitated endocytosis or fusion, and viral DNA is uncoated inthe nucleus (Fig. 1). DNA replication starts about 6 h post-infection (pi). In both in vivoand in vitro conditions, the Baculovirus infection cycle can be divided into two differ-ent phases, early and late. During the early phase, the infected insect cell releases extra-cellular virus particles (ECV) by budding off from the cell membrane of infected cells.During the late phase of the infection cycle, occluded virus particles (OV) are assem-bled inside the nucleus. The OV are embedded in a homogenous matrix made pre-dominantly of a single protein, the polyhedrin protein.8, 9 OV are released when theinfected cells lyse during the last phase of the infection cycle. Whereas the first ECVare detectable 10 h pi, the first viral occlusion bodies of wild-type AcNPV virus develop3 days pi but continue to accumulate and reach a maximum between 5-6 days pi.Occlusion bodies are visible under light microscopy where they appear as dark poly-gonal-shaped bodies filling up the nucleus of infected cells. Not all known Bac-uloviruses form occlusion bodies; AcNPV is representative of the group of occlusionbody-positive Baculoviruses. The polyhedrin protein, the major component of occlu-sion bodies, has a molecular weight of 29 kD.1 During late phases of infection, thepolyhedrin protein accumulates to very high levels. Up to 1 mg of polyhedrin protein† vEHuni and vECuni Baculovirus DNAs allow for direct cloning of heterologous genes into the BV genome (Chapter 6).

2

may be synthesized per 1–2 × 106 infected cells accounting for 30-50% of the totalinsect protein. Although the polyhedrin protein seems to be one of the most abundantproteins in infected insect cells, it is not essential for the Baculovirus life cycle in tis-sue culture. However, in vivo viral occlusion bodies are an important part of the Bac-ulovirus life cycle, essential for its dissemination into the environment (Fig. 1).

Deletional or insertional inactivation of the polyhedrin gene in AcNPV results in the pro-duction of occlusion body-negative viruses, a phenomenon which simplifies the identifica-tion of recombinant viruses. The plaques formed by occlusion body-negative viruses are dis-tinctly different from those of occlusion body-positive wild-type viruses. Newer modifiedAcNPV allow either color selection to identify recombinants or permit positive survival selec-tion for recombinants (BaculoGold™ Cat. No. 21001K), rendering the occlusion body-basedvisual screening method obsolete.

A variety of Baculovirus Transfer Vectors have been constructed for use with AcNPVDNA (Appendix E). Each vector contains: 1) an E. coli origin of replication, 2) an ampi-cillin resistance marker, 3) a promoter from the polyhedrin, p10 or basic protein AcNPVgene,10 4) a cloning region downstream from promoter in which to insert foreign genesand 5) a large tract of AcNPV sequence flanking the cloning region to facilitate homolo-gous recombination. Purified recombinant vectors containing the gene of interest may beco-transfected with AcNPV Baculovirus DNA into insect cells. After several days, recom-binant viruses, which arise by homologous recombination between the transfer vectorand AcNPV DNA, are selected.

Figure 1. The Baculovirus life cycle in vivo and in vitro. A) In vivo. Two distinct viral populations areformed in infected insect cells, occluded and budded virions. Occluded virions are protected from desiccationin the environment, allowing primary infection in susceptible larva. Once ingested, the occlusion body is solu-bilized in the gut, releasing virions which fuse with midgut cells. The virion nucleocapsid migrates through thecytoplasm to the nucleus. The core is uncoated from the capsid structure in the nucleus. Here replication takesplace. Secondary infection is mediated by the budded form of the virus entering adjacent cells via adsorptiveendocytosis. B) In vitro. The Baculovirus genome is too large to directly insert foreign genes easily. Hence, theforeign gene is cloned into a transfer vector that contains flanking sequences which are homologous (5’ and 3’to your insert) to the Baculovirus genome. BaculoGold™ DNA and the transfer vector containing your clonedgene are co-transfected into Sf9 insect cells. Recombination takes place within the insect cells between thehomologous regions in the transfer vector and the BaculoGold™ DNA. Recombinant virus produces recombi-nant protein and also infects additional insect cells thereby resulting in additional recombinant virus.

BaculoGold®DNA

Your Gene

Homologous Recombination

Recombinant Protein

BuddingRecombinant VirusSecondary Infection

of Insect Cells

GST/6xHISTag

Your Gene

BaculoGold® DNA

Co-transfection

Transfer VectorB

Uncoating

Replication

ViralOcclusion

BuddingVirus

Secondary Infectionof Insect Cells

Endocytosis

A

Primary Infection of Host Insect

Fusion(Midgut Cells)

Soluble in Gut

Ingestion

3

2 Advantages of using the Baculovirus Expression Vector System

Choosing the right system for foreign gene expression can be particularly importantin obtaining biologically active recombinant protein. Several unique features of theBEVS have made it the system of choice for many applications (Table 1). This chapterhighlights the advantages of using BEVS to express recombinant proteins. Often,recombinant proteins expressed in bacterial systems are insoluble, aggregated andincorrectly folded.11 In contrast, proteins expressed in BEVS are, in most cases, solu-ble and functionally active.

Table 1. Comparison of BEVS and bacterial expression systems. The insect cell, unlike bac-terium, is capable of performing many of the processing events that are required for forming biologi-cally active, foreign proteins.

1) Functional activity of the recombinant proteinThe BEVS typically produces overexpressed recombinant proteins containing properfolding, disulfide bond formation and oligomerization.2 Additionally, this system iscapable of performing several post-translational modifications. This leads to a proteinthat is similar to its native counterpart, both structurally and functionally. However, incases where the authentic protein functions as a heterodimer or relies on tissue- orspecies-specific modifications, the recombinant Baculovirus-expressed protein may notbe functionally active, unless its binding partner or modifying enzyme is co-expressed.

2) Post-translational modificationsSeveral post-translational modifications have been reported to occur in the BV, includ-ing N- and O-linked glycosylation, phosphorylation, acylation, amidation, car-boxymethylation, isoprenylation, signal peptide cleavage and proteolytic cleavage.12-14

The sites where these modifications occur are often identical to those of the authenticprotein in its native cellular environment. However, the BEVS can express the gene ofinterest at a high expression rate which may overwhelm the ability of the cell to mod-

Features BEVS Bacterial

Simple to use ✓ ✓Protein size unlimited <100 kD

Multiple gene expression ✓Signal peptide cleavage ✓Intron splicing ✓Nuclear transport ✓Functional protein ✓ sometimes

Phosphorylation ✓ sometimes

Glycosylation ✓Acylation ✓

4

ify the protein product. This often results in lower levels of glycosylation or phospho-rylation of the target protein than in the native cell line. Also, tissue- or species-specificpost-translational modifications will not be performed in the BV, unless the modifyingenzyme is being co-expressed.

3) High expression levelsCompared to other higher eukaryotic expression systems, the most distinguishing fea-ture of the BEVS is its potential to achieve high levels of expression of a cloned gene.The BV system has proven particularly useful in the generation of large quantities ofproteins for structural analysis.15-20 The highest expression level reported is 50% of thetotal cellular protein of an infected insect cell corresponding to approximately 1g ofrecombinant protein per 1 × 109 cells. However, many recombinant proteins are notproduced at such high amounts and it is usually difficult to predict the amount of pro-tein expression. There are some guidelines one can follow to optimize protein produc-tion. Of primary importance is optimizing the design of the recombinant BaculovirusTransfer Vector (Chapter 4.1).

4) Capacity for large insertsThe expandability of the capsid structure of Baculoviruses allows the packaging andexpression of very large genes. There is no known upper size limit for the insertion offoreign sequences into the BV genome.

5) Capacity to express unspliced genesInsect cells have the capability to perform intron/exon splicing. However, certainvirus-, tissue- or species-specific splicing patterns will not be obtained if they requirethe presence of particular splicing factors which are not available in the infected insectcell environment. In general, for high protein expression levels, a cDNA insert ratherthan a genomic DNA fragment is recommended.

6) Simplicity of technologyBaculoGold™ technology has made expression of full-length proteins fast, easy and reli-able. Recombinant Baculovirus can be obtained in two simple steps–cloning andco-transfection–in as little as 5 days. The ease of use now rivals that of bacterial expres-sion systems and BEVS technology does not require that the recombinant protein beexpressed as a fusion protein. With the addition of several vectors containing genesencoding the green fluorescent protein from the jellyfish Aquorea victoria (Appendix E),protein expression can easily be monitored.

7) Simultaneous expression of multiple genesBEVS has the capability to express two or more genes simultaneously within singleinfected insect cells. Protein complexes that depend on dimer or multidimer formationfor activity can be assembled. A well known example is the formation of complete viruscapsids from a variety of viruses which have been assembled in vitro, using BEVS, by co-expressing the capsid subunits simultaneously. To this end, several multiple promoterplasmids have been constructed and are described in Appendix E.

5

8) Localization of recombinant proteinsBaculovirus-expressed recombinant proteins are usually localized in the same sub-cellular compartment as the authentic protein. Nuclear proteins will be transported tothe insect nucleus, membrane proteins will be anchored into the cell membrane, andsecreted protein will be secreted by infected insect cells.

9) Ease of purificationPharMingen has developed the 6xHis and glutathione S-transferase (GST) BaculovirusExpression and Purification Kits, designed for easy and reliable single-step purificationof recombinant proteins. The 6xHis purification system (Cat. No. 21474K) relies on thehigh specificity of the 6xHis tag for Ni-NTA Agarose. The GST purification system (Cat. No. 21475K) takes advantage of the high affinity of glutathione agarose beads forreduced glutathione. These kits combine the advantages of expressing functional andsoluble recombinant proteins using BEVS technology with the convenience of a GSTor 6xHis affinity purification system. Even under the highest expression levels, mostGST and 6xHis fusion proteins expressed in insect cells remain predominantly soluble.An extensive line of vectors has been developed for use in these systems. When usingthe GHLT, HLT or G series of vectors, the inserted gene will be produced as a fusionprotein with an affinity tag on the amino terminus. Vectors in the GHLT series producea fusion protein composed of both a 6xHis and a GST tag. Vectors in the pAcG and thepAcHLT series produce proteins with a GST or a 6xHis tag respectively. The GST and6xHis tags can be removed by incubating the protein in the presence of a site-specificprotease (Chapter 5).

10) Direct cloning Generally, heterologous genes are cloned into transfer vectors, which homologouslyrecombine with the BV genome in insect cells. vEHuni and vECuni Baculovirus DNAallow the direct cloning of heterologous genes into the BV genome (Chapter 6).21

7

3 AcNPV Baculovirus DNAs

Infection of susceptible insect cells AcNPV wild-type virus results in the production ofocclusion bodies. These opaque, light-refractive particles can be easily visualized underthe light microscope (Chapter 4.5). This phenomenon aids in the identification ofrecombinant Baculoviruses in which the polyhedrin gene has been replaced by acloned gene of interest. Recombinant viruses expressing the protein of interest ratherthan the polyhedrin protein fail to produce occlusion bodies and can be visually iden-tified as occlusion body-negative plaques. However, in the past, non-recombinantswere the vast majority over recombinants, usually 1,000:1. Modified AcNPV DNA (Bac-uloGold™, AcRP23.lacZ and AcUW1.lacZ DNA) revolutionized the BV technology andmade the occlusion body-based visual screen method obsolete. To improve recombi-nation efficiencies, a single restriction site was added behind the polyhedrin or p10promoter (AcRP23.lacZ or AcUW1.lacZ, respectively) so that the modified BaculovirusDNA can be linearized. Co-transfecting the linearized AcNPV DNA with a BaculovirusTransfer Vector shows an improved recombination frequency of 30%. The addition ofthree restriction sites in the polyhedrin locus of BaculoGold™ allows for the deletion ofessential portions of the virus genome. Co-transfecting BaculoGold™ with a Bac-ulovirus Transfer Vector rescues the lethally deleted virus at recombination frequenciesgreater than 99% (Table 2).

Table 2. Analysis of recombination frequencies by plaque assays. Plaque assays were done usingviral inoculum from wild-type high titer viral stock (A), and 5-day transfection supernatants from Sf9 cellsco-transfected with either AcRP23.LacZ Baculovirus DNA and pVL1392-XylE plasmid DNA (B) or Baculo-Gold™ Baculovirus DNA and pVL1392-XylE plasmid DNA (C) on X-gal plates. After 7 days the plates wereanalyzed and the number of recombinant (R) (yellow in the presence of Catechol) versus non-recombinant(NR) (blue in the presence of β-gal) plaques were noted above. Recombination frequencies were deter-mined by the number of R versus NR plaques. Each lot of BaculoGold™ Baculovirus DNA undergoes test-ing to insure that the recombination efficiency is greater than 99%.

To improve selection and screening methods, a polyhedrin-driven lacZ genecoding for β-galactosidase was inserted into the virus genome. Preparation of lin-earized BaculoGold™ DNA removes the lacZ gene. Non-recombinant, lacZ positiveplaques stain blue and recombinant, lacZ negative plaques are colorless. Recombi-nant virus are selected as colorless in a plaque assay overlayed with agar contain-ing X-gal: (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside).

Volume Virus Used Number of Plaques Recombination Viral Stock 10 ml 1 ml 0.1 ml 0.01 ml Frequency

A. AcNPV wt high titer stock solution >5,000 >5,000 >5,000 1,096 NA

B. AcRP23.lacZ R 1,500 200 52 6 Baculovirus DNA ~34%+1392-XylE NR >5,000 398 87 11

C. BaculoGold™ R >5,000 885 105 9Baculovirus DNA +1392-XylE NR 3 0 0 0

~99.9%

R = Recombinant NR = Nonrecombinant

8

AcNPV C6 Wild-type Baculovirus DNAThe wild-type AcNPV DNA is a super-coiled, double-stranded, circular DNA moleculewith a molecular weight of 130 kb BaculoGold™ DNA, AcRP23.lacZ DNA andAcUW1.lacZ DNA are all derivatives of the AcNPV wild-type DNA. Originally, AcNPVwild-type DNA was widely used for co-transfection with recombinant BaculovirusTransfer Vectors to obtain recombinant BV particles. However, the identification ofrecombinants is time-consuming, requires considerable skills, and the recombinationfrequency is only 0.1%. Wild-type AcNPV DNA has no advantages over BaculoGold™,AcRP23 DNA or AcUW1.lacZ DNA. PharMingen sells purified ready-to-use AcNPV C6Wild-type Baculovirus DNA (Cat. No. 21103D).

BaculoGold™ Linearized Baculovirus DNABaculoGold™ DNA22, 23 is a modified AcNPV Baculovirus DNA which contains a lethaldeletion and does not code for viable virus (Fig. 2). Co-transfection of the BaculoGold™

DNA with a complementing Baculovirus Transfer Vector rescues the lethal deletion byhomologous recombination. Since only the recombinant BaculoGold™ produces viablevirus, recombination frequencies exceed 99%.

The flanking sequences of the complementing vector’s promoter region must bederived from the polyhedrin locus of the AcNPV wild-type DNA. p10 locus derivedvectors (pAcUW1, pAcUW41, pAcUW42, pAcUW43) will not recover the lethal dele-tion of BaculoGold™. Furthermore, not all polyhedrin derived vectors are compatiblewith BaculoGold™ DNA. The lethal deletion in BaculoGold™ spans 1.7 kb downstreamof the polyhedrin gene. Small streamlined vectors may not contain the entire regionand will not rescue the lethal deletion. PharMingen sells purified ready-to-use linearized BaculoGold™ DNA (Cat. No. 21100D).

Figure 2. Design of AcNPV BaculoGold™ DNA. The polyhedrin gene locus of AcNPV DNA has beenaltered in the following ways: (1) a lacZ gene has replaced the viral polyhedrin gene; (2) three Bsu36I cut-ting sites have been added, in ORF 603, 1629 and in lacZ, which do not alter the amino acid sequences oftheir coding regions. The modified AcNPV DNA is linearized at the Bsu36I cutting sites deleting essentialportions of the ORF 1629.

AcNPV wt DNA

p10 genepolyhedrin gene

Uncut BaculoGold™ DNA

Cleaved BaculoGold™ DNAcontaining lethal deletion∆ORF1629∆ORF603

LacZ ORF1629ORF603

Bsu36I Bsu36I Bsu36I

polyhedrin promoter

∆LacZ

9

Linearized AcRP23.lacZ Baculovirus DNALinearized AcRP23.lacZ DNA is a modified AcNPV Baculovirus DNA in which the viralpolyhedrin gene was replaced by a lacZ gene (Fig. 3). AcRP23.lacZ is linearized at a sin-gle Bsu36I site introduced downstream of the polyhedrin promoter. Homologousrecombination occurs during co-transfection of polyhedrin locus derived BaculovirusTransfer Vectors with AcRP23.lacZ. Approximately 30% of the resulting virus will behomologously recombined Baculovirus DNA. Since recombination disables the lacZgene, recombinant Baculoviruses can be selected by plaque assay on X-gal plates. Non-recombinant virus express the lacZ gene and plaques will appear blue. Recombinantvirus do not express lacZ and plaques appear colorless. PharMingen sells purified ready-to-use linearized AcRP23.lacZ DNA (Cat. No. 21101D).

Note: When using AcRP23.lacZ DNA, a plaque assay is necessary to iden-tify and isolate recombinants from non-recombinant Baculovirus.

Figure 3. Design of AcRP23.lacZ DNA. The polyhedrin gene locus of AcNPV DNA has been alteredin the following ways: (1) a lacZ gene has replaced the viral polyhedrin gene; (2) a single Bsu36I cuttingsite has been added downstream of the polyhedrin promoter. The modified AcNPV DNA is linearized atthe Bsu36I site.

AcNPV wt DNA


Uncut AcRP23.lacZ DNA

Bsu36I

ORF1629LacZORF603

Linearized AcRP23.lacZ DNA

ORF1629LacZORF603

polyhedrin promoter

polyhedrin promoter

10

Linearized AcUW1.lacZ Baculovirus DNALinearized AcUW1.lacZ DNA is a modified AcNPV Baculovirus DNA which contains a p10promoter driven lacZ gene (Fig. 4). AcUW1.lacZ DNA is linearized at a single single Bsu36Iintroduced downstream of the p10 promoter. Homologous recombination occurs duringco-transfection of p10 locus derived Baculovirus Transfer Vectors with AcUW1.lacZ.Approximately 30% of the resulting virus will be homologously recombined BaculovirusDNA. Since recombination disables the lacZ gene, recombinant AcUW1.lacZ DNA can becolor-selected by plaque assay on X-gal plates. Non-recombinant virus express the lacZ geneand plaques appear blue. Recombinant virus do not express the lacZ gene and plaquesappear colorless. Both non-recombinant and recombinant virus are occlusion body posi-tive. PharMingen sells purified ready-to-use linearized AcUW1.lacZ DNA (Cat. No. 21102D).

Note: When using AcUW1.lacZ DNA, a plaque assay is necessary to iden-tify and isolate recombinants from non-recombinant Baculovirus.

Figure 4. Design of AcUW1.lacZ DNA. The p10 gene locus of AcNPV DNA has been altered in thefollowing way: (1) a lacZ gene has replaced the viral p10 gene; (2) a single Bsu36I cutting site wasadded downstream of the p10 promoter. The modified AcNPV DNA is linearized at the single Bsu36Icutting site.

AcNPV wt DNA


p10 promoter

Linearized AcUW1.lacZ DNA

p26

p10 promoter

LacZ

LacZp26 p74

p74Uncut AcUW1.lacZ DNA

Bsu36I

11

4 General Methods

The steps necessary to construct recombinant Baculoviruses using BaculoGold™ DNA(Cat. No. 21100D) are outlined in Figure 5. Protocols for each step are given within thischapter.

Figure 5. Experimental scheme using BEVS. Choose the appropriate transfer vector and clone in theforeign gene. Propagate the transfer vector containing the foreign gene using competent cells and purifyby suitable means. Co-transfect BaculoGold™ DNA and recombinant transfer vector into Sf9 insect cells.Amplify the resultant recombinant virus in Sf9 insect cells. Use the amplified viral stock to produce protein.Purify your protein using appropriate methods.

4.1 Selecting an Appropriate Baculovirus Transfer VectorThe BaculoGold™ Starter Package and Transfection Kit (Cat. No. 21001K and No.21100K) both contain the Transfer Vector Set pVL1392/1393 (Cat. No. 21201P) (Appen-dix A). The pVL1392 and pVL1393 vectors are based on the polyhedrin locus and con-tain an extended MCS downstream of a polyhedrin promoter (Appendix E). These vectorshave been used extensively to express a variety of proteins and should be adequate inmost cases (Chapter 4.6). However, your protein expression needs may require that youuse a specialized vector. For this reason, a variety of different Baculovirus Transfer Vec-tors have been constructed. The choice of vector will be determined by the applicationof the purified recombinant protein and in some cases by the nature of the protein itself.This section is intended as a guide to help researchers choose a vector which best fitstheir needs.

First, decide whether to clone the gene of interest into a Baculovirus Transfer Vectorthat will produce the authentic protein encoded by its own ATG, or into a fusion-proteinvector providing an N-terminal tag. The BEVS allows the expression of full length authen-tic proteins and does not require the expression of an N-terminal fusion sequence. This isa major advantage over many other expression systems, although, for certain applicationsit may be desirable to express a fusion protein. The tag may provide a sequence which canbe used to label or modify the protein in a desired way that may not be available with the

BaculoGold™ DNA Clone foreign gene into transfer vector

Propagate and purify vector containing foreign genes

Co-transfect into insect cells

Amplify recombinant virus

Produce recombinant protein

Purify recombinant protein

12

authentic protein. The pAcGP67 and pAcSecG2T vectors incorporate a secretion signalsequence fused to the desired protein to force the recombinant protein into the secretorypathway. A fusion tag may also ease purification of non-secreted proteins. The pAcGHLTand pAcHLT vectors contain GST and 6xHis tags which can be purified on glutathioneand Ni-NTA Agarose beads, respectively.

Secondly, a suitable promoter must also be chosen. Baculovirus encoded promoterscan be divided into the following classes according to the time, the viral infection cycleand conditions under which they are activated. PharMingen’s vectors contain eitherlate or very late promoters.

Immediate Early Promoters: Baculovirus promoters, activated due to the action ofinsect encoded transcription factors, control early viral transcription factors.

Early Promoters: Baculovirus promoters, activated before viral DNA synthesis occurs,usually control genes necessary for the onset of viral replication (not usually used forforeign gene expression).

Late Promoters: Baculovirus promoters, active during and after viral DNA synthesis,when the cell is producing Baculovirus components, control genes necessary to assem-ble the virus particles (e.g., 39K protein promoter, basic protein promoter).

Very Late Promoters: Baculovirus promoters, activated very late during the infectioncycle, well after virion assembly has been completed, control genes involved in theformation of occlusion bodies and cell lysis. Most genes controlled by very late pro-moters are non-essential under tissue culture conditions (e.g., p10 promoter, poly-hedrin promoter).

The early and immediate early promoters are generally very weak and are notroutinely used in Baculovirus Transfer Vectors. The late promoters (the 39K and basicprotein promoters) are moderately strong promoters which express their products latein the infection cycle when enzymes needed for post-translationally modified proteinsare still present. The pAcMP2 and pAcMP3 transfer vectors (Cat. No. 21209P) containthe basic protein promoter and should be considered when the foreign protein is gly-cosylated, phosphorylated, etc. The polyhedrin and the p10 protein promoters are verystrong promoters expressed during the very late phase of viral infection. They areessentially non-competitive and have been used together to construct multiple pro-moter vectors. The polyhedrin promoter is most commonly used and has been clonedinto a variety of Baculovirus Transfer Vectors.

Third, consider whether you want to use a single or multiple promoter vector. Multi-ple promoter vectors are useful for expressing subunits of heterodimers or for expressinga cell type- or tissue type-specific modifying enzyme along with your protein of interest.

Table 3 is designed to help you to decide which Baculovirus Transfer Vector may bemost appropriate for your work.

13

Vector Compatibility Promoter Type Fusion Protein Features Cat. #

Polyhedrin locus-based

BaculoGold™ DNA

AcRP23.lacZ DNA

AcUW1.lacZ DNA

AcNPV wild

-type D

NA

Single Promoter Plasmids

pVL1392/3 (set) • • • Polyhedrin very late no Standard polyhedrin locus vectors 21201PpAcSG2 • • • Polyhedrin very late site dependent Recommended for large inserts, has an ATG 21410PpAcMP2/3 (set) • • • Basic protein late no Facilitates post-translational modifications 21209PpAcUW21 • • • p10 very late no Allows for in-larval expression, F1 origin 21206PpAcGHLT-A, -B, -C (set) • • • Polyhedrin very late yes GST-tag, 6xHis-tag thrombin cleavage site 21463PpAcHLT-A, -B, -C (set) • • • Polyhedrin very late yes 6xHis-tag, thrombin cleavage site 21467PpAcG1 • • • Polyhedrin very late yes GST-tag 21413PpAcG2T • • • Polyhedrin very late yes GST-tag, thrombin cleavage site 21414PpAcG3X • • • Polyhedrin very late yes GST-tag, factor Xa cleavage site 21415PBioColors™ BV Control (set) • • • Polyhedrin very late yes BioColors™ Genes 21518PBioColors™ His (set) • • • Polyhedrin very late yes BioColors™ Genes, 6xHis tag, thrombin 21522P

cleavage site

SecretorypAcGP67 A, B, C (set) • • • Polyhedrin very late yes Signal sequence 21223PpAcSecG2T • • • Polyhedrin very late yes Signal sequence, GST-tag 21469P

Multiple Promoter PlasmidspAcUW51 • • • Polyhedrin, p10 very late no Simultaneous expression of 2 foreign genes; 21205P

F1 originpAcDB3 • • • Polyhedrin, p10 very late no Simultaneous expression of 3 foreign genes; 21532P

F1 originpAcAB3 • • • Polyhedrin, p10 very late no Simultaneous expression of 3 foreign genes 21216PpAcAB4 • • • Polyhedrin, p10 very late no Simultaneous expression of 4 foreign genes 21412P

p10 locus-based

Single Promoter Plasmids

pAcUW1 • • p10 very late no Standard p10 locus vectors 21203P

Multiple Promoter Plasmids

pAcUW42/43 (pair) • • Polyhedrin, p10 very late no Simultaneous expression of 2 foreign genes; 21208PF1 origin

Table 3. Vector Selection. The Vector Selection Chart gives a comprehensive overview of the vectors available for use withthe BEVS. Please refer to Appendix E for vector maps and descriptions.

14

4.2 Optimizing Gene ExpressionOnce the vector is chosen, the gene of interest is cloned into a restriction enzyme sitedownstream of the BV promoter. The efficiency of heterologous gene expression in theBV System can differ by approximately 1000 fold due to the intrinsic nature of the geneand the encoded protein. Modifying the heterologous gene will generally influencegene expression by only 2-5 fold. Researchers should not feel compelled to excessivelymodify their gene. However, there are some general rules for improving gene expres-sion. Since translation will start at the first ATG initiation codon downstream of thechosen BV promoter, there should be no additional ATG codons upstream of the gene.Additionally, the 5’ untranslated sequence between the promoter and the start ATGshould be kept to a minimum. In some cases, genes have been efficiently expressedfrom constructs with around 150 nucleotides between the promoter and the start ATG.However, it is advisable to trim down 5’ untranslated sequences to less than 50nucleotides. The 3’ untranslated region downstream of the stop codon is of minorimportance. There have been conflicting results regarding the importance of thepolyadenylation signal. We have found that the expression level is generally notaffected by the sequence downstream of the stop codons.

4.3 Cloning your Gene into a Baculovirus Transfer VectorThe techniques required for inserting a foreign sequence into a Baculovirus TransferVector and preparing high quality plasmid DNA for co-transfections are described inthis chapter. Most of the techniques are not unique to BEVS and we suggest referring tomolecular biology manuals for supplementary cloning information.24, 25

Preparing Vector and InsertExamine the endonuclease restriction map for both the transfer vector and your geneof interest. Identify restriction site(s) common to the cloning site of the vector and toyour gene of interest. The 5’ cloning site of your insert should be as close as possibleto the ATG start codon of your gene (not more than 100 bases upstream). A polyadeny-lation sequence for the 3’ cloning site is optional and has not been shown in this sys-tem to improve stability or expression of recombinant protein. Both the insert andBaculovirus Transfer Vector DNA should be digested with appropriate restrictionenzymes to generate compatible ends for cloning. If a single restriction enzyme isused to prepare the vector, the DNA must be treated with calf intestinal alkalinephosphatase (CIAP) to remove 5’ phosphate groups and prevent recirculation of thevector during ligation. When preparing the insert DNA, the correct restriction frag-ment (gene of choice) should be purified from an agarose gel by electroelution orDNA purification using glass-milk beads. PCR products should be similarly purified.

15

1. Prepare insert and Baculovirus Transfer Vector DNA by restriction endonucleasedigestion. The following 20 µl reaction is provided as an example:

5 µl plasmid DNA (1 µg/µl)1 µl appropriate restriction enzyme (e.g. BamHI, 20 U/µl)2 µl appropriate restriction buffer (10X)

12 µl sterile deionized water20 µl final volume

2. Incubate sample(s) at the appropriate temperature (depending on the restrictionendonuclease used, usually 37°C) for 2–4 h.

3. If the vector has been digested with a single restriction endonuclease, the DNAshould be treated with CIAP. Thus, add the following components directly to therestriction endonuclease digest after the incubation time has been completed:

20 µl previous volume3 µl CIAP 10X buffer1 µl CIAP6 µl sterile deionized water

30 µl new final volume

4. Incubate for 20 min at 37°C.

5. Add 1 volume of TE-saturated phenol/chloroform. Vortex each sample for 10 sand centrifuge samples for 5 min at 12,000 × g in a microcentrifuge.

6. Transfer the upper, aqueous phase to a fresh tube and add 1 volume of chloro-form:isoamyl alcohol (24:1). Vortex each sample for 10 s and centrifuge samplesfor 2 min at 12,000 × g in a microcentrifuge.

7. Transfer the upper aqueous phase to a fresh tube and add 0.5 volumes of 7.5 Mammonium acetate and 2.5 volumes of ice-cold 100% ethanol. Mix carefully byslowly inverting tubes several times by hand. Precipitate DNA by placing for 1 hat –20°C or 20 min on dry ice.

8. Collect the DNA pellets by centrifugation at 12,000 × g for 5 min.

9. Carefully remove the supernatant, wash the pellet with 1 ml of 70% ethanol, drybriefly in a 37°C oven or in a vacuum desiccator. Resuspend pellet in 20 µl TEbuffer. Determine the approximate DNA concentration by agarose gel elec-trophoresis with comparison to known amounts of DNA standards.

Agarose minigel (agarose concentration depends on the size range of the fragments)0.5 M EDTATE-saturated phenol/chloroformChloroform:isoamyl alcohol (24:1)7.5 M ammonium acetateEthanol (100% and 70%)TBE gel electrophoresis bufferCIAP [(0.01 U/pmol of ends) if vector has been digested w/single endonuclease]TE buffer

Materials Needed✓

16

Ligating Vector and Insert An insert DNA:vector molar ratio of 1:3, 1:1 and 3:1 should be used to determine opti-mal insert:vector ratios. The total amount of DNA for recessive-end cloning per 10 µlvolume should be 200 ng.

assuming: si is the size of the insertsv is the size of the vectorriv is the molar ratio of insert:vectort is the amount of total DNA (insert plus vector)i is the amount of insert needed in the DNA ligation reactionv is the amount of vector needed in the DNA ligation reaction

the formula for this is as follows:

1. Set up a ligation reaction as described below. This example assumes an insert:vector ratio of 3:1.

Therefore, riv = 3. We define sv = 10 kb and si = 3.3 kb. The total DNA for recessiveend cloning should be 200 ng. Therefore, t = 200 ng. If we insert these values intothe formula above we calculate that we need 100 ng of vector DNA and 100 ng ofinsert DNA. Thus, our sample ligation looks as follows:

1 µl vector DNA (100 ng/µl, 10 kb)1 µl insert DNA (100 ng/µl, 3.3 kb)1 µl T4 DNA ligase (1 Weiss unit)1 µl 10X ligase buffer6 µl sterile deionized water

10 µl final volume2. Incubate the mixture at 16°C overnight.

3. Following the ligation reaction, transform the ligated plasmid DNA (usually1 µl of the ligation mixture) into competent cells of an appropriate hoststrain (e.g., HB101, DH5α).

Note: To monitor the efficiency of the ligation and transformationsteps, competent cells should also be transformed with uncut non-recombinant vector DNA as well as cut vector DNA which has been lig-ated in the absence of an insert.

T4 DNA ligase10X ligase buffer containing 10 mM ATP

Materials Needed✓

Sterile deionized water

i = v = (si x t)

[(sv/riv) + si] sv+ (si x riv)

t x sv and

17

Propagating VectorsThere are many different E. coli strains available which are suitable for preparation ofcompetent cells used in transformations, e.g., DH5α or HB101. Many of these strainsare available as commercially prepared competent cells. Several comprehensive manu-als containing procedures for preparation of competent cells are listed in the Referencesection of this manual. PharMingen’s transfer vectors are “high copy number” vectorsand should generate yields of up to several milligrams per liter.

Transforming bacterial strains

Before starting:

Place DNA and 5 ml culture tubes on ice.Place culture plates in 37°C incubator to dry.Make SOC medium: (1 ml for each transformation)

to each ml of SOB medium, add:• 10 µl 2M Glucose solution and• 10 µl 2M Mg solution

Place SOC medium in the 37°C water bath.Make β-mercaptoethanol dilution - 1:20 dilution in sterile water.

1. Thaw competent cells on ice (100 µl/ transformation).

2. Gently thaw cells by hand. Aliquot 100 µl into pre-chilled 15 ml polypropylenetubes (Falcon Cat. No. 2097).

3. Add 1.7 µl of the fresh β-mercaptoethanol dilution to the 100 µl of bacteria, result-ing in a final concentration of 25 mM. Gently swirl.

4. Incubate on ice for 10 min, swirling every 2 min.

5. Add 0.1-50 ng of recombinant plasmid DNA (1 µl) to cells and swirl gently. As apositive control, add 1 ng of pBR322 to another 100 µl of cells.

6. Incubate on ice for 30 min. Heat shock at 42°C for 45-55 seconds (critical!).Return to ice for 2 min.

7. Add 0.9 ml of SOC medium preheated to 37°C. Incubate at 37°C for 1 h, shakingat 225 RPM on an orbital shaker.

SOB Medium (liter):20 g Bactotryptone 5 g yeast extract

0.5 g NaClAutoclave solution

2 M Mg solutionMix equal volumes of 2 M MgCl2 and 2 M MgSO4

Filter sterilize solution2 M Glucose

Filter sterilize solutionLB/Amp (150 µg/ml) platesCompetent cellsβ-mercaptoethanol

Materials Needed✓

18

8. Spin down bacteria using a table-top centrifuge at 10,000 × g for 5 min. Removeall media except for 100 µl. Resuspend bacteria in remaining 100 µl and spreadthin on an LB-Amp plate.

9. Incubate plates at 37°C overnight.

10. The next day, pick up several colonies for miniprep DNA isolation to confirm thepresence of the recombinant plasmid. Perform restriction endonuclease analysisto confirm the presence and orientation of the insert.

Note: After transformation of a suitable E. coli host strain (e.g., HB101,DH5α, etc.) by a Baculovirus Transfer Vector and plating the bacteria onselective medium, cells harboring recombinant plasmid DNA will growinto colonies. Since all current Baculovirus Transfer Vectors contain anampicillin resistant gene, the selection should be done on LB plates con-taining 50 µg/ml ampicillin.

Purifying VectorsThe quality of both the vector and viral DNA is critical for successful co-transfections.Sf cells are sensitive to some contaminant’s found in crude plasmid preparations,which cannot be removed by phenol/chloroform extraction or ethanol precipitation.Vector DNA purified by CsCl-EtBr density gradient centrifugation, anion exchangechromatography (QIAEX resin, QIAGEN Inc) or by extraction with glassmilk will besufficiently pure for co-transfection. Refer to molecular biology manuals for compre-hensive purification techniques.24, 25

4.4 Insect Cell LinesSeveral established insect cell lines are highly susceptible to AcNPV virus infection. Thetwo most frequently used insect cell lines are Sf9 and Sf21 (Cat. No. 21300L and No. 21301L). Both cell lines were originally established from ovarian tissues of Spodopterafrugiperda larvae and are highly recommended for use in the BEVS. Healthy insect cellsattach well to the bottom of the plate forming a monolayer and double every 18–24 h.Infected cells become uniformly round, enlarged, develop enlarged nuclei, don’t attachas well and stop dividing. Sf9 and Sf21 cells may also be grown in suspension. Antibioticsare not required, but gentamicin sulfate (50 µg/ml) and Amphotericin B (“Fungizone”)(2.5 µg/ml) are often added to the media. CO2 supplementation is not required. We rou-tinely use Sf9 cells and will refer to them from here on; however, Sf21 cells may be sub-stituted.

We commonly receive questions concerning cell confluency and BEVS assays. Table 4was designed to help new users determine accurate cell densities per desired assay.

Table 4. Recommended cell numbers and approximate densities for various assays. Thesenumbers are routinely used for Sf9 insect cell cultures in PharMingen’s laboratories. Individual users maywant to further optimize these numbers for their own experimental systems.

Transfection 60 mm 2.0 x 106 ~60% 3.2 x 106

Dilution Assay 12 well 1.0 x 105 ~30% 3.0 x 105

Plaque Assay 100 mm 6.2 x 106 ~70% 8.8 x 106

Viral Amplification 150 mm 2.0 x 107 ~70% 2.9 x 107

Protein Production 150 mm 2.0 x 107 ~70% 2.9 x 107

Plate # Cells % # Cells/ Assay Size per Assay Confluent Confluent Plate

19

General Handling Techniques

The following information is helpful when handling insect cells.

• Healthy Sf9 cells generally double every 18–24 h when grown in TNM-FHmedia (Cat. No. 21227M).

• To maintain healthy cultures, Sf9 cells should be subcultured 1:3 when theyreach confluency on plates (three times a week). They will grow reasonablywell at temperatures between 26-28°C. However, after infection it is importantto keep the temperature at 27°C ±0.5°C; otherwise, recombinant protein pro-duction may be poor, although cells will look infected.

• An adjustment period ranging from a few days to several weeks should beallowed when transferring Sf9 cells between monolayer and suspension cultures.

• Always equilibrate insect cell culture medium to RT before using.

• When removing liquid from a plate of cells, tip the flask at a 30–60° angle sothe liquid pools toward the bottom of the flask. Remove the liquid withouttouching the cell monolayer using a sterile pipette.

• When seeding cells into a tissue culture plate or flask, be sure the vessels areplaced on a flat surface to ensure homogenous cell density.

• It is extremely important when doing a plaque assay to provide the proper celldensity for plaque formation (Table 4). Rock the plate back and forth to evenlydistribute the cells over the surface of the plate.

• To pellet cells, gently dislodge cells from monolayers and transfer the cell sus-pension to a sterile centrifuge tube of appropriate size. Spin the suspension for2–5 min at 1,000 × g. Carefully remove the supernatant without disrupting thecell pellet. To resuspend the cell pellet for culture, add the desired volume offresh medium to the side of the tube and gently resuspend the pellet by pipet-ting the suspension up and down several times.

• Insect cells are sensitive to centrifugal forces. For resuspension, cells should becentrifuged for 2–5 min at 1,000 × g in a GH-3.7 Beckman GPR horizontal rotoror equivalent.

• TNM-FH and Grace’s medium do not contain pH color indicators. These mediausually have a pH around 6.2.

• Cell viability may be checked using trypan blue. To 1 ml of cells add 0.1 ml ofa 0.4% stock solution of trypan blue (in PBS or other isotonic salt solution).Non-viable cells will take up trypan blue. Healthy, log-phase cultures shouldcontain more than 97% unstained viable cells.

• To minimize centrifugation cells may be transferred to a new tissue culture plateusing the old medium. Once cells have adhered (10 min), change to fresh media.

Insect cells grow well both in suspension and as monolayer cultures and can betransferred from one to the other with minimal adaptation (Fig. 6). Small-scale propa-gation of cells can be maintained on plates; however, for large scale it is time-consum-ing and costly to use plates. Spinner flasks are ideal for scaling up insect cell cultures.Both monolayer and suspension cultures should be evaluated for optimal levels ofprotein expression.

20

Monolayer Cultures

• In monolayer cultures, you may notice loosely attached cells or cells floating inthe medium. These floaters are especially frequent in cultures that are overgrown.In healthy cultures, floaters should be less than 2% of the cell population.

• When resuspending attached cells, use a stream of medium from a 10 mlpipette or a Pasteur pipette and gently dislodge the cells from the surface.Strongly attached cells may require persistence and more forceful pipetting.Try to minimize foaming. Trypsin and other enzymes are not recom-mended to dislodge Sf9 cells. Cell scrapers should be used only if absolutelynecessary, as scraping may damage cells.

• Initiate new plate or flask cultures by adding one volume of the cell suspensionto two volumes of fresh medium. Confirm that initial cell density is approxi-mately 30%.

2 × 107 Sf9 cells (Cat. No. 21300L) per plateTNM-FH media (Cat. No. 21227M)15 cm tissue culture plate (Falcon Cat. No. 3025)Sterile 10 ml pipets (Falcon Cat. No. 7551)Hemocytometer (Fisher Cat. No. 0267110)

Materials Needed✓

27°C incubator

Figure 6. Monolayer and suspension Sf cultures. A) Monolayer cultures. 150 mm tissue cultureplates (Falcon Cat. No. 3025) used at PharMingen for protein production. B) Suspension cultures. 2 L,1 L and 5 L (not shown) spinner flasks (Techne) used at PharMingen for cell propagation. Sf9 cells aresuspended in Techne spinner flasks by a magnetic arm that spins at ~60 rpm. The culture volume shouldalways remain less than half of the full volume of the flask. For example, a 1-liter flask should contain<500 ml suspended culture.

A B

21

• Cells may be grown in BaculoGold™ Protein-free medium (Cat. No. 21228M).Sf9 and Sf21 cells may attach more firmly in Protein-free medium and dou-bling time may vary.

• Sf9 cells can be adapted to Protein-free medium by slowly decreasing the ratioof TNM-FH to Protein-free medium (Cat. No. 21227M and No. 21228M). Splita confluent plate of Sf9 cells, and allow them to attach to a fresh tissue cultureplate. Remove the medium and replace it with a 1:2 ratio of Protein-free:TNM-FH media. Incubate cells until confluent. Split the cells and allow them toattach. Remove the media and replace with a 1:1 ratio of Protein-free:TNM-FHmedium. Incubate cells until confluent. Split the cells and allow them toattach. Remove the media. and replace with a 2:1 ratio of Protein-free:TNM-FHmedium. For the next medium change use pure Protein-free medium.

Suspension Cultures

• Sf9 and Sf21 cells grow well in suspension cultures. A spinner culture shouldbe started at an initial density of 5 × 105 cells/ml. The cell density can be eas-ily determined using a hemocytometer.

• Incubate spinner flasks at 27°C under constant stirring at 50-60 rpm.

• Routine maintenance of spinner cultures requires subculturing when the celldensity reaches approximately 2 × 106 cells/ml (2-3 times a week). Remove65-75% of the cell suspension and replace with fresh medium. Re-seed the cul-ture in a clean sterile spinner flask at least every 2 weeks to prevent build-upof by-products or other contaminants.

• Aeration may be required for large cultures or during infections. Optimum con-ditions depend on the particular setup and should be determined empirically.1

• Sf9 cells grown in suspension culture may be adapted to serum-free mediumby slowly decreasing the ratio of TNM-FH to Protein-free medium. Remove2/3 of a healthy suspension culture containing 2 × 106 cells/ml and replacewith Protein-free media. When a density of 2 × 106 cells/ml is reached (usually3–4 days) replace 2/3 of the culture with Protein-free media. Repeat these stepsuntil the culture is 100% Protein-free.

TNM-FH media (Cat. No. 21227M)

Spinner flask (Techne)

5 × 105 Sf9 cells per ml of culture (Cat. No. 21300L)

Hemocytometer (Fisher Cat. No. 0267110)

Materials Needed✓

27°C Incubator

Spinner apparatus

22

Freezing and Thawing Insect CellsFreezing and storing insect cells:Spin down Sf9 or Sf21 cells from a healthy, log-phase culture at 1,000 × g for 10 min.(2,000 rpm in a GH-3.7 Beckman GPR horizontal rotor or equivalent). Decant super-natant and keep cell pellet on ice. Resuspend pellet in 90% media, 10% DMSO. The celldensity should be at least 4 × 106 cell/ml. Dispense 1 ml aliquots quickly into freezingvials, keeping the cells cold at all times. Freeze cells slowly placing them at –20°C forone hour, then place them at –80°C overnight. Transfer to liquid nitrogen the next day.Cells should maintain viability when properly frozen and stored in liquid nitrogen.

Thawing insect cells:Thaw frozen cells quickly after removing them from liquid nitrogen by gentle agitation ina 37°C water bath. When thawed (30-40 seconds), quickly rinse the outside of the vial with70% ethanol and proceed aseptically in a sterile hood. Transfer the cells to a centrifuge tube,add 20 ml of TNM-FH and spin at 600 × g for 2–5 min to pellet the cells. Decant the super-natant and resuspend cells in 10 ml fresh TNM-FH medium. Seed the entire suspension intoa single flask or tissue culture plate and incubate at 27°C. After 12–24 h, remove the oldmedium and replace with fresh TNM-FH. The cells should be dividing within two days.

4.5 Producing and Maintaining AcNPV-derived BaculovirusesWhen producing recombinant virus it is important to include both positive and nega-tive controls. Figure 7 shows the negative control uninfected Sf9 cells (A), the positivecontrol wild-type infected Sf9 cells (B) and the positive control Recombinant XylE-Bac-ulovirus infected Sf9 cells before (C) and after (D) the addition of catechol. The use ofthese controls is particularly valuable for the end-point dilution and plaque assays.Cells expressing XylE or polyhedrin protein are easy to identify to determine if yourassay was successful. Negative control uninfected cells continue to proliferate; there-fore, relative cell number is a criterion for degree of infection.

Figure 7. Comparison of unin-fected and infected Sf9 cellmonolayers. Sf9 cells uninfected(A), infected with wild-type(AcNPV) Baculovirus (B), orinfected with recombinant Bac-ulovirus containing the XylE gene(C and D). Cells infected with wild-type Baculovirus are occlusionbody positive (B), whereas cellsinfected with recombinant virusare not (C and D). Cells expressingrecombinant XylE turn yellow inthe presence of Catechol (D).

Uninfected Sf9 cells Sf9 cells infected withwild-type Baculovirus

Visualization of XylE protein in Sf9 cells

A B

C D

Sf9 cells infected withrecombinant XylE Baculovirus

23

Generating Recombinant Baculoviruses by Co-TransfectionPrepare at least 10 µg of highly purified plasmid DNA for co-transfection. Sf cells aresensitive to some contaminants found in crude plasmid preparations that are notremoved during phenol/chloroform extraction or ethanol precipitation. Impurepreparations of plasmid DNA are toxic to the cells, and many cells may lyse shortlyafter transfection. The result is a lower viral titer. At about 24 h post-transfection,Sf9/Sf21 cell viability should be greater than 97%.

1. Prepare and label three tissue culture plates, one each for the experimental co-transfection, positive co-transfection control, and negative control. Seed 2 × 106

Sf9 cells onto each 60 mm tissue culture plate. Initial cell density should be50–70% confluent. Cell attachment should be done on a flat and even surface,allowing the cells to attach firmly, usually about 5 min. If cells don’t attach afterthat time, they are either not healthy or the wrong plates have been used (e.g., non-coated petri dishes).

Note: A fourth tissue culture plate may be seeded with 2 x 106 Sf9 cells forinfection with wild-type AcNPV virus as a positive control for infection.

2. Experimental co-transfection: Combine 0.5 µg BaculoGold™ DNA and 2–5 µgrecombinant Baculovirus Transfer Vector, containing your insert, in a microcen-trifuge tube. Mix well by gentle vortexing or by flicking the tube. Let mixture sitfor 5 min before adding 1 ml of Transfection Buffer B.

3. Positive control co-transfection: Combine 0.5 µg BaculoGold™ DNA and 2 µgpVL1392-XylE Control Transfer Vector DNA in a microcentrifuge tube. Mix wellby gentle vortexing or by flicking the tube. Let mixture sit for 5 min before adding1 ml of Transfection Buffer B.

4. Aspirate the old medium from the cells on the experimental co-transfection plateand replace with 1 ml of Transfection Buffer A. Make sure that the entire surface ofplate is covered to prevent the cells from drying out.

5. Aspirate the old medium from the positive control co-transfection plate andreplace it with 1 ml of Transfection Buffer A as in Step 4.

6. Aspirate the old medium from the negative control plate and replace it with 3 mlfresh TNM-FH medium. Nothing else will be added to this plate.

7. Add the 1 ml of Transfection Buffer B/DNA solution from Step 2, drop by drop tothe experimental co-transfection plate. After every 3–5 drops, gently rock the plate

6 × 106 Sf9 cells (Cat. No. 21300L), 2 × 106 cells per plate

Three 60 mm tissue culture plates (Falcon Cat. No. 3802)

15 ml TNM-FH insect medium (Cat. No. 21227M)

1.0 µg BaculoGold™ DNA (Cat. No. 21100D) (0.5 µg per co-transfection)

2-5 µg Recombinant Baculovirus Transfer Vector DNA containing your insert

Wild-type AcNPV Virus supernatant (Cat. No. 21103E)

2 µg pVL1392-XylE Control Vector (Cat. No. 21484P)

Transfection Buffer A and B Set (Cat. No. 21483A) 100 µl catechol solution (500 µM catechol, 50 µM sodium bisulfate solution)

Materials Needed✓

24

back and forth to mix the drops with the medium. During this procedure, a finecalcium phosphate/DNA precipitate should form. This precipitate is characterizedby a fine white milky color.

8. Add 1 ml of the Transfection Buffer B/XylE Positive Control DNA solution from Step3 drop by drop to the positive control co-transfection control plate, as in Step 7.

9. Incubate all three plates at 27°C for 4 h.

10. After 4 h, remove the medium from the experimental and positive control co-transfection plates. Add 3 ml fresh TNM-FH medium and rock the plates back andforth several times before once again removing all the medium. Add 3 ml of freshTNM-FH medium and incubate the plates at 27°C for 4–5 days. It is not necessaryto change the medium of the negative control plate.

11. After 4 days, check the three plates for signs of infection. Compare the negativeand positive controls to the experimental co-transfection plate. Infected cells willappear much larger than uninfected ones, will have enlarged nuclei, will stopdividing, and will often float in the medium.

12. After 5 days, collect the supernatant of the positive control and experimental co-transfection plates. Assess co-transfection efficiencies by end-point dilution assayor identify recombinant viruses by plaque assay. Transfection supernatants shouldbe amplified to produce high titer virus stocks that are used for recombinant pro-tein production. Alternatively, single recombinant viruses, obtained by plaquepurification or end-point dilution assay, may be used for virus amplification. Tocheck the expression of your protein of interest, lyse the transfected cells or usean aliquot of the supernatant (depending whether the recombinant protein issecreted or not) and spin down debris. Transfected cells expressing the XylE pro-tein can be assayed by adding 10-100 µl catechol solution to the cells after the co-transfection supernatant has been removed and replaced with fresh media.Infected cells will turn bright yellow in approximately 5 min.

End-point Dilution AssayThe end-point dilution assay (EPDA) is a versatile assay that is useful for a variety ofscreens. A 96-well plate EPDA may be used to replace the plaque assay and plaquepurification as a method for either determining viral titer or identifying and purifyingrecombinant virus.1 We use a modified 12-well plate EPDA on a routine basis. In the12-well EPDA, individual wells containing equal amounts of insect cells are inoculatedwith 100, 10, 1 or 0 µl aliquots of the original transfection supernatant, wild-type virus,or recombinant XylE positive control viral (typically pVL1392-XylE) supernatant(Fig. 8). This modified EPDA is useful for determining the efficiency of the initial co-trans-fection, identifying infected cells, approximating viral titers, and amplifying viral titer.

Cells are visually inspected for signs of infection following an initial co-transfectionin tissue culture plates. However, it may be difficult to identify infected cells as signs ofinfection are not always visually apparent, particularly if the transfection efficiency islow. The EPDA is then used to amplify the viral titer, and a visual comparison betweencells inoculated with 100, 10, 1 and 0 µl of the original transfection supernatant is usedto ascertain whether or not the initial co-transfection was successful. For example, if cellsreceiving 100 µl of the initial co-transfection supernatant look infected in the EPDA, butcells receiving 10, 1 and 0 µl do not, then it is likely that the viral titer is low and shouldbe amplified to produce a high titer stock. If the EPDA is used as an amplification step togenerate a high titer stock, care should be taken to avoid cross-contamination between

25

wells containing different viruses. Wild-type virus is highly infectious and can contami-nate wells containing recombinant virus. If cells receiving 100 µl of the original co-trans-fection supernatant look similar to those receiving 0 µl, it is likely that the original co-transfection did not result in a significant viral titer, and must be amplified.

Two types of positive EPDA controls are recommended. The supernatant from apVL1392–XylE transfection is a particularly useful positive control. Cells expressing thegene turn yellow in the presence of catechol and are easily identifiable (Fig. 8B). A smallnumber of infected cells may not turn yellow. For example, cells which are newlyinfected will show signs of infection (stop dividing, become enlarged and float) but maynot yet be producing protein. Additionally, cells near the 5th day of infection may havebegun to lyse and much of their protein may be dispersed throughout the media. Thewild-type virus, a viral stock of known titer, can be used as a standard against which toapproximate viral titers. Cells infected with wild-type virus will, in addition to showingtypical visual signs of infection, contain occlusion bodies. This criterion is particularlyuseful for first-time users who have not previously visualized infected cells. The wild-type virus can also be used to verify the health and infectivity of the cells.

Viral titers may be approximated by performing the 12-well EPDA with your trans-fection supernatant and a viral stock of known titer. A high titer stock at 2 X 108 pfu/ml(wild–type viral stock, Cat. No. 21103E) will show equal signs of infection in all three(100, 10 and 1 µl) infected wells, 3 days pi. Each well of the 12-well plate contains3 X 105 cell and a high titer stock contains 2 X 105 virus/µl, resulting in nominal cellproliferation and total cell infection three days pi.

If your transfection supernatant shows a 10-fold decrease in the number of infected cellsbetween dilutions, you should amplify the virus once or twice more to generate a high titerstock for protein production. High titer virus stocks are used for infection of cells at optimalmultiplicity of infection (MOI = No.virus/No.cells) resulting in maximum protein produc-tion.

Figure 8. 12-well End-point Dilution Assay. A twelve-well tissue culture plate was seeded at 30%confluency with Sf9 cells and infected with 100, 10, 1 and 0 µl aliquots of viral inoculum from AcNPVwild-type high titer stock (A), Recombinant AcNPV-XylE transfection supernatant (B) and recombinantAcNPV-IL2 transfection supernatant (C). Photographs of each well were taken 3 days pi.

100 µl

A. AcNPV wild-type viral stock

C. Recombinant AcNPV-IL-2

10 µl 1 µl 0 µl

B. Recombinant AcNPV-XylE

26

1. Seed 1 × 105 Sf9 cells per well on a 12-well plate. Allow cells to attach firmly.Replace medium with fresh TNM-FH.

2. Add 100, 10, 1 and 0 µl of the recombinant virus supernatant (usually obtained5 days after the start of transfection) to separate wells. Do the same for the posi-tive control, e.g., pVL1392-XylE supernatant or wild-type viral stock.

3. Incubate the cells at 27°C for three days. Examine the cells for signs of infection.

4. A successful transfection should result in uniformly large infected cells in the 100,10, and 1 µl experimental wells. The cells in the 0 µl control wells should not lookinfected because they were not inoculated with virus.

5. If only the 100 µl and 10 µl wells seem to have infected cells and the 1 µl welllooks more like the control, then the titer of your virus supernatant is low.Amplify the virus an additional time before you proceed with protein production.

6. The cells from the 100 µl well can be harvested and lysed in lysis buffer (Chapter 5.6.1). The desired protein production may be checked by western blotanalysis (if antibodies are available) or by Coomassie blue-stained SDS-PAGE gel.

7. It is recommended that the virus supernatant from the 100 µl well is kept asthe first viral amplification stock, however care should be taken to avoid cross-contamination between wells containing different virus.

8. To further purify the virus population, a plaque assay purification may be per-formed. It is optional for BaculoGold™ DNA users, but required if any otherAcNPV DNA was used for co-transfection.

Plaque AssayThe plaque assay can be used to plaque purify virus or to determine viral titer inplaque-forming units per ml (pfu/ml) so that known amounts of virus can be used toinfect cells during subsequent experimental work. In this assay, cell monolayers areinfected with a low ratio of virus, such that only isolated cells become infected. Anoverlay of agarose keeps the cells stable and limits the spread of virus. When eachinfected cell produces virus and eventually lyses, only the immediate neighboring cellsbecome infected. Each group of infected cells is referred to as a plaque. Uninfected cellsare dispersed throughout the culture, surrounding the plaques. After several infectioncycles, the infected cells in the center of the plaques begin to lyse and the peripheralinfected cells remain surrounded by uninfected cells. This phenomenon causes thelight passing through the infected cells to refract differently than the surroundinguninfected cells, and the plaque can be visualized either by the naked eye or by lightmicroscopy. Each plaque represents a single virus. Therefore, clonal virus populationsmay be purified by isolating individual plaques. Individual plaques obtained fromvarying dilutions of a viral stock can be counted to determine the viral titer (pfu/ml)

1.2 × 106 Sf9 cells (Cat. No. 21300L)

One 12-well tissue culture plate (Falcon Cat. No. 3043)

12 mls TNM-FH insect medium (Cat. No. 21227M)

Baculovirus transfection supernatant

Materials Needed✓

27

of a given transfection amplification supernatant. The condition of the cells and theireven distribution over the surface of the tissue culture plate is important to the successof a plaque assay. Cells should be healthy and in log growth phase at the time of theassay and at least 90% viable. Clumpy cells, cells that are not evenly distributed at thecorrect density (~ 70%) over the plate, and cells that do not adhere to the tissue cul-ture dishes within about 2 h after plating are detrimental to the assay.

Figure 9. Western blot analysis of Retinoblastoma protein (Rb) in plaques. 10 randomly pickedplaques were amplified from plates inoculated with pVL1392-Rb rescued BaculoGold™ virus (A) and platesinoculated with BaculoGold™ alone (B). As expected, Rb expression was only detected in plaques obtainedfrom cultures inoculated with pVL1392-Rb (A), and not in the background plaques in non-rescued cultures(B). Rb was detected using an anti-human Rb monoclonal antibody (clone G3-245, Cat. No. 14001A).

1. Seed Sf9 cells at 7 × 106 cells on a 100 mm plate. Allow the cells to attach firmlyto the plate (10 min). It is important that this is done on a level surface to allowthe cells to spread evenly over the bottom of the plate.

2. Replace medium with 10 ml fresh TNM-FH.

3. Add virus inoculum to the plate. Commonly, serial dilutions of the viral transfec-tion supernatant (10–3, 10–4, 10–5) are made and 100 µl of each dilution is addedto the medium of each plate. Mix gently by rocking the plate. Larger dilutions willbe necessary for high titer stock solutions.

4. Incubate the plates at 27°C for 1 h to allow virus particles to infect the cells.

2.1 × 107 Sf9 cells (Cat. No. 21300L)

Three 100 mm tissue culture plates (Falcon Cat. No. 3003)

130 ml TNM-FH insect medium (Cat. No. 21227M)

Baculovirus transfection supernatant

1-2 g Agarplaque-Plus™ Agarose (Cat. No. 21403A)

100 ml protein-free insect medium (Cat. No. 21228M)

Materials Needed✓

110

68

1 2 3 4 5 6 7 8 9 10

1 2 3 4 5 6 7 8 9 10

A

B

110

68

kD

kD

28

5. While the cells are incubating, prepare a 2% agarose solution using Agarplaque-Plus™ Agarose (low melting point agarose) in protein-free medium. Heat the solu-tion in a microwave until the agarose is dissolved; allowing the solution to reachboiling will help to ensure its sterility. Take care that all the agarose is melted but donot overheat. High heat will cause precipitation of certain nutrients. Cool to 45°Cin waterbath. Prewarm 1X volume of TNM-FH to room temperature (RT).

6. Mix equal volumes of the agarose solution and pre-warmed TNM-FH medium. Thefinal agarose solution should be between 0.8% and 1%. A final agarose concentra-tion less than 0.8% will not solidify well, whereas concentrations over 1% willcause damage to the cells. Remove plates from incubator and remove medium.Overlay cells with 10 ml of the Agarplaque-Plus™ Agarose solution by carefullyadding agarose to the side of the tilted plate. Allow plates to sit undisturbed on alevel surface until agarose hardens (about 20 min). If color selection is required(e.g., AcRP23.lacZ or AcUW1.lacZ), add 100 µl of an X-gal stock solution (25 mg/mlX-gal in DMF) to 10 ml of agarose solution before pouring it onto the plates.

7. Plates should be kept in a humid atmosphere at 27°C until visible plaques develop(6-10 days). Plaques can be visualized by inverting the plates on a dark back-ground and illuminating them with a strong light source from the side of theplate, or by holding them at a 45° angle into a light source. Plaques can be usedto determine virus titer or for screening to identify recombinant virus.

Plaque PickupTo ensure proper isolation, it is best that plaques are picked from plates containingfewer than 50 plaques. Plate several dilutions of the virus to ensure that a sufficientlylow number of plaques are obtained. Plaques maybe picked up using sterilemicropipette tips (1,000 µl) or microcapillary tubes.

1. Mark the plate under the plaque with a marker. Using a sterile pipette tip, removean agarose plug directly over the plaque; pick up between 10 and 100 plaques inthis manner.

2. Place each agarose plug in separate microcentrifuge tubes containing 1 ml tissueculture medium. Elute the virus particles out of the agarose by rotating the tubeovernight at 4°C.

3. Add 200 µl of each plaque pickup to separate wells of a 12-well tissue culture plateseeded with 2 × 105 cells per well in 1 ml fresh TNM-FH media. Incubate the platesfor 3 days at 27°C.

4. The virus supernatant of this passage one stock can be collected and centrifugedfor 5 min at 1,000 × g at 4°C to remove debris. Store at 4°C.

2.4 × 106 Sf9 cells (Cat. No. 21300L), 12-well tissue culture plate

(Falcon Cat. No. 3043)

100 mm tissue culture plate (Falcon Cat. No. 3003)


Sterile micropipette tips or capillary tubes

Microcentrifuge tubes

Materials Needed✓

29

5. Seed a 100 mm tissue culture plate with 5 × 106 cells for each plaque pickup. Allowcells to attach and replace medium with 10 ml fresh TNM-FH media.

6. Add 200 µl of the passage one stock to the 100 mm plate and incubate at 27°C for4 days. Save the remaining 800 µl passage one stock at 4°C as a backup.

7. Harvest the viral supernatant and centrifuge to remove debris. Determine the titerof this passage two stock. If the titer remains below 2 × 108 pfu/ml, proceed toAmplifying Virus.

Amplifying VirusPrepare large stocks of virus by infecting insect cells at a low MOI (<1) and harvestingsupernatant 4–5 days pi. It is critical to use a low MOI because passaging the virus athigh MOI increases the number of virus with extensive mutations in their genome.1

The number of mutant virus is also increased by serial passage, and it may be advan-tageous to maintain a low passage seed stock from which larger working stocks areamplified. Eventually the titer in the seed stock will be reduced through storage, andit becomes necessary to generate a new passage seed stock.

Since BaculoGold™ recombinants are greater than 99% of the total virus population, itis not generally necessary to initially prepare all stocks from a clonal viral population. How-ever, if there is a reduction in protein production after multiple passages of a viral stock, itmay be necessary to isolate clonal viral populations by EPDA or plaque purification.1 Afterverification of protein production, the clonal virus population can be amplified to producea high titer stock. The viral stock is then ready for large-scale protein production.

1. Seed 2 × 107 Sf9 cells on a 15 cm plate. Allow them to attach for 15 min andchange to fresh TNM-FH.

2. Add 100 µl-1 ml of your low titer recombinant stock to the plate. If you know thevirus titer of your stock solution, make sure that the MOI is below one. Repetitiveinfections with an MOI of substantially higher than one will select for deletionmutants which may no longer express your gene.

3. Incubate the cells at 27°C for 3 days. Check for signs of infection 2 days pi.

4. Harvest the supernatant from the plate, then spin down the cellular debris in atable-top centrifuge 10,000 × g.

5. Store the virus supernatant in a sterile tube at 4°C for up to 6 months. For longerstorage periods, virus supernatant should be frozen at –80°C. Store in a dark area;the viral titer appears to decrease when exposed to fluorescent light for prolongedperiods of time.

6. Determine the viral titer of your amplification solution using the plaque assayprocedure. Amplification typically is done 2 or 3 times to attain a high viral titer(2 × 108pfu/ml).

2 × 107Sf9 cells (Cat. No. 21300L) per plate

15 cm tissue culture plate (Falcon Cat. No. 3025)

100 mls TNM-FH insect medium (Cat. No. 21227M)

Baculovirus low titer virus stock

Materials Needed✓

30

Storing Virus ParticlesSupernatants containing Baculovirus particles may be stored at 4°C for up to 6 monthsor frozen at –80°C for a longer period of time. If frozen, avoid multiple freeze and thawcycles. Upon freezing, the viral titer may decrease and should be reamplified whenthawed. Store viral stocks in the dark; titers appear to decrease when exposed to fluo-rescent light for prolonged periods of time. The best way to preserve a recombinantvirus is to isolate its DNA and store it at –80°C.

Isolating AcNPV ParticlesFor long-term storage, you may want to isolate the recombinant Baculovirus particlesand purify the viral DNA.

1. Produce several liters of high titer Baculovirus stock solution. Remove cell debrisby spinning the stock solution at 10,000 × g for 5 min.

2. Transfer the supernatant to ultracentrifuge tubes (Nalgene Cat. No. 3114-0050)and pellet the virus particles by spinning the supernatant at 40,000 × g for 30 min(18,000 rpm in an SS34 rotor). A bluish-white pellet should be visible at the bot-tom of each tube.

3. Decant the supernatant and invert the centrifuge tubes on a paper towel for a fewminutes. Wipe the residual medium from the inside of the tubes. Carefully avoidtouching the virus pellet.

4. Resuspend virus pellet in 10 ml of TE buffer.

5. Prepare a 5%/40% sucrose step gradient in an ultracentrifuge tube: pipette severalmilliliters of a 40% sucrose cushion into the tube, and carefully layer 3 ml of a 5%sucrose cushion on top of it.1 Finally, place a layer of the resuspended viral parti-cles on top of the 5% layer.

6. Spin the tubes at 40,000 × g for 30 min. During that time, the viral particles willmove through the 5% sucrose layer and will be collected at the interface betweenthe 5% and 40% sucrose solution layers. It will appear as a white band. Most con-taminants will either float on top of the 5% sucrose layer or precipitate to the bot-tom of the tube.

7. Use a sterile 9-inch Pasteur pipette to harvest the virus particles located betweenthe two layers.

8. Transfer the harvested virus to a new ultracentrifuge tube and fill up the tube withTE buffer. If necessary, this resuspension can be stored at 4°C for a few days.

9. Spin the tube at 40,000 × g for 30 min to pellet the virus.

10. Decant the supernatant and invert the centrifuge tubes on a paper towel for a fewminutes. Wipe the residual buffer from inside of the tubes. Avoid touching thevirus pellet.

11. Resuspend the virus pellet in an appropriate volume of TE buffer (1 ml per 1 × 1010

viruses).

12. If necessary, store the virus resuspension for a few days at 4°C. Otherwise, proceedwith DNA isolation.

31

Isolating AcNPV DNA1. Digest the resuspended virus particles with RNase A (10 µg/ml final concentra-

tion) for 30 min at 37°C.

2. Add 10% SDS to the resuspended virus particles such that the final SDS concen-tration is 0.5%.

3. Digest with Proteinase K (10 µg/ml final concentration) for 30 min at 37°C.

4. Extract once with phenol/chloroform:

• Add one volume of phenol to the solution, mix well but avoid vortexing. Spinmixture in a table-top centrifuge to separate the organic and aqueous layers,and then transfer the upper layer to a new tube.

• Add one volume of phenol/chloroform (1:1 mixture) to the aqueous layer. Mixwell and spin tubes in a table-top centrifuge to separate the organic and aque-ous layer. Transfer the upper aqueous layer to a new tube.

• Add one volume of chloroform to the aqueous layer. Mix well and spintubes in a table-top centrifuge to separate the organic and aqueous layers.Remove the top aqueous layer to a new tube, being careful not to removeany chloroform.

5. Dialyze the aqueous layer against TE (pH 8.0) to eliminate traces of chloroform.We recommend 3 dialysis changes: 2 × 2 h, 1 × overnight.

6. Measure the A260 of the obtained viral DNA solution to determine concentration andmeasure the 260/280 ratio to verify purity. The 260/280 for DNA ~1.8. Run a 0.5%agarose gel to verify that the purified DNA is intact and of high molecular weight.

7. The DNA should be aliquoted and stored at 4°C. Since the Baculovirus genome islarge, avoid freezing which may shear the DNA.

4.6 Expressing Recombinant ProteinsRecombinant proteins have been produced in the Baculovirus system at levels rang-ing between 0.1% and 50% of the total insect cell protein. For optimal protein pro-duction, the MOI should be between 3 and 10. Researchers should test different MOIto empirically determine optimum levels for protein production. The supernatant fromprotein production should not be used as a viral stock. Since the MOI used was muchhigher than one, a considerable portion of the virus population may contain deletionmutations. Several variables influence protein levels, functional activity and post-trans-lational modifications of Baculovirus-expressed protein (refer to Chapter 4.2). An exam-ple of the variation of expression levels between four proteins–cyclin A, cdk2, TR2orphan receptor, and androgen receptor–is seen in Fig. 10(A). The percent of proteinexpressed in the system is highly dependent on the intrinsic property of the protein. Thecyclin A from Fig. 10(A) was not visible by Coomassie blue-staining, and was analyzedby western blot analysis Fig. 10(B). Figure 10(C) shows post-translationally modifiedIL–4. A comparative analysis of phosphorylated Baculovirus-expressed and nativeretinoblastoma protein (Rb) is shown in Fig. 11. Figure 12 shows assays used to mea-sure the functional activity of Baculovirus-expressed granulocyte macrophage colonystimulated factors (GM-CSF) and Interleukin-4 (IL-4). We suggest that the user consultthe literature pertinent to their recombinant protein to gain information regardingexpected post-translational modifications and levels of functional activity.

32

Figure 10. Examples of recombinant protein expression levels in Baculovirus-infected Sf9 cells.A) Protein expression levels. Amido black SDS-PAGE of total insect cell lysate (20 µg/lane) containing Bac-ulovirus-expressed cyclin A (lane 1), Cdk2 (lane 2), TR2 (lane 3), or androgen receptor (lane 4). B) Westernblot analysis of Baculovirus-expressed cyclin A. Anti-cyclin A monoclonal antibody (clone BF683, Cat. No.14531A) (lane 1). Isotype (negative) IgE control (lane 2). C) SDS-PAGE analysis of Baculovirus-expressed,purified mouse IL-4. IL-4 was purified using an anti-mouse IL-4 monoclonal antibody (clone BVD6-24G2,Cat. No. 18041D). The gel was stained with coomassie blue. Note that although Baculovirus-expressedcyclin A was not visible by staining (A, lane 1) it was readily visible by western blot analysis (B, lane 1). IL-4migrates as two bands due to differential glycosylation (C).

For large-scale protein production, we have found that cell propagation in spinnerflasks and protein production on tissue culture plates is optimal. Protein may be pro-duced in suspension, but often the levels are lower than on plates.

Monolayer Cultures

1. Seed several individual 15 cm tissue culture plates with 2.0 × 107 Sf9 cells per plate.Add fresh TNM-FH medium to make up a total of 30 ml media per plate.

2. Calculate the amount of virus needed using the formula: ml of inoculumneeded = MOI (pfu/cell) × number of cells/titer of virus per ml.

3. Infect seeded cells with high titer recombinant Baculoviruses stock solution (virustiter should be 1-2 × 108 pfu/ml). For optimal protein production, the MOI shouldbe between 3 and 10. Often researchers will test different MOIs to empiricallydetermine the optimum level of infection.

15 cm tissue culture plate (Falcon Cat. No.3025)

High titer viral stock (1.2 × 106 pfu/ml)

2.0 × 107 Sf9 cells (Cat. No. 21300L) per plate


Materials Needed✓

27°C Incubator

kD

116

66

36

3 4

Cyclin

A

Cdk2TR

2Andro

gen RA

1 2

B

IL-4

116

66

36

21

14

6

C

Anti-Cyc

lin A

Contro

l

IL-4

1 2SDS-PAGE SDS-PAGEWestern Blot

116

66

36

kD kD

33

4. Incubate the cells for 3 days at 27°C. Check for signs of infection 2–3 days afterinoculation. Cells should be enlarged in size (about 2 fold) and a large nucleusshould be visible.

5. Harvest the cells and supernatant from the plates and spin down the cells at10,000 × g for 5 min using a table-top centrifuge. Non-secreted proteins will befound in the cell pellet, which can be stored at –80°C. Secreted proteins will befound in the supernatant, which can be stored at –80°C. When purifying secretedprotein, the cell pellet should be tested to determine the amount of protein, if any,that remains in the cells.

Suspension Cultures

1. Seed approximately 2 × 106 Sf9 cells/ml in a spinner flask. The cells should behealthy (98% viable).

2. Calculate the amount of virus needed using the formula: ml of inoculum needed= MOI (pfu/cell) x number of cells/titer of virus per ml. The desired MOI for pro-tein production is 3–10.

3. Add the inoculum to the flask. Incubate the flask at 27°C with stirring for 2-4days. Check the progress of the infection by examining aliquots of the cultureunder the microscope.

4. To harvest, pellet cells by centrifugation. For secreted protein, store the super-natant in sterile tubes. For non-secreted proteins, store the cell pellet at –80°C anddiscard the supernatant.


Spinner flask (Techne)

5 × 105 Sf9 cells per ml of culture (Cat. No. 21300L)

Hemocytometer (Fisher Cat. No. 0267110)

Materials Needed✓

27°C Incubator

Spinner apparatus

34

Figure 11. Characterization of native and Baculovirus-expressed Retinoblastoma protein (Rb).A) Western blot analysis of native Rb during different stages of the MOLT-4 (a human leukemia cell line)cell cycle. Native Rb migrates as multiple bands due to varying degrees of phosphorylation. Cell cyclestages are denoted as Q (quiescent), G1, S, and M. B) SDS-PAGE analysis of recombinant Rb. Rb is detectedin Baculovirus-infected (lane 1) but not in mock-infected (lane 2) Sf9 cell lysates. The gel was stained withCoomassie blue. C) Comparative analysis of native and Baculovirus-expressed Rb by western blot. Rbexpressed in MOLT-4 cells (lane 1) is more highly phosphorylated than Rb expressed in Baculovirus-infectedSf9 cells (lane 2). D) Analysis of phosphorylation in Baculovirus-expressed Rb. Baculovirus-infected Sf9 cellswere labeled with 32P orthophosphate and treated or not treated with placental alkaline phosphatase(PAP). Both untreated (lanes 1a and 1b) and treated (lanes 2a and 2b) lysates were immunoprecipitatedwith anti-Rb antibody (clone G3-245, Cat. No. 14001A). Detection by autoradiography (top gel) showsthat the radioactive label (lane 1a) is greatly reduced (lane 2a) following PAP-treatment. Western blot analy-sis of the autoradiographs (bottom gel) show that Rb in untreated lysates migrated at a higher molecularweight (lane 1b) than Rb in PAP-treated lysates (lane 2b). Collectively, the data indicate that Baculovirus-expressed Rb is phosphorylated, although at a lower level than native Rb. Abbreviations: pRb, under-phosphorylated Rb. ppRb, phosphorylated and highly phosphorylated Rb species.

1 2

110

ppRbpRb

Rb

1 2Sf

9

Sf9

+ Rb

Rb

A B

C D

116

97

pRb

+ ppRb

1 32 4

Q G1 S MMOLT-4

MO

LT-4

Sf +

Rb

– PA

P

+ PA

P

Rb

1b 2b

1a 2a

Sf9 + Rb

kD

35

Figure 12. Functional activity of Baculovirus-expressed recombinant protein. hGM-CSF andmIL-4 were cloned into pVL1393 and expressed in Sf9 cells. A) hGM-CSF assay. hGM-CSF activity wasmeasured using the continuous cytokine dependent human cell line, TF-1.26 hGM-CSF, at 10 µg/ml, wasserially diluted 3 fold in 12 wells across a 96-well flat-bottom microtiter plate in 50 µl. 50 µl of TF-1 cellsat 2 × 105 cells/ml were then added to each well for a final cell density of 2 × 104/ml. After a 44 h incu-bation at 37°C in the presence of 5% CO2, the cultures were pulsed with 0.5 µCi tritiated thymidine(20 Ci/mM) for an additional 4 h. The cultures were then harvested and the incorporated thymidine mea-sured by scintillation counting. The data shown represent the cpm of thymidine incorporation versus3 fold serial dilutions of hGM-CSF. Each point represents the mean of three replicates. B) Western blotanalysis of hGM-CSF. Recombinant human GM-CSF (Cat. No. 19741V) loaded at 100 ng/lane and testedby Western blot analysis against purified anti-human GM-CSF (Cat. No. 18591D) at 1 µg/ml (lane 1) andnormal rat serum at 1:500 dilution (lane 2). C) mIL-4 assay. IL-4 activity was measured using the contin-uous IL-2 dependent murine cell line, CTLL-2.27,28 mIL-4 at 10 µg/ml was serially diluted 3 fold in 12wells across a 96-well flat-bottom microtiter plate in 50 µl. 50 µl of CTLL-2 cells at 2 × 105 cells/ml werethen added to each well for a final cell density of 2 × 104/ml. After a 20-h incubation at 37°C in the pres-ence of 5% CO2, the cultures were pulsed with 0.5 µCi tritiated thymidine (20 Ci/mM) for an additional4 h. The cultures were then harvested and the incorporated thymidine measured by scintillation count-ing. The data shown represents the cpm of thymidine incorporated versus 3 fold serial dilutions of mIL-4.Each point represents the mean of three replicates. D) Western blot analysis of mIL-4. Recombinant mIL-4 lysate (Cat. No. 19231N) was loaded at 100 ng/lane and tested by Western blot analysis using purifiedanti-mouse IL-4 (Cat. No. 18031D) at 5 µg/ml (lane 1), and normal rat serum at 1:500 dilution (lane 2).

A

C D

55

200

97116

66

3631

21

14

1 2

55

200

97

116

66

36

31

21

14

1 2

BTF-1 Based GM-CSF Assay

hGM-CSF

CPM

TdR

Inco

rpor

ated

0

10000

20000

30000

40000

50000

60000

70000

1 2 3 4 5 6 7 8 9 10 11 12Serial 3-Fold Dilutions

CPM

Td

R In

corp

ora

ted

CTLL-2 Based mIL-4 Assay

mIL-4

0

2000

4000

6000

8000

10000

12000

1 2 3 4 5 6 7 8 9 10 11 12Serial 3-Fold Dilutions

kD

kD

36

4.7 Purifying Recombinant ProteinsProteins expressed in the BEVS may be either secreted or non-secreted proteins. Proteinsmay be isolated by any conventional means including polyacrylamide gel elec-trophoresis and affinity columns. The purification of GST and 6xHis tagged proteinsusing affinity columns is described in Chapter 5. Additional protein purification meth-ods are beyond the scope of this manual and are described in specialized manuals.24

Non-secreted Recombinant ProteinsNon-secreted proteins will remain in the cells. Cells should be pelleted and lysed torelease the protein.

Cell Lysate Preparation1. Harvest cells infected with recombinant virus 3 days pi.

2. Spin down cells at 2,500 × g for 5 min.

3. Resuspend cell pellet in ice-cold Insect Cell Lysis Buffer (Cat. No. 21425A)containing reconstituted Protease Inhibitor Cocktail (Cat. No. 21426Z). Use 1 ml of lysis buffer per 2 × 107 cells. Lyse cells on ice for 45 min.

4. Clear lysate from cellular debris by centrifuging at 40,000 × g for 45 min, or fil-ter lysate through a 0.22 µm filter.

5. Harvest clear supernatant, which should contain your recombinant protein.

6. Run an SDS-PAGE gel to determine the amount of your recombinant proteinin the total insect lysate.

Note: Insect cells infected with either wild-type AcNPV or with XylErecombinant virus should be lysed as a negative control for western blotanalysis. This lysate should lack the protein band derived from yourcloned gene of interest. If occlusion body-positive virus particles are usedfor infection, an additional intense band of 29 kD should be visible,which represents the polyhedrin protein of the wild-type virus. If XylEinfected cells are used, an additional band should be visible at 35 kD.

37

Secreted Recombinant ProteinsIn general, secreted recombinant proteins are much easier to purify than non-secretedproteins. The ratio between the recombinant protein and host proteins in the mediumis much higher than in lysates, especially when protein-free medium has been used.The general strategy for purifying secreted protein from the medium depends on thenature of the recombinant protein. If an antibody against the desired protein is avail-able in large quantities, it can be used for affinity purification. Otherwise, conventionalion-exchange chromatography matrices may perform equally well. Oftentimes, a per-centage of the protein tagged for secretion will remain cell-bound due to the intrinsicnature of the protein. Researchers should assay the cell lysate as well as the supernatantto determine the effectiveness of the secretion sequence with their protein. The poly-hedrin promoter driven gp67 secretion sequence (contained in PharMingen vectorspAcSecG2T and pAcGP67, Cat. No. 21469P and No. 21223P) may be useful in forcinga recombinant protein to secrete. Even non-secreted proteins are secreted using thissecretion sequence, unless the proteins are insoluble or are structural componentsinside the cell. The following protocol is suggested for harvesting protein-containingtissue culture supernatant:

1. If infected cells are growing attached to tissue culture plates, the supernatant can beharvested without detaching the cells from the plates. For infected cells maintainedin spinner culture bottles, the whole cell suspension should be harvested and cellsremoved by centrifuging the suspension at 5,000 × g for 10 min.

2. Clear the supernatant by centrifuging it at 10,000 × g for an additional 10 min.

3. This cleared medium will still contain a high titer stock of recombinant virusparticles which will most likely not influence the protein purification. If you areconcerned, you can pellet the virus by centrifuging the solution for 1 h at50,000 × g. A bluish-white pellet will appear, which represents the virus parti-cles. If protein-free insect medium is used, the supernatant will contain only afew secreted viral proteins, including the recombinant protein of your choice.However, it will still contain other contaminants including amino acids, sugarsand lipids.

39

5 Purification Systems

PharMingen has developed two Baculovirus Expression and Purification Kits, one usingthe 6xHis affinity tag and the other using the glutathione S-transferase (GST) affinitytag (Appendices B and C for kit components and product descriptions). The 6xHis and theGST Expression and Purification Kits (Cat. No. 21474K and No. 21475K) combine theadvantages of expressing functional and soluble recombinant proteins using Bac-ulovirus expression technology with the purification power of the 6xHis and GST affin-ity purification systems. Even under the highest expression levels, most 6xHis and GSTfusion proteins expressed in insect cells remain predominantly soluble.

5.1 6xHis Expression and Purification KitThe 6xHis Purification Kit contains the pAcHLT-A,B and C transfer vectors whichencode an N-terminal 6xHis tag followed by a proteolytic thrombin cleavage site, andan extended MCS. The MCS is in a different reading frame in each of the vectors tosimplify cloning (Appendix E). A protein kinase A site is present downstream of the6xHis tag which allows efficient in vitro phosphorylation of the recombinant proteinwithout destroying the proper folding and functional properties of the protein. Theexpressed recombinant protein will be a 6xHis fusion protein suitable for affinitypurification on Ni-NTA Agarose. Approximately 1 to 2 mg of 6xHis recombinant fusionprotein is routinely obtained per liter of insect cell culture.

The 6xHis purification method is based upon the high affinity of recombinant pro-teins equipped with a 6xHis affinity tag for Ni-NTA Agarose (a metal chelating agent).29

PharMingen selected Ni-NTA Agarose over other metal chelating resins due to its supe-rior affinity purification for 6xHis-tagged proteins. Ni-NTA Agarose has an extremelyhigh affinity for 6xHis residues. The binding affinity is approximately Kd=10–13, whichis higher than most antibody/antigen or enzyme/substrate interactions.30 The 6xHis-Ni2+NTA interaction can tolerate stringent washing conditions needed to remove non-specifically bound host proteins. Since the 6xHis tag is very small in size anduncharged under physiological pH conditions, it is not immunogenic and does notalter the folding, compartmentalization or biochemical properties of the recombinantprotein. Therefore it is usually not necessary to remove the 6xHis tag. However, the6xHis tag can be proteolytically cleaved from the recombinant protein at the throm-bin site located between the affinity tag and the MCS (Chapter 5.4).

6xHis fusion proteins may be purified using either the batch or column proceduresdetailed below. Batch binding for an extended time may be preferable when purifyingdilute proteins. We recommend including insect cell lysate or supernatant from aninfection using pAcHLT-XylE recombinant virus as a positive control in the affinitypurification procedure.

40

Figure 13. Expression, purification and cleavage of fusion proteins. A) Single-step protein purifi-cation methodology. Recombinant GST or 6xHis fusion protein is expressed in Sf cells, and total cell lysate ismixed with the appropriate affinity matrix. After centrifugation, the supernatant containing all untagged pro-teins is discarded, and the GST- or 6xHis-tagged proteins are eluted from the affinity matrix. B) The fusion tagscan be proteolytically cleaved from the recombinant protein at the thrombin cleavage site downstream of boththe GST and 6xHis sequences. C) Purification of Baculovirus-expressed GST-XylE and 6xHis-XylE from Sf9 cells.SDS-PAGE analysis of total cell protein containing GST-XylE (lane 1), purified GST-XylE (lane 2), total cell pro-tein containing 6xHis-XylE (lane 3) and purified 6xHis-XylE (lane 4). D) SDS-PAGE analysis of Baculovirus-expressed GST-XylE before and after thrombin cleavage. Total Sf9 cell protein containing GST-XylE (lane 1) waspurified (lane 2), and then incubated with thrombin, yielding GST and XylE (lane 3). GST was removed withglutathione agarose beads, resulting in purified XylE (lane 4). Residual GST was eluted from the glutathioneagarose beads (lane 5). GST-XylE (black arrow). XylE (gray arrow). GST (white arrow). “XylE” is a Pseudomonasputrida gene. GST-XylE was purified using glutathione agarose beads. 6xHis-XylE was purified using Ni-NTAagarose beads. Gels were stained with Coomassie blue.

Affin

ityM

atrix

GST

or 6xHIS

Your

ProteinGST or6xHis

Tag

Insect

Protein

A B

DC

Your

Protein

GST or

6xHisTag

GST or

6xHisTag

Your

Protein

Thrombin

Sf9+

GST

-Xyl

E

GST

-Xyl

E

Sf9+

6xH

is-X

ylE

6xH

is-X

ylE

kD

97

66

36

21

1 2 3 4

55

31

36

21

66

1 2 3 4 5

55

31

14

97

GST

-Xyl

E

Xyl

E +

GST

Xyl

E

GST

Sf9+

GST

-Xyl

E

kD

41

Batch Purification

1. Bead preparation for batch purification.

• Gently resuspend the Ni-NTA Agarose.

• Transfer the Ni-NTA slurry into a sterile tube.

• Centrifuge the slurry at 500 × g for 3–5 min to sediment the matrix.

• Carefully remove the supernatant by pouring off or aspirating.

• Wash the beads two times with 5–10 bead volumes of 6xHis Wash Buffer (Cat. No. 21472A) to remove the ethanol preservative.

• After each wash, centrifuge the slurry at 500 × g for 3–5 min to sediment thematrix.


• Add enough 6xHis Wash Buffer to the beads to make a 50% slurry.

2. Add lysate to equilibrated Ni-NTA Agarose for batch purification.

• Mix 10 volumes of insect cell lysate containing the recombinant 6xHis fusionprotein with 1 volume of the Ni-NTA Agarose. One ml of Ni–NTA Agarose willbind approximately 5–10 mg of 6xHis fusion protein.

• Incubate the slurry for 1 h at 4°C on a rocking platform.


• Draw off the supernatant. Keep the supernatant fractions to run on SDS- PAGEto determine whether the binding capacity of the Ni-NTA Agarose wasexceeded and whether all of the 6xHis fusion protein bound to the matrix.

Note: The 10:1 volume ratio mentioned above is an approximation.Expression levels should be empirically determined by the researcher.

3. Wash.

• Wash the bead slurry in 10 bead volumes of 6xHis Wash Buffer (Cat. No. 21472A).


• Discard the washes.

• Repeat wash steps until the wash A280 is less than 0.01 (approximately 4 washes).

Note: For stringent washes, 20-40 mM imidazole should be added to the6xHis Wash Buffer.

4. Elute the fusion protein with imidazole.

• Add the desired amount of imidazole to the 6xHis Elution Buffer (note below).

• Add 1 bead volume of the 6xHis Elution Buffer (Cat. No. 21476A) containingimidazole to the Ni-NTA Agarose from Step 3.

Please see Appendix B for kit components and product descriptions.

Materials Needed✓

42

• Gently mix the slurry.

• Incubate the slurry for 2 min at RT on a rocking platform.


• Collect the eluted fraction.

• Repeat this step two more times (each time increase the amount of imidazole)or use a linear gradient.

• Pool the three eluted fractions.

• Determine the purity and amount of protein by SDS-PAGE and spectropho-tometry.

Note: The optimal amount of imidazole (0.1 M to 0.5 M) needed for elu-tion will vary based upon the properties of the bound protein andshould be empirically determined by the researcher.

Column Purification1. Bead preparation for column purification.

• Gently resuspend the Ni-NTA Agarose.

• Place the Ni-NTA Agarose into a suitable chromatography column. One ml ofNi-NTA Agarose will bind approximately 5-10 mg of 6xHis fusion protein.

• Allow the beads to settle and the column to drain.

• Wash the beads two times with 3-5 bead volumes of 6xHis Wash Buffer(Cat. No. 21472A) to remove the ethanol preservative.

• Allow the column to drain but not to dry out.

Note: The 10:1 volume ratio mentioned above is an approximation.Expression levels should be empirically determined by the researcher.

2. Add lysate to equilibrated Ni-NTA Agarose for column purification.

• Apply the clarified lysate to the column.

• Adjust the column flow rate to a maximal 5 column volumes per hour. Keepthe flow-through fraction to run on SDS-PAGE to determine whether the bind-ing capacity of the Ni-NTA Agarose was exceeded.

3. Wash.

• Wash the column with 10 bead volumes of 6xHis Wash Buffer (Cat. No. 21472A).

• Allow the column to drain.

• Repeat wash step until the wash A280 is less than 0.01 (approximately 4 washes).


• Add the desired amount of imidazole to the 6xHis Elution Buffer (note below).

Note: For stringent washes, 20–40 mM imidazole should be added to the6xHis Wash Buffer.

43

4. Elute the fusion protein with imidazole.

• Add 3 bead volumes of the 6xHis Elution Buffer (Cat. No. 21476A), includingimidazole either as a step or a linear gradient, to the column.

• Adjust the column flow rate to a maximum of 1 ml/min per ml of beads.

• Allow the column to drain completely.

• Collect the eluted fractions.

Note: The optimal amount of imidazole (0.1 M to 0.5 M) needed for elu-tion will vary based upon the properties of the bound protein andshould be empirically determined by the researcher.

5.2 GST Expression and Purification Kit The GST Expression and Purification Kit (Cat. No. 21475K) contains the pAcGHLT-A, Band C transfer vectors which encode N-terminal GST and 6xHis tags followed by anextended MCS. The MCS is in a different reading frame in each of the vectors to sim-plify cloning (Appendix E). A protein kinase A site follows the 6xHis tag for convenientlabeling of the recombinant fusion protein. Because the GST vectors also contain a6xHis sequence the expressed recombinant protein will be a 6xHis-containing GSTfusion protein. The recombinant fusion protein can be affinity purified using eitherglutathione or Ni-NTA agarose beads. The GST tag can be proteolytically removed fromthe recombinant protein at the thrombin site located between the affinity tag and theMCS (Chapter 5.4).

The GST purification method is based on the remarkable selectivity and affinity ofrecombinant proteins equipped with a GST affinity tag for glutathione immobilized ona resin.31 The expressed GST fusion proteins may be purified without the use of deter-gents under completely non-denaturing conditions. Purifications to greater than 90%homogeneity are easily achieved in a single step by affinity chromatography using glu-tathione agarose beads. The affinity of GST for glutathione is so strong that it allows ahighly efficient separation of GST fusion proteins from contaminating polypeptideseven under non-denaturing conditions. GST fusion proteins may be purified usingeither batch or column procedures detailed below. Batch binding for an extended timemay be preferable when purifying dilute proteins. We recommend including insect celllysate from an infection using pAcGHLT-XylE recombinant virus as a positive controlin the affinity purification procedure.

Please see Appendix C for kit components and product descriptions.

Materials Needed✓

44

Batch Purification

1. Bead preparation for batch purification.• Determine the amount of glutathione agarose beads needed. One ml of glu-

tathione beads will bind approximately 5 mg of GST fusion protein. 1–2 mg ofGST fusion protein is routinely obtained per liter of insect cell culture.

• Gently resuspend the glutathione agarose beads (Cat. No. 21427B).

• Place the glutathione agarose beads as a slurry in a sterile tube.



• Wash the beads two times with 5–10 bead volumes of PBS Wash Buffer(Cat. No. 21428A) to remove the 20% ethanol preservative.

• After each wash, centrifuge the slurry at 500 × g for 3–5 min to sediment the matrix.

• Draw off the supernatant.

2. Add lysate to equilibrated glutathione beads for batch purification.

• Add the clarified lysate to the beads.

• Mix 10 volumes of insect cell lysate containing the recombinant GST fusionprotein of choice with 1 volume glutathione agarose beads.

• Incubate the slurry for 30 min at 4°C on a rocking platform.


• Draw off the supernatant. Keep the supernatant fractions to run on SDS-PAGEto determine whether the binding capacity of the glutathione beads wasexceeded and whether all of the GST fusion protein bound to the matrix.

Note: The 10:1 ratio mentioned above is an approximation. Expressionlevels should be empirically determined by the researcher.

3. Wash.

• Wash the slurry beads twice with 5 bead volumes of PBS Wash Buffer Cat. No. 21428A).



4. Elute the fusion protein with reduced glutathione.

• Add 1 bead volume of the reconstituted GST Elution Buffer (Cat. No. 21429Z andNo. 21455A) to the bead matrix. (See Appendix C for reconstitution of GST ElutionBuffer).

• Gently mix the slurry.

• Incubate the slurry for 2 min at RT.



• Repeat the elution steps two more times.

• Pool the three eluted fractions.

45

Column Purification1. Bead preparation for column purification.

• Determine the amount of glutathione agarose beads needed. One ml of glu-tathione beads will bind approximately 5 mg of GST fusion protein. 1–2 mg ofGST fusion protein is routinely obtained per liter of insect cell culture.

• Gently resuspend the glutathione agarose beads (Cat. No. 21427B).

• Repeat the elution steps two more times.

• Place the glutathione beads as a slurry in a suitable chromatography column.

• Allow the beads to settle and the column to drain.

• Wash the beads two times with 3–5 bead volumes of PBS Wash Buffer (Cat. No. 21428A)to remove the 20% ethanol preservative.

• Allow the column to drain but not to dry out.

2. Add lysate to equilibrated glutathione beads for column purification.

• Apply the clarified lysate to the column.

• Adjust the column flow rate to a maximal 5 ml/min per ml of beads. Keep theflow-through fraction to run on SDS-PAGE to determine whether the bindingcapacity of the glutathione beads was exceeded.

3. Wash.

• Wash the column twice with 5 bead volumes of PBS Wash Buffer (Cat. No. 21428A).

• Allow the column to drain.


4. Elute the fusion protein with reduced glutathione.

• Add 3 bead volumes of the GST Elution Buffer (Cat. No. 21429Z and No. 21455A)to the column.

• Adjust the column flow rate to a maximal 1 ml/min per ml of beads.

• Allow the column to drain completely.


Note: The addition of 150 mM NaCl, 5 mM CaCl2 (or for some proteins

5 mM MgCl2) or 0.1% β-mercaptoethanol to the GST Elution Buffer is

optional but may be required for the solubility of some proteins.

Dialyzing GST-Fusion Protein • Remove the free glutathione by dialysis against 100 volumes of 50 mM Tris-

HCl (pH 8.0) at 4°C.

• Dialyze a minimum of 4 h. Change dialysis buffer after 2 h.

Alternative GST Purification Procedure

In the aforementioned purification protocols, a second purification step is used to dis-sociate the GST from the protein of interest.31 In an alternate approach, only one affin-ity step is required.32, 33 Briefly, the affinity resin-bound GST fusion protein is equili-brated in thrombin cleavage buffer (1 wash) followed by the addition of 2 µg thrombin

46

per mg fusion protein. The reaction mixture is gently shaken on a rocking platform atRT for 20 min. In this reaction, the protein of interest is cleaved from the GST carrierprotein and can be recovered in the supernatant after brief centrifugation. The entireprocedure can be completed within a few hours and results in highly purified protein.Recovered protein should be stored at –80°C.

5.3 Checking Purity and Recovery of Recombinant Protein• Add 2 volumes of 3X SDS sample buffer to 1 volume of the clarified lysates.

• Run the samples on a 5–15% SDS-PAGE.

• Stain the gel with Coomassie blue.

• Check for the amount of recombinant fusion protein in the sample.Note: PharMingen’s monoclonal antibody to GST (clone G172-11381, Cat. No. 21481A) may be used to detect recombinant GST-fusion proteins.

5.4 Cleavage Fusion Proteins using Site-specific ProteasesThe His vector set (pAcHLT-A, -B, -C), the GST vector set (pAcGHLT-A, -B, -C) and sev-eral individual GST vectors (pAcG2T, pAcSecG2T) contain a thrombin cleavage site andpAcG3X contains a factor Xa cleavage site. These sites enable the proteolytic cleavageof the recombinant protein from the fusion partner. Removal of the fusion partner isoptional for many applications.

Thrombin Cleavage• Mix 1 mg of purified GST or 6xHis fusion protein containing a thrombin cleav-

age site with 200 µg (10 thrombin units) of bovine thrombin (Cat. No. 21430Zand No. 21454A).

• Incubate at RT for up to 12 h (in many cases a 20-60 min incubation will besufficient).

• GST and uncleaved GST fusion protein may be removed by directly adding2 volumes of glutathione-coupled resin (50% v/v) at the end of the cleavagereaction. Similarly, the 6xHis and uncleaved 6xHis fusion protein may beremoved by directly adding Ni-NTA Agarose at the end of the cleavage reac-tion. The sample is then incubated for 30 min at 4°C and centrifuged for10 min at 5,000 × g. The supernatant will contain the purified protein as wellas thrombin and can be stored frozen at –80°C. Some proteins may require theaddition of BSA or glycerol (final concentration 50%) for stability.

Note: Thrombin cleaves in 50 mM Tris-HCl buffer and does not requirespecific metal ions for its activity.34 However, Guan and Dixon have rec-ommended using a buffer containing 50 mM Tris-HCl (pH 8.0), 150 mMNaCl, 2.5 mM CaCl2 and 0.1% β-mercaptoethanol for efficient cleav-age.32 An efficient thrombin cleavage primarily depends on thesequence of the thrombin consensus site and the three-dimensionalstructure surrounding that site.

47

Design of the Thrombin Cleavage Site

The thrombin cleavage consensus site is XXP(K/R)*BB7, where X stands for hydrophobicapolar amino acids, P stands for proline, (K/R) symbolizes that either lysine or arginineworks in this position, and B stands for non-acidic amino acids. The asterisk (*) repre-sents the cleavage position which is at the carboxy-terminal side of the arginine or thelysine residue. The thrombin site used in the pAcGHLT and pAcHLT vectors is LVPR*GS.The desired gene is inserted at the BamHI site (at the amino acids GS), thrombin codingsequence. Cleavage by thrombin will then release the nearly authentic protein.

Factor Xa Cleavage• Mix 1 mg of your purified GST fusion protein containing a factor Xa cleavage

site with 10 mg factor Xa (factor Xa is not available from PharMingen).• Incubate at RT for up to 12 h.

5.5 Generating 32P-Labeled GST or 6xHis Fusion ProteinsPurified, radiolabelled 6xHis or GST fusion proteins can be generated using the pAcHLT(Cat. No. 21467P) and pAcGHLT (Cat. No. 21463P) vectors. Fusion proteins encodedby these vectors contain the peptide recognition sequence (RRASV) for the catalyticsubunit of cAMP-dependent protein kinase from heart muscle, between the 6xHis orGST tag gene and the foreign protein.36 The gene of interest should be cloned intoeither the pAcHLT or pAcGHLT vector and purified on either Ni-NTA or glutathioneagarose beads, respectively, as described earlier in this chapter.

In this protocol, the fusion protein is radiolabelled while it is still bound to the glutathione or Ni-NTA agarose beads.

1X HMK Buffer:20 mM Tris [pH 7.5]100 mM NaCl12 mM MgCl2

1X HMK STOP Buffer10 mM sodium phosphate [pH 8.0]10 mM sodium pyrophosphate10 mM EDTA

1 mg/ml BSANETN Buffer

20 mM Tris [pH 8.0]100 mM NaCl1 mM EDTA0.5% NP 40

cAMP-dependent protein kinase

1 mM dithiothreitol (DTT)

1 µCi/ml [γ-32P] ATP

23 G needle

Materials Needed✓

48

All steps should be carried out at 4°C.

1. Wash the agarose beads (coupled with the fusion protein of interest from thesections listed above) once with HMK buffer.

2. Centrifuge the slurry at 500 × g for 3–5 min to sediment the matrix.

3. Aspirate off the supernatant with a 23 G needle.

4. Resuspend the agarose beads in 2–3 volumes of HMK buffer containing 1 U/µl con-centration of the catalytic subunit of cAMP-dependent protein kinase, 1µCi/µl [γ-32P]ATP (6,000 Ci/mMol, 10 mCi/ml) and 1 mM DTT.

5. Allow the kinase reaction to proceed for 30 min.

6. Terminate the reaction by adding 1 ml of HMK Stop buffer.

7. Remove the supernatant.

8. Wash the agarose beads five times with NETN.

9. After the final wash, aspirate the residual supernatant.

10. Elute the labelled fusion protein according to the elution protocol in Step 4 ofChapters 5.1 or 5.2.

49

6 Generating Recombinant Baculovirusby Direct Cloning

The Baculovirus genome is generally too large to easily clone foreign genes directlyinto the genome. A newly developed method allows for the generation of recombi-nant Baculovirus by direct cloning of heterologous genes into the Baculovirus genome(Fig. 14). This method may be especially useful in generating high diversity expressionlibraries in Baculovirus.21 Two modified AcNPV DNAs, vEHuni and vECuni, have beenconstructed containing either the hsp70 promoter of Drosophila melanogaster, or ahybrid minimal synthetic late/enhanced polyhedrin promoter PcapminXIV, respectively,(Fig. 15).21 Cleavage of Bsu36I sites produces linear Baculovirus DNA with overhang-ing TTA ends, which after incubation with dTTP and the Klenow fragment of DNApolymerase I leaves TT overhanging ends. The gene to be cloned must be flanked byEcoRI sites which must be partially filled in with dATP and the Klenow fragment togenerate AA overhanging ends. The gene can then be cloned directly into vEHuni orvECuni and transfected into insect cells to generate recombinant virus. PharMingensells vEHuni and vECuni reagent sets containing either undigested, untreated vEHunior vECuni DNA with vEHuni or vECuni high titer stock, respectively (Appendix D).

Prepare vEHuni or vECuni Baculovirus DNA

1. Digest the vEHuni or vECuni Baculovirus DNA with Bsu36I for 16 h at 37°C.

2. Incubate 0.1–1 µg of digested vEHuni or vECuni with 5 U Klenow DNA poly-merase I and 0.5 mM dTTP in the presence of 20 µl Reaction Buffer A for 15 minat 30°C.

3. Stop the reaction by heating to 75°C for 10 min or by adding 1 µl of 0.5 M EDTA.

4. Extract with one volume of 25:24:1 phenol/chloroform/isoamyl alcohol (1:1),ethanol precipitate and resuspend DNA in 10 µl of 1 X TE buffer.

0.5 µg vEHuni (Cat. No. 21524P) or vECuni (Cat. No. 21527P) linearized,

partially filled-in Baculovirus DNA

0.5 mM dTTP

0.5 mM dATP

0.1-1 µg purified gene of interest, flanked by EcoRI sites

Reaction buffer A, (10 mM Tris-HCl buffer, pH7.5, 10 mM MgCl2)

10 U large fragment of DNA Polymerase I (Klenow)

25:24:1 phenol/chloroform/isoamyl alcohol

1X TE buffer

2 U T4 DNA ligase

Materials Needed✓

50

Figure 14. Strategy for directly cloning EcoRI fragments into the AcNPV genome. On the left,an AcNPV DNA has been altered to contain 2 Bsu36I sites (vEHuni or vECuni). After digestion with Bsu36I,the DNA is treated with dTTP and Klenow DNA polymerase I to generate a linear viral DNA with TT over-hanging ends. On the right, a foreign gene (PCR or cDNA synthesis product) with flanking EcoRI sites isdigested with EcoRI. The resultant overhanging ends are digested with dATP and Klenow DNA polymerase Ito generate AA overhanging ends. The compatible viral DNA and heterologous gene DNA are then com-bined, ligated and transfected into insect cells.

vEHuni or vECuni

Bsu36ICCTAAGGGGATTCC

CCTTAGGGGAATCC

CCGGATT

TTAGG CC

Vector

GeneGAATTCCTTAAG

EcoRI

AATTC G

GCTTAAGene

GAACTTAA AAG

ligate

TTAGG TCC

Incubate w/dTTP + Klenow

Bsu36I

Digest w/Bsu36IGAATTCCTTAAG

EcoRI

Digest w/EcoRI

Incubate w/dATP + Klenow

CCTGGATT

CCTAATTCGGATTAAG

GAATTAGGCTTAATCC

Gene

Gene

AATTC

vEHuni or vECuni

vEHuni or vECuni

vEHuni or vECuni

egtpromoter egt

egt

egt promoter

egt

egt promoter

egt promoter egt

51

Prepare gene fragment

1. Digest the vector containing the gene, PCR or cDNA synthesis product of interestwith EcoRI for 16 h at 37°C. Isolate the gene of interest containing flanking sites,EcoRI, by gel purification.24

Note: If gene of interest doesn’t contain flanking EcoRI sites, a PCR withspecific EcoRI containing primers can be used to insert flanking EcoRIsites. Alternatively, EcoRI linkers can be ligated to the purified gene.24

2. Incubate 0.1–1 µg EcoRI-digested and purified gene, PCR or cDNA synthesis prod-uct of interest in presence of 5 U Klenow DNA polymerase I and 0.5 mM dATP in20 µl 1X Reaction buffer A for 15 min at 30°C.

3. Stop the reaction by heating to 75°C for 10 min or by adding 1 µl of 0.5 M EDTA.

4. Extract with one volume of 25:24:1 phenol/chloroform/isoamyl alcohol (1:1),ethanol precipitate and resuspend DNA in 10 µl of 1 X TE buffer.

Ligate vEHuni or vECuni with treated gene fragment

Mix 0.5 µg of vEHuni or vECuni DNA with 0.1–1 µg of the treated gene fragmentfrom above (about 1:60 molar ratio of viral DNA to gene fragment) and 2 units ofT4 DNA ligase overnight at 15°C. The ligated AcNPV DNA is now ready to betransfected into susceptible insect cells.

Figure 15. Baculovirus vectors for direct cloning. A) Design of vEHuni direct cloning vector: An hsp70promoter from Drosophila melanogaster, and a multiple cloning site (MCS) containing two Bsu36I restriction siteswere inserted into the nonessential Ecdysteroid UDP-glucosyltransferase (egt) gene of the wild-type AcNPVgenome. Recognition sites within the MCS are bracketed. B) Design of vECuni direct cloning vector. A PcapminXIVhybrid late/very late promoter and a MCS containing 2 different Bsu36I restriction sites were inserted into thenonessential egt gene of the wild-type AcNPV genome. Recognition sites within the MCS are bracketed.

CCTAAGGCCTGCAGGCCCGGGCCTTAGGCCTGCAGG

Srfl

Bsu36I Sse8387I Bsu36I Sse8387I

hsp 70promoteregt egt

vEHuni

vECuni

CCTAAGGCCTGCAGGCCCGGGCCTTAGGCCTGCAGG

Srfl

Bsu36I Sse8387I Bsu36I Sse8387I

CAPminXIV egtegt

A

B

53

7Troubleshooting

Throughout this manual, protocols and suggestions have been made to optimize theoutcome during the various steps involved in using BEVS. However, as in all experi-mental systems, difficulties do occur. This trouble-shooting guide has been developedboth from our laboratory experience with BEVS, and from questions received by ourtechnical service department. We always welcome additional suggestions from users.

7.1 Cloning Inserts into Baculovirus Transfer VectorsIn any cloning system, there may be occasional problems with inserting foreign DNAinto a vector. This may be due to the reagents or to the characteristics of the DNA frag-ment itself which may make it difficult to insert into a particular restriction enzymesite. Refer to references 24 and 25 for comprehensive cloning manuals.

Most common problems arising from inserting foreign DNA.

• Suboptimal purity of Baculovirus Transfer Vector stock: Purify vector DNAaccording to one of the methods described in Chapter 4.3. Check that DNA isreadily cleavable with restriction enzymes and appears clean on an agarose gel.DNA should have a 260/280 nm ratio of 1.6-1.8.

• High background of non-recombinant bacterial colonies after transforma-tion of E. coli: Incomplete restriction digestion of transfer vector will maskrecombinants. Use agarose gel electrophoresis to check transfer vector afterrestriction digestion.

Be sure to use an optimized molar ratio of insert:vector.

• Difficulty in cloning in large inserts: Use a smaller transfer vector likepAcSG2 rather than a larger vector like pVL1392/1393.

7.2 Insect Cell CultureHealthy insect cell cultures are essential to obtaining success with BEVS. Several types ofproblems may be encountered with cell culture, fortunately most are easily addressed.

Cells do not double every 24 h. Cells are floating or enlarged. Cells havereduced protein production.

• Cells are unhealthy.

– Cells that have been left too long between passages do not thrive well, andmay be floating in the medium. Cells can still be subcultured at this stage,but may not be optimal for experimental work. To obtain healthy, logphase cells for experimental work, subculture when cells have just formeda confluent monolayer.

– Cells continuously passaged for more than 6 months may show reducedprotein production. Retrieve low passage cells from liquid nitrogen.

54

– Check for microbial contamination under high magnification (40X).Mycoplasma contamination can be easy to miss in the early stages. Cellswill have a grainy appearance with motile organisms inside. Cells that arecontaminated with wild-type virus will have polyhedra inside.

• The medium or temperature may not be correct.

• The cells are infected by Baculovirus particles.

Cells are sticking to the plate and it is difficult to remove them for subculture.

• Cells may be seeded too thinly. Sf9 cells rely on growth factors from one anotherfor healthy logarithmic growth and tend to adhere more tightly when dilute. Ini-tial cell density should not be less than 30%. Allow cells to overgrow. Usually over-grown cells will be easier to remove. Pipet streams of media over cells to dislodgethem.

• The media may contain non-heat inactivated FBS. It has been reported thatnon-heat inactivated FBS may increase how vigorously Sf cells stick to tissueculture plates. PharMingen’s TNM-FH media (Cat. No. 21227M) contains heatinactivated FBS.

Cells are growing very slowly in protein-free media.

• Attempt to wean only healthy, log phase cells into protein-free media. It may beeasier to wean cells of lower rather than higher passage.

• An adjustment period ranging from a few day to several weeks should be allowedfor when Sf9 cells are subjected to any variation in environmental conditions.

• In protein-free medium, cells may attach more firmly and doubling time may vary.

7.3 Co-transfectionWe strongly recommend using PharMingen’s Transfection Buffer Set (Cat. No. 21483A)for co-transfections. Each batch is rigorously tested in a co-transfection using Baculo-Gold™ DNA and a Baculovirus Transfer Vector.

There is no precipitate.

• Check pH of Transfection Buffer B. It should be pH 7.1 ± 0.05. If buffer is oldor stored improperly the pH may be altered.

Note: The precipitate may not be identifiable under a microscope. A suc-cessful precipitation is characterized by a milky white color which isvisible to the naked eye.

All the cells die within a day after transfection.

• Check purity of your plasmid DNA. Dirty DNA can cause cell death. See “purifying vectors” in Chapter 4.3.

• For a suspected microbial contamination, ethanol precipitate the DNA againand resuspend in freshly prepared, sterile, TE buffer.

When using lipid-based transfection kits.

• Check that you did not use media with FBS. Use serum-free medium forlipofection.

• There may be lot-to-lot variations between different batches of lipofection.

55

7.4 Plaque AssayThis technique often poses a challenge for new users. It is essential to use cells that areexponentially growing and at least 90% viable. Cells should adhere to tissue culturedishes within about 2 h after plating. Otherwise, discard dishes and obtain fresh cells.Use extreme caution to avoid dislodging cells when replacing media prior to inoculat-ing cells with viral dilutions.

Cells are dead.

• The temperature of the agarose overlay may have been too high when it wasadded to cells. Make sure the agarose is cooled to 45°C prior to adding to RT(22-27°C) medium.

• FBS may have been omitted from the overlay medium.

There are cracks in the agarose overlay.

• Make sure that the medium containing the virus inoculum is totally removedafter the 1 h incubation period. Any liquid remaining on the cells will inter-fere with the gelling process and produce cracks. Cells underneath the crackswill not be properly covered.

No visible plaques.

• Check to be sure your agarose has a low sulfide concentration and is low-melt-ing-point.

• Be sure you did not initially plate out too many cells which could cause thecells to overgrow the plaques. Initial density should be 70%.

• Has enough time passed to give the plaques time to grow? Plaques take6–10 days to appear.

• Does your viral dilution have a sufficient titer? A viral dilution that is too lowwill not yield visible plaques. Repeat assay with lower dilutions of virus.

• The viral dilution may be too high, resulting in a complete cell lysis.

Plaques are so small that they are barely visible.

• Cells seeded too densely will reach confluence too quickly and inhibit virusreplication resulting in small plaques. Try reducing initial cell plating densityfrom 70% to 50% confluent.

• Wait several days to see if plaques become larger.

Plaques are mostly on the perimeter of the cell culture dish.

• Make sure that the virus inoculum is added to the center of the cell monolayer.Gently rock dishes 3 or 4 times during the 1 h viral incubation period to makesure that virus evenly covers the monolayer.

There are holes in the cell monolayer that resemble plaques and it is difficultto distinguish holes from plaques.

• Holes result in damage to the cell monolayer. Use caution to avoid dislodgingcells when removing medium from the plates, adding fresh medium to theplates, inoculating plates with virus and adding the overlay medium.

56

7.5 Virus Amplification

No visible signs of infection.

• Check the initial density of cells. Cells seeded too densely will overgrow, mask-ing the signs of the infection.

• Virus titer may be low. Amplify the viral stock to increase titer.

7.6 Recombinant Protein ProductionThe most common methods for verifying protein expression include analysis ofCoomassie blue-stained polyacrylamide gels, Western blot analysis, immunoprecipita-tion, and indirect immunofluorescence. When protein is not detected, users may needto analyze the viral DNA to check the integrity of the foreign insert, analyze RNAlevels, use a more sensitive protein detection method, and/or optimize their experi-mental system.

Low expression.

• Analyze the recombinant Baculovirus genome by Southern hybridization or bysequencing the DNA across cloning junctions to verify that the foreign proteincoding sequences have been inserted correctly.

• Check whether the protein coding sequence of the insert is cloned in theproper translational frame by DNA sequence analysis.

• Conduct a concurrent time course of mRNA expression levels by Northernhybridization and of protein expression to determine the correlation betweenthe mRNA and protein expression.

• A point mutation in the polyhedrin promoter may reduce mRNA levels.

• Amplify your recombinant Baculovirus infection stock to at least 108 pfu/ml.

• Optimize the infection period to maximize protein expression.

• Conduct a time course to determine the optimal time for harvesting yourrecombinant protein.

• Optimize the multiplicity of infection (MOI). For protein expression, the MOIshould generally be between 3–10. MOIs that are too high or too low mayaffect protein expression.

• Grow and infect insect cells on plates instead of spinner bottles.

• Seed cells at a different cell density.

• Try a different insect cell line for protein expression.

• Use a different insect cell culture medium.

• Maintain a constant temp of 27°C during protein expression.

• Multiple passages of viral stock may cause a loss of your gene of interest.Always keep a stock of low passage virus to use as an infection source.

• Sf9 cells in continuous culture for more than a year may lose the ability toexpress foreign proteins efficiently.

• Your protein may be mildly toxic to the insect cell. Harvest the infected cellsearlier in the infection cycle.

57

• Some proteins may not be stable in virus-infected cells. Compare mRNA andprotein levels.

• Membrane-bound glycoproteins and secreted proteins may be produced at lowerlevels than proteins that remain in the cytoplasm or are targeted to the nucleus.

• Check for your protein in both the cell pellet and supernatant.

7.7 6xHis Expression and Purification SystemWe recommend using the pAcHLT-XylE control vector (Cat. No. 21471P) as a positivecontrol in this system.

6xHis protein does not bind to the Ni-NTA Agarose.

• Check pH of all buffers and solutions.

• Check that imidazole concentrations are not too high.

• Check that reducing agents such as DTT and DTE were not used. They reducethe Ni2+ ions and cause them to dissociate from the Ni-NTA Agarose.

• Reduce or eliminate the use of chelating agents such as EDTA and EGTA. Theymay chelate the Ni2+ ions and cause them to dissociate from the Ni-NTA Agarose.

• Check that the 6xHis tag is present and in the correct reading frame bysequencing the ligation junctions.

• The 6xHis tag may be hidden due to folding of the protein.

6xHis protein is insoluble or nonfunctional.

• Review the biological properties of your protein. Does your protein require acell-specific or tissue-specific modification enzyme not present in insect cells?

• If your protein requires post-translational modifications, using a late promoter(the basic protein or 39K protein promoters) may result in better modificationsand possibly active protein.

• Harvest infected insect cells earlier in the infection cycle.

• Infect insect cells with a lower MOI.


• Co-express a dimerization partner which may keep your protein in solution.

• Try to solubilize your protein with detergents or by denaturing.

6xHis protein precipitates during purification.

• Check that the temperature is not too low. Purification may be repeated at RT.

• To prevent the purified protein from aggregating, solubilizing reagentssuch as Triton™-X100 (0.1-2.0%), Tween™-20 (0.1–2.0%), β-mercaptoethanol(10 mM) or NaCl (0.1–1 M) may be added to all buffers.

58

Inefficient or no elution of 6xHis.

• The elution conditions may be too mild. Determine optimal elution conditionsby varying pH (generally 4.5–5.9) and imidazole concentrations (0.1–0.5 M).

• Protein has precipitated on the agarose. Elute under denaturing conditions. Avoidhigh local concentrations of proteins by binding and eluting using a batch format.

6xHis eluate contains contaminating proteins.

• Reduce the amount of Ni-NTA Agarose to decrease non-specific binding ofcontaminating proteins.

• Determine optimal elution conditions by varying pH (generally 4.5–5.9)and imidazole concentrations (0.1–0.5M).

• Increase salt (0.1–1 M NaCl) or detergent (0.1–1% Triton™-X100) levels to disrupt non-specific protein interactions.

• Increase glycerol concentrations up to 30% in the 6xHis Elution Buffer toreduce hydrophobic interactions.

• Add 1–10 mM β-mercaptoethanol to the lysis buffer. This will reduce disulfidebonds which may link contaminating host proteins to the 6xHis fusion protein.

6xHis protein elutes in the wash buffer.

• The wash stringency may be too high. Increase the pH or lower the concen-tration of imidazole.

• Check pH of all buffers and solutions.

• Check that reducing agents such as DTT and DTE were not used. They mayreduce the Ni2+ ions and cause them to dissociate from the Ni-NTA Agarose.

• Reduce or eliminate the use of chelating agents such as EDTA and EGTA. Theymay chelate the Ni2+ ions and cause them to dissociate from the Ni-NTA Agarose.

• The 6xHis tag may be partially hidden due to protein folding. Reduce thewashing stringency. Purify under denaturing condition.

7.8 GST Expression and Purification SystemWe recommend using the pAcGHLT-XylE control vector (Cat. No. 21470P) as a positivecontrol in this system.

GST Fusion Protein expression is low or absent.

• Refer to Troubleshooting Section 7.6.

GST Fusion Protein is insoluble36 or non-functional.

• Harvest infected insect cells earlier in the infection cycle.

• Infect insect cells with a lower MOI.


• Review the biochemical properties of your protein.

• Co-express a dimerization partner which may keep your protein in solution.

59

GST Fusion Protein does not bind to glutathione agarose beads.

• Check binding of the unfused GST protein and the GST-XylE protein.

• In case of weak binding due to altered conformation of the GST fusion protein,lower binding temperature to 4°C and limit the number of washes.

Inefficient elution of GST Fusion Protein.

• Make fresh elution buffer from dry reduced glutathione.

• Increase incubation time of beads in elution buffer.

• Increase volume of elution.

• Increase glutathione concentration in elution buffer.

• Add increasing amounts of NaCl to elution buffer (100-500 mM).

• Try an overnight elution at RT or at 4°C.

7.9 Thrombin Cleavage

Inefficient thrombin cleavage.

• Increase the amount of thrombin (up to 100 cleavage units/mg of fusion pro-tein). We recommend 50 cleavage units/mg of fusion protein.

• Add heparin (1–20 mM) to your thrombin cleavage buffer. Heparin has beenshown to enhance thrombin cleavage.37

• Increase the incubation temperature. If your protein is stable at 37°C and thepreparation is low in proteases, try a 4-16 h incubation at 37°C.

• Increase the incubation time. If your protein is not degraded by extensive incuba-tion in presence of thrombin, increase the reaction time (up to 24 h).

• Verify the existence of a functional thrombin site by sequencing. Ensure thatyour cloning strategy has not altered the consensus thrombin site. If thecloning of your desired protein destroyed the thrombin site, you will need toreclone your gene using a different cloning site or strategy.

• The heat shock step is critical. Heat shock at 42°C for 45–55 seconds.

61

8References

1. O’Reilly, D., L.K. Miller and V.A. Luckow. 1992. Baculovirus expression vectors: A labora-tory manual. W.H. Freeman and Company, New York, NY.

2. Kidd, I.M. and V.C. Emery. 1993. The use of baculoviruses as expression vectors. AppliedBiochemistry and Biotechnology. 42:137-159.

3. Matthews, R.E.F. 1982. Classification and nomenclature of viruses. Fourth report of theinternational committee on taxonomy of viruses. Karger, Basel.

4. Burgess, S. 1977. Molecular weights of lepidopteran baculovirus DNAs: Derivation byelectron microscopy. J. Gen. Virol. 37:501-510.

5. Ayres, M.D., S.C. Howard, J. Kuzio, M. Lopez-Ferber and R.D. Possee. 1994. The com-plete DNA sequence of Autographa californica nuclear polyhedrosis virus. Virology.202:586-605.

6. Kool, M. and J.M. Vlak. 1993. The structural and functional organization of the Auto-grapha californica nuclear polyhedrosis virus genome. Arch. Virol. 130:1-16.

7. Harrap, K.A. 1972. The structure of nuclear polyhedrosis viruses. The inclusion body.Virology. 50: 114-123.

8. Rohrmann, G.F. 1986. Polyhedrin structure. J. Gen. Virol. 67: 1499-1513.9. Summers, M.D., and G.E. Smith. 1978. Baculovirus structural polypeptides. J. Virol.

84:390-402.10. Smith, G.E., M.J. Fraser and M.D. Summers. 1983. Molecular engineering of the Auto-

grapha californica nuclear polyhedrosis virus genome: Deletion mutations within thepolyhedrin gene. J. Virol. 46:584-593.

11. Marston F.A. 1986. The purification of eukaryotic polypeptides synthesized inEscherichia coli. J. Biochem. 240:1-12.

12. Hoss, A., I. Moarefi, K.H. Scheidtmann, L.J. Cisek, J.L. Corden, I. Dornreiter, A.K.Arthur and E. Fanning. 1990. Altered phosphorylation pattern of simian virus 40T antigen expressed in insect cells by using a baculovirus vector. J. Virol. 64:4799-4807.

13. Kloc, M,. B. Reddy, S. Crawford and L.D. Etkin, 1991. A novel 110-kDa maternal CAAXbox-containing protein from Xenopus is palmitoylated and isoprenylated whenexpressed in baculovirus. J. Biol. Chem. 266:8206-8212.

14. Kuroda, K., M. Veit and H.D. Klenk, 1991. Retarded processing of influenza virushemagglutinin in insect cells. Virology. 180:159-165.

15. Baixeras, E., S. Roman-Roman, S. Jitsukawa, C. Genevee, S. Mechiche, E. Viegas-Pequig-not, T. Hercend and F. Triebel. 1990. Cloning and expression of a lymphocyte activa-tion gene (Lag-1). Mol. Immunol. 27:1091-1102.

16. Brandt-Carlson, C. and J.S. Butel. 1991. Detection and characterization of a glycopro-tein encoded by the mouse mammary tumor virus long terminal repeat gene. J. Virol.65:6051-6060.

17. Caroni, P., A. Rothenfluh, E. McGlynn and C. Schneider. 1991. S-cyclophilin. J. Biol.Chem. 266:10739-10742.

18. Christensen, J., T. Storgaard, B. Bloch, S. Alexandersen and B. Aasted. 1993. Expressionof Aleutian mink disease parvovirus proteins in a baculovirus vector system. J. Virol.67:229-238.

19. Mattion, N.M., D.B. Mitchell, G.W. Both and M.K. Estes. 1991. Expression of rotavirusframes of genome segment 11. Virology. 181:295-304.

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20. Hsu, C.Y., D.R. Hurwitz, M. Mervic and A. Zilberstein. 1991. Autophosphorylation ofthe intracellular domain of the epidermal growth factor receptor results in differenteffects on its tyrosine kinase activity with various peptide substrates. J. Biol. Chem.266:603-608.27.

21. Lu, A. and L.K. Miller. 1996. Generation of recombinant baculoviruses by directcloning. Biotechniques. 21:63-68.

22. Serrano, M.,G.J. Hannon and D. Beach. 1993. A new regulatory motif in cell-cycle con-trol causing specific inhibition of cyclin D/CDK4. Nature. 366:704-707.

23. Frankel, A., H. Roberts, L. Afrin, J. Vesely and M. Willingham. 1994. Expression of ricinB chain in Spodoptera frugiperda. Biochem. J. 303:787-794.

24. Ausubel, F.M., R. Brent, R. E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith andK. Struhl, eds. 1994. Current protocols in molecular biology. (2 vols: 1. Molecular biology-technique, 2. Molecular biology-laboratory manuals). Current Protocols. Greene Pub-lishing Associates, Inc. and John Wiley and Sons, Inc. USA.

25. Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular cloning: A laboratory manualsecond edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

26. Kitamura, T., T. Tange, T. Terasawa, S. Chiba, T. Kuwaki, K. Miyagawa, Y. Piao, K. Miya-zono, A. Urabe and F. Takaku. 1989. Establishment and characterization of a uniquehuman cell line that proliferates dependently on GM-CSF, IL-3, or Erythropoietin. J.Cell Physiol. 140:323-334.

27. Gillis, S., M.M. Ferm, W. Ou and KA. Smith. 1978. T cell growth factor: Parameters ofproduction and a quantitative microassay for activity. J. Immunol. 120:2027-2032.

28 . Grabstein, K., J. Eisenman, D. Mochizuki, K. Shanebeck, P. Conlon, T. Hopp, C. Marchand S. Gillis. 1986. Purification to homogeneity of B cell stimulating factor. J. Exp. Med.163:1405-1414.

29. Janknecht, R., G. de Martynoff, J. Lou, R. Hipskind, A. Nordheim and H.G.Stunnenberg. 1991. Rapid and efficient purification of native histidine-tagged proteinexpressed by recombinant vaccinia virus. Proc. Natl. Acad. Sci. USA. 88:8972-8976.

30. Crowe, J. and K. Henco. 1992. The QIAexpressionist, QIAexpress: The high level expression& protein purification system. QIAGEN GmbH, QIAGEN Inc.

31. Smith, D.B. and K.S. Johnson. 1988. Single-step purification of polypeptides expressedin Escherichia coli. as fusions with glutathione S-transferase. Gene. 67:31-40.

32. Guan, K. and J.E. Dixon. 1991. Eukaryotic proteins expressed in Escherichia coli: Animproved thrombin cleavage and purification procedure of fusion proteins withglutathione S-transferase. Anal. Biochem. 192 262-267.

33. Gearing, D.P., N.A. Nicola, D. Metcalf, S. Foote, T.A. Willson, N.M. Gough andR.L. Williams. 1989. Production of leukemia inhibitory factor in Escherichia coli by anovel procedure and its use in maintaining embryonic stem cells in culture. Bio. Tech-nol. 7:1157-1161.

34. Wu, Hai-Feng, G.C. White II, E.F. Workman, Jr., J.W. Jenzano and R.L. Lundblad. 1992.Affinity chromatography of platelets on immobilized thrombin: Retention of catalyticactivity by platelet-bound thrombin. Thrombosis Res. 67:419-427.

35. Kaelin Jr, W.G., W. Krek, W.R. Sellers, J.A. DeCaprio, F. Ajchenbaum,C.S. Fuchs, T. Chittenden, Y. Li, P.J. Farnham, M.A. Blanar, D.M. Livingston andE.K. Flemington. 1992. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 70:351-364.

36. Frangioni, J.V. and B.G. Neel. 1993. Solubilization and purification of enzymatically ac-tive glutathione S-transferase (pGEX) fusion proteins. Analytical Biochem. 210:179-187.

37. Chang, J.Y. 1985. Thrombin specificity. Requirement for apolar amino acids adjacentto the thrombin cleavage site of polypeptide substrate. Eur. J. Biochem. 151:217-224.

38. Smith, G.E., G. Ju, B.L. Ericson, J. Moschera, H-W Lahm, R. Chizzonite, and M.D. Sum-

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mers. 1985. Modification and secretion of human interleukin 2 produced in insectcells by a baculovirus expression vector. Proc. Natl. Acad. Sci. USA. 82:8404-8408.

39. Vaughn, J.L., R.N. Goodwin, G.J. Thompkins and P. McCawley. 1977. The establish-ment of two cell lines from the insect Spodoptera frugiperda. In Vitro. Cell Devel. Biol.13:213-217.

40. Davies, A.H., J.B.M. Jowett and I.M. Jones. 1993. Recombinant baculovirus vectorsexpressing glutathione-S-transferase fusion proteins. Bio. Technol. 11:933-936.

41. Hill-Perkins, M.S. and R.D. Possee. 1990. A baculovirus expression vector derived fromthe basic protein promoter of Autographa californica nuclear polyhedrosis virus. J. Gen.Virol. 71:971-976.

42. Livingstone, C. and I. Jones. 1989. Baculovirus expression vectors with single strandcapability. Nucl. Acids Res. 17:2366.

43. Whitford, M., S. Stewart, J. Kuzio and P. Faulkner. 1989. Identification and sequenceanalysis of a gene encoding gp67, an abundant envelope glycoprotein of the bac-ulovirus Autographa californica nuclear polyhedrosis virus. J. Virol. 63 (3):1393-1399.

44. Stewart L.M.D., M. Hirst, M.L. Ferber, A.T. Merryweather, P.J. Cayley and R.D. Possee.1991. Construction of an improved baculovirus insecticide containing an insect-specific toxin gene. Nature. 352:85-88.

45. Kain, S.R., M. Adams, A. Kondepudi, T.-T. Yang, W.W. Ward and P. Kitts. 1995. Greenfluorescent protein as a reporter of gene expression and protein localization. BioTech-niques. 19:650-655.

46. Chalfie, M., Y. Tu, G. Euskirchen, W.W. Ward and D.C. Prasher. 1994. Green fluores-cent protein as a marker for gene expression. Science. 263:802-805.

47. Heim, R., D.C. Prasher and R.Y. Tsien. 1994. Wavelength mutations and posttrans-lational autoxidation of green fluorescent protein. Proc. Natl. Acad. Sci. USA.91:12501-12504.

48. Delagrave, S., R.E. Hawtin, C.M. Silva, M. M. Yang and D.C. Youvan. 1995. Red-shiftedexcitation mutants of the green fluorescent protein. Bio. Technol. 13:151-154.

49. Crossen, R.E., C. Torres, H. Liu, L.S. Stein, S. Singh and S. Gruenwald. 1996. Separationof baculovirus-expressed green fluorescent protein (GFP) variants using fluorescence-activated cell sorting. J. NIH Res., Application Note. 8:64.

50. Wu, C., H. Liu, R. Crossen, S. Gruenwald and S. Singh. 1997. Novel green fluorescentprotein (GFP) baculovirus expression vectors. Gene 190:157-162

51. Belyaev, A.S. and P. Roy. 1993. Development of baculovirus triple and quadrupleexpression vectors: Co-expression of three or four bluetongue virus proteins andthe synthesis of bluetongue virus-like particles in insect cells. Nucleic Acids Res.21:1219-1223.

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Appendix ABaculoGold™ Starter Package and Transfection Kit

Contents of the BaculoGold™ Starter Package (Cat. No. 21001K)

Description: The BaculoGold™ Starter Package (Cat. No. 21001K) contains the crit-ical components necessary to begin using the BEVS. The package provides sufficientmaterials for five co-transfections.

Contents of the BaculoGold™ Transfection Kit (Cat. No. 21100K)

Description: The BaculoGold™ Transfection Kit (Cat. No. 21100K) contains thecritical basic components necessary to use the BEVS. Supplementary components maybe purchased separately. The kit provides sufficient materials for five co-transfections.

Description of Provided Reagents

Linearized BaculoGold™ DNA (Cat. No. 21100D) 2.5 µg in 25 µl

BaculoGold™ DNA is a modified linearized AcNPV Baculovirus DNA which contains a lethal dele-tion and does not code for viable virus. Co-transfection of BaculoGold™ DNA with a complement-ing plasmid construct, including pVL1393/1392, rescues the lethal deletion of this virus DNA andresults in production of viable virus particles in transfected insect cells. When using BaculoGold™

DNA for co-transfection, more than 99% of all virus particles will be recombinant and will expressthe gene of interest. For one transfection, 5 µl (500 ng) of linearized BaculoGold™ DNA should beused in combination with 2-5 µg of purified recombinant Baculovirus transfer DNA (e.g., pAcGHLT,pAcHLT). Store at 4°C.

Cat. No. Component Contents

21100D Linearized BaculoGold™ Baculovirus DNA 2.5 µg21201P pVL1392/1393 Baculovirus Transfer Vector Set 5 µg each21484P pVL1392-XylE Baculovirus Control Vector 5 µg21103E AcNPV Wild-Type High Titer Stock Solution 1 ml21483A Transfection Buffer A & B Set 5 ml eachN/A Baculovirus Procedures & Methods Manual 1 manual


21100D Linearized BaculoGold™ Baculovirus DNA 2.5 µg21201P pVL1392/1393 Baculovirus Transfer Vector Set 5 µg each21484P pVL1392-XylE Baculovirus Control Vector 5 µg21103E AcNPV Wild-Type High Titer Stock Solution 1 ml21227M PharMingen's TNM-FH Medium 1 liter21300L Live Sf9 Insect Cells (1 × 107) 1 flask21483A Transfection Buffer A & B Set 5 ml each21403A Agarplaque-Plus™ Agarose 50 gN/A Baculovirus Procedures & Methods Manual 1 manual

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pVL1329/1393 Transfer Vector Set (Cat. No. 21201P) 5 µg in 50 µl, each vector

The pVL1392 (Cat. No. 21485P) and pVL1393 (Cat. No. 21486P) Baculovirus Transfer Vectors aresold only as a set (Cat. No. 21201P). pVL1392 and pVL1393 contain an extended MCS in oppositeorientation for simplified cloning. The plasmid DNA has been purified on silica and dissolved in TEbuffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA). Store at –20°C. Refer to Appendix E for detailed infor-mation and restriction maps.

pVL1392-XylE Baculovirus Control Vector (Cat. No. 21484P) 5 µg in 50 ml

The pVL1392-XylE Control Vector is a purified Baculovirus Transfer Vector which can be used as a pos-itive control in co-transfection with PharMingen’s BaculoGold™ Baculovirus DNA (Cat. No. 21100D).In this vector a Pseudomonas putrida gene “XylE” was cloned into the pVL1392 Baculovirus TransferVector. Co-transfection with BaculoGold™ DNA will generate recombinant Baculoviruses that expressthe XylE protein which runs as a 35 kD protein on SDS-PAGE. Infected insect cells producing the XylEprotein will turn yellow in the presence of catechol (500 µM catechol, 50 µM bisulfate). Store at –20°C.

AcNPV Wild-Type High Titer Viral Stock Solution (Cat. No. 21103E) 1 ml

AcNPV wild-type high titer stock contains 1 × 108 pfu/ml. It is an excellent choice for an occlusionbody positive control for insect cell infection. Store at 4°C.

TNM-FH Insect Medium (Cat. No. 21227M) 1 liter

TNM-FH medium is fully supplemented Grace’s medium including trace metals, lactalbuminhydrolysate, yeastolate, 10% heat inactivated FBS, and gentamicin (50 µg/ml). This medium is ide-ally suited for growth, infection, and protein expression of invertebrate cell lines derived from theFall armyworm, Spodoptera frugiperda (Sf). This medium may be used for suspension or monolayercultures. Store at 4°C.

Live Sf9 Insect Cells (1x107) (Cat. No. 21300L) 1 flask

The Sf9 cell line was cloned by Gale E. Smith and Carol L. Cherry in 198338 from the parent line,IPLB-Sf21 AE39, which was derived from pupal ovarian tissue of the Fall armyworm, Spodopterafrugiperda. The Sf9 cell line is highly susceptible to infection with AcNPV and other Baculoviruses,and can be used with all Baculovirus expression vectors. Sf21 (Cat. No. 21301L) cells are availableupon request. Propagate immediately upon arrival.

Transfection Buffer A & B Set (Cat. No. 21483A) 5 ml each

Transfection Buffer A contains Grace’s Medium supplemented with 10% FBS and should be at pH6.0–6.2. It should be used for co-transfections of Baculovirus DNA and Baculovirus transfer plas-mids as specified in the Baculovirus Manual. Store at 4°C.

Transfection Buffer B contains 25 mM Hepes, pH 7.1; 125 mM CaCl2; 140 mM NaCl. It shouldbe used for co-transfections of Baculovirus DNA and Baculovirus transfer plasmids as specified inthe Baculovirus Manual. Store at 4°C.

Agarplaque-Plus™ Agarose (Cat. No. 21403A) 50 g

Agarplaque-Plus™ Agarose is an optimized agarose with low-melting temperature (≤65°C), lowgelling temperature (29°C), no toxic metals, and low sulfate content (≤0.08%). These featuresenable Agarplaque-Plus™ Agarose to be used for maximum plaque size, safe plating, and optimalgrowth of plated Sf9, Sf21, or other insect cells. Agarplaque-Plus™ Agarose is easy to use and guar-anteed to provide consistent results. This agarose is recommended for applications includingplaque assay for determination of Baculovirus titers as well as viral purification by singleplaque pick-up. Store at RT.

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Appendix B6xHis Kits

PharMingen sells a novel Baculovirus affinity tag expression and purification kit, the6xHis Expression and Purification Kit (Cat. No. 21474K).

Contents of the 6xHis Expression and Purification Kit (Cat. No. 21474K)

PharMingen’s 6xHis VectorsEach of the pAcHLT vectors contain an N-terminal 6xHis tag with an extended multi-ple cloning region in which to insert the desired gene (Appendix E). The resultant pro-tein will be a 6xHis-containing fusion protein which can be affinity purified using Ni-NTA Agarose. Ni-NTA Agarose has an extremely high affinity for 6xHis residues. Thebinding affinity is approximately Kd=10–13, which is higher than most antibody/anti-gen or enzyme/substrate interactions.30 Approximately 1 to 2 mg of 6xHis fusion pro-tein is routinely obtained per liter of insect cell culture. The 6xHis-Ni2+NTA interactioncan tolerate stringent washing conditions needed to remove non-specifically boundhost proteins. Since the 6xHis tag is very small in size and uncharged under physio-logical pH conditions, it is not immunogenic and does not alter the folding, compart-mentalization or biochemical properties of the recombinant protein. Therefore it isusually not necessary to remove the 6xHis tag. However, if desired, the 6xHis tag canbe removed by incubating the 6xHis fusion protein in the presence of thrombin.

Description of Provided Reagents Linearized BaculoGold™ DNA (Cat. No. 21100D) 2.5 µg in 25 µl

BaculoGold™ DNA is a modified linearized AcNPV Baculovirus DNA which contains a lethal dele-tion and does not code for viable virus. Co-transfection of BaculoGold™ DNA with a complement-ing plasmid construct (e.g., pAcHLT) rescues the lethal deletion of this virus DNA and results in pro-duction of viable virus particles in transfected insect cells. When using BaculoGold™ DNA forco-transfection, more than 99% of all virus particles will be recombinant and will express the geneof interest. For one transfection, 5 µl (500 ng) of linearized BaculoGold™ DNA should be used incombination with 2-5 µg of purified recombinant Baculovirus transfer DNA (e.g., pAcHLT). Refer toPharMingen’s Baculovirus Manual for a detailed description of all the protocols necessary to useBaculoGold™ DNA for constructing recombinant AcNPV Baculoviruses. Store at 4°C.


21100D Linearized BaculoGold™ DNA 2.5 µg21467P pAcHLT-A, B, C Baculovirus Transfer Vector Set 20 µg each21471P pAcHLT-XylE Control Vector 5 µg21430Z Thrombin Powder 20 mg/1000U21454A Thrombin Dilution Buffer 1 ml21426Z Protease Inhibitor Cocktail lyophilized21425A 1X Insect Cell Lysis Buffer 50 ml N/A Ni-NTA Agarose 10 ml21476A 6xHis Elution Buffer 40 ml21472A 6xHis Wash Buffer 250 ml21473Z 3M Imidazole Solution 125 ml21483A Transfection Buffer A & B 5 ml eachN/A Baculovirus Procedures & Methods Manual 1 manual

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pAcHLT-A, -B, -C Transfer Vector Set (Cat. No. 21467P) 20 µg in 20 µl; each vector

Individual Cat. Nos. 21464P, 21465P and 21466P for pAcHLT-A, -B and -C, respectively. The pAcHLTvectors contain an N-terminal 6xHis tag with an extended multiple cloning region. The vectorDNA has been purified on silica and dissolved in TE buffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA).The 6xHis vectors should be kept at –20°C for long-term storage. Refer to Appendix E for detailedinformation and restriction maps.

pAcHLT-XylE Control Vector (Cat. No. 21471P) 5 µg in 50 µl

The pAcHLT-XylE Control Vector is purified Baculovirus transfer DNA for control transfection exper-iments. In this construct a Pseudomonas putrida gene “XylE” was cloned in frame with the 6xHis tagcloning region. Co-transfection with BaculoGold™ DNA will generate recombinant Baculovirusesthat express the 6xHis-XylE fusion protein which runs as a 40 kD protein on SDS-PAGE. Infectedinsect cells producing the 6xHis-Xyle fusion protein will turn yellow in the presence of catechol(500 µM catechol, 50 µM bisulfate). This protein can be purified using the 6xHis purification system(Cat. No. 21474K) and the authentic XylE protein can be recovered by cleaving away the 6xHisfusion tag with thrombin. Store at –20°C.

Thrombin Powder† (Cat. No. 21430Z) 20 mg (1,000 U)

20 mg (1,000 U) of bovine thrombin is provided as a lyophilized powder. One unit of thrombindigests 100 µg of recombinant protein containing a single thrombin site within 1 h under standardassay conditions. Before usage, dissolve the Thrombin Powder (20 mg, 1,000 U, Cat. No. 21430Z)in 1 ml of Thrombin Dilution Buffer (Cat. No. 21454A). The resulting thrombin solution is readyto use and should be stored at –20°C. †Warning: Thrombin may be fatal if it enters the blood stream and is a possible sensitizer. Targetorgans include the vascular system. Do not use if skin is cut or scratched, wash thoroughly afterhandling.

Thrombin Dilution Buffer (Cat. No. 21454A) 1 ml

The Thrombin Dilution Buffer is used to reconstitute the Thrombin Powder (Cat. No. 21430Z) above.Dissolve 20 mg Thrombin Powder (Cat. No. 21430Z) in 1 ml Thrombin Dilution Buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). The resulting thrombin solution is ready to use and should be stored at–20°C.

Ni-NTA Agarose 10 ml beads

10 ml of Ni-NTA Agarose beads are shipped as a 50% slurry in 10 mM NaOAc with 30% ethanol as apreservative. Ni-NTA Agarose contains the Nitrilo-tri-acetic-acid (NTA) chelating ligand charged withNi2+ ions bound to Sepharose CL-6B. The agarose has a high affinity to proteins which contain a 6xHistag. The Ni2+ ion has six coordination sites, four of which interact with NTA ligand leaving two sitesfree for the binding of the 6xHis tag.29 The Ni-NTA Agarose is very stable and will retain full activitythrough prolonged storage. For long-term storage, the agarose can be stored at either RT or 4°C in 30%ethanol to inhibit microbial growth. The agarose resuspended in 6xHis Wash Buffer may be kept at RTfor up to one week. Additional Ni-NTA Agarose can be purchased from QIAGEN and its distributors.

6xHis Elution Buffer (Cat. No. 21476A) 40 ml

6xHis Elution Buffer contains 50 mM Na-phosphate, 300 mM NaCl, 10% glycerol, pH 6.0. Imida-zole (Cat. No. 21473Z) should be added to the 6xHis Elution Buffer to make either a step or lineargradient for elution. A maximum concentration of 0.5 M imidazole is recommended for the finalelution using either the step or linear gradient. The optimal concentration of imidazole must beempirically determined by the researcher. Store at 4°C.

6xHis Wash Buffer (1X) (Cat. No. 21472A) 2 bottles of 125 ml each

6xHis Wash Buffer contains 50 mM Na-phosphate, 300 mM NaCl, 10% glycerol, pH 8.0. For a morestringent wash the pH may be adjusted to 6.0 and 0.8-40 mM imidazole may be added. The optimalconcentration of imidazole must be empirically determined by the researcher. Store at 4°C.

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3M Imidazole Solution (Cat. No. 21473Z) 125 ml

Imidazole is an organic crystalline base, C3H4N2, that competes with histidine residues to bind toNi-NTA Agarose. It should be added to the 6xHis Elution Buffer (Cat. No. 21476A) at concentrationsbetween 0.1 M to 0.5 M to displace 6xHis tagged proteins. Imidazole may also be added to the6xHis Wash Buffer (Cat. No. 21472A) at concentrations between 0.8-40 mM to facilitate the elutionof nonspecific contaminating host proteins. Store at RT.

Protease Inhibitor Cocktail (Cat. No. 21426Z) lyophilized

The Protease Inhibitor mix is provided as lyophilized powder. Before use, add 1 ml of pure ethanolto obtain a 50X Protease Inhibitor Cocktail. The reconstituted 50X Protease Inhibitor Cocktail willhave the following ingredients: 800 µg/ml benzamidine HCl, 500 µg/ml phenanthroline, 500µg/ml aprotinin, 500 µg/ml leupeptin, 500 µg/ml pepstatin A, 50 mM PMSF. Always store thereconstituted protease inhibitor cocktail at –20°C.

Insect Cell Lysis Buffer (1X) (Cat. No. 21425A) 50 ml

Insect Cell Lysis Buffer contains 10 mM Tris, pH 7.5; 130 mM NaCl; 1% Triton™

-X-100; 10 mM NaF;10 mM NaPi, 10 mM NaPPi. Store at 4°C.


Transfection Buffer A contains Grace’s Medium supplemented with 10% fetal bovine serum andshould be pH 6.0–6.2. It should be used for co-transfections of Baculovirus DNA and Baculovirustransfer plasmids as specified in the Baculovirus Manual. Store at 4°C.

Transfection Buffer B contains 25 mM Hepes, pH 7.1, 125 mM CaCl2, 140 mM NaCl. It shouldbe used for co-transfections of Baculovirus DNA and Baculovirus transfer plasmids as specified inthe Baculovirus Manual. Store at 4°C.

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Appendix CGST Kits

PharMingen sells a novel Baculovirus affinity tag GST Expression and Purification Kit(Cat. No. 21475K). Refer to Table 1 below for the kit components.

Contents of the GST Expression and Purification Kit (Cat. No. 21475K).

PharMingen’s GST Vectors

The GST fusion vectors are designed for high-level expression of genes or gene frag-ments as fusion proteins with glutathione S-transferase from Schistosoma japonicum(Sj).40 PharMingen sells a novel set of Baculovirus GST Transfer Vectors directing theexpression of cloned genes as GST fusion proteins in insect cells (Appendix E). ExpressedGST fusion proteins are easily purified to near homogeneity from the cell lysate byaffinity chromatography using glutathione agarose. The expressed GST fusion proteinsare authentically processed and may be purified without the use of detergents undercompletely non-denaturing conditions. This system eliminates the insolubility prob-lem often encountered with overexpressed heterologous proteins in bacterial expres-sion systems.

Description of Provided ReagentsLinearized BaculoGold™ DNA (Cat. No. 21100D) 2.5 µg in 25 µl

BaculoGold™ DNA is a modified linearized AcNPV Baculovirus DNA which contains a lethal deletionand does not code for viable virus. Co-transfection of BaculoGold™ DNA with a complementing plas-mid construct (e.g., pAcGHLT) rescues the lethal deletion of this virus DNA and results in produc-tion of viable virus particles in transfected insect cells. When using BaculoGold™ DNA for co-trans-fection, more than 99% of all virus particles will be recombinant and will express the gene ofinterest. For one transfection, 5 µl (500 ng) of linearized BaculoGold™ DNA should be used in com-bination with 2-5 µg of purified recombinant Baculovirus transfer DNA (e.g., pAcGHLT, pAcHLT).Refer to PharMingen’s Baculovirus Manual for a detailed description of all the protocols necessary touse BaculoGold™ DNA for constructing recombinant AcNPV Baculoviruses. Store at 4°C.


21100D Linearized BaculoGold™ DNA 2.5 µg21463P pAcGHLT-A, B, C Baculovirus Transfer Vector Set 20 µg each21470P pAcGHLT-XylE Control Vector 5 µg21430Z Thrombin Powder 20 mg/1000U21454A Thrombin Dilution Buffer 1 ml21426Z Protease Inhibitor Cocktail lyophilized21425A 1X Insect Cell Lysis Buffer 50 ml 21429Z Glutathione Powder 62 mg21427B Glutathione Agarose Beads 10 ml21455A GST Elution Buffer 40 ml21428A 1X PBS Wash Buffer 375 ml21483A Transfection Buffer A & B 5 ml eachN/A Baculovirus Procedures & Methods Manual 1 manual

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pAcGHLT-A, -B, -C Transfer Vector Set (Cat. No. 21463P) 20 µg in 20 µl, each vector

Individual Cat. No. 21460P, No. 21461P and No. 21462P for pAcGHLT-A, -B and -C respectively. ThepAcGHLT vectors encode N-terminal 6xHis and GST tags followed by an extended MCS. The plasmidDNA has been purified on silica and dissolved in TE buffer (10 mM Tris-HCl, pH 7.5; 1 mM EDTA).The GST vectors should be kept at –20°C for long-term storage. Refer to Appendix E for detailed infor-mation and restriction maps.

pAcGHLT-XylE Control Vector (Cat. No. 21470P) 5 µg in 50 µl

The pAcGHLT-XylE Control Vector is purified Baculovirus transfer plasmid DNA for control transfec-tion experiments. In this construct a Pseudomonas putrida gene “XylE” was cloned in-frame with theglutathione S-transferase coding sequence in the pAcGHLT vector. Co-transfection with BaculoGold™

DNA will generate recombinant Baculoviruses that express the GST-XylE fusion protein which runsas a 60 kD protein on SDS-PAGE. Infected insect cells producing the XylE-GST fusion protein will turnyellow in the presence of catechol (500 µM catechol, 50 µM bisulfate). This protein can be purifiedusing either the GST or 6xHis purification system (Cat. No. 21475K or No. 21574K) and the authen-tic XylE protein can be recovered by cleaving away the fusion tag with thrombin. Store at –20°C.

Thrombin Powder† (Cat. No. 21430Z) 20 mg (1,000 U)

20 mg (1,000 U) of bovine thrombin is provided as a lyophilized powder. One unit of thrombindigests 100 µg of recombinant protein containing a single thrombin site within 1 h under standardassay conditions. Before usage, dissolve the Thrombin Powder (20 mg, 1,000 U, Cat. No. 21430Z)in 1 ml of Thrombin Dilution Buffer (Cat. No. 21454A). The resulting thrombin solution is readyto use and should be stored at –20°C. †Warning: Thrombin may be fatal if it enters the blood stream and is a possible sensitizer. Targetorgans include the vascular system. Do not use if skin is cut or scratched, wash thoroughly afterhandling.

Thrombin Dilution Buffer (Cat. No. 21454A) 1 ml

The Thrombin Dilution Buffer is used to reconstitute the Thrombin Powder (Cat. No. 21430Z) above.Dissolve 20 mg thrombin powder (Cat. No. 21430Z) in 1 ml Thrombin Dilution Buffer (10 mM Tris-HCl, pH 8.0; 1 mM EDTA). The resulting thrombin solution is ready to use and should be stored at –20°C.

Glutathione Agarose Beads (Cat. No. 21427B) 10 ml beads

10 ml of Glutathione Agarose Beads are shipped in PBS with 20% ethanol as a preservative. Theirbinding capacity is approximately 5 mg of recombinant GST fusion protein (e.g., GST-XylE) per mlbead volume. No significant loss of binding capacity is detected when glutathione agarose beadsare exposed to 100 mM acetate (pH 4.0), 0.1 N NaOH (pH 13), 70% ethanol, 6 M guanidinehydrochloride or 6 M urea. However, the agarose should never dry out, be stored frozen, be exposedto excessive heat, boiled nor autoclaved. For long-term storage, store the beads at 4°C. For short-term storage, the beads may be stored in 1X PBS Wash Buffer at 4°C. To inhibit bacterial growthadd 0.1% sodium azide. Glutathione Agarose beads are stored in 20% ethanol.—Highly Flammable,Keep container tightly closed and in a well-ventilated place. Keep away from sources of ignition -No smoking. If swallowed, seek medical advice immediately and show this container or label.

Glutathione Powder (Cat. No. 21429Z) 62 mg

Glutathione is provided as lyophilized powder because reduced glutathione is not stable in solu-tion. Dissolve the powder in 40 ml of GST Elution Buffer (Cat. No. 21455A) to obtain a 5 mM glu-tathione solution. Store dry glutathione at 4°C and dissolved glutathione at –20°C.

73

GST Elution Buffer (Cat. No. 21455A) 40 ml

Dissolve the lyophilized Glutathione Powder (Cat. No. 21429Z) in 40 ml of the GST Elution Buffer(50 mM Tris-HCl, pH 8.0). Store the new glutathione solution at –20°C. For best results make smallaliquots of the glutathione elution buffer. This will allow you to avoid multiple freeze-thaw cycleswhich may oxidize glutathione and inactivate the capacity of this buffer to compete with the GSTfusion protein for binding to the glutathione beads.

Protease Inhibitor Cocktail (Cat. No. 21426Z) lyophilized

The protease inhibitor mix is provided as lyophilized powder. Before use, add 1 ml of pure ethanolto obtain a 50X Protease Inhibitor Cocktail. The reconstituted 50X Protease Inhibitor Cocktail willhave the following ingredients: 800 µg/ml benzamidine HCl, 500 µg/ml phenanthroline, 500µg/ml aprotinin, 500 µg/ml leupeptin, 500 µg/ml pepstatin A, 50 mM PMSF. Always store thereconstituted protease inhibitor cocktail at –20°C.

Insect Cell Lysis Buffer (1X) (Cat. No. 21425A) 50 ml

Insect Cell Lysis Buffer contains 10 mM Tris pH, 7.5; 130 mM NaCl; 1% Triton™

X-100; 10 mM NaF;10 mM NaPi; 10 mM NaPPi. Store at 4°C.

PBS Wash Buffer (1X) (Cat. No. 21428A) 3 bottles of 125 ml each

PBS Wash Buffer contains 140 mM NaCl; 2.7 mM KCl; 10 mM Na2HPO4; 1.8 mM KH2PO4 dis-solved in distilled, autoclaved water. The pH has been adjusted to 7.4 using hydrochloric acid.Store at 4°C.


Transfection Buffer A contains Grace’s Medium supplemented with 10% FBS and should be pH6.0–6.2. It should be used for co-transfections of Baculovirus DNA and Baculovirus transfer plas-mids as specified in the Baculovirus Manual. Store at 4°C.

Transfection Buffer B contains 25 mM Hepes, pH 7.1, 125 mM CaCl2, 140 mM NaCl. It shouldbe used for co-transfections of Baculovirus DNA and Baculovirus transfer plasmids as specified inthe Baculovirus Manual. Store at 4°C.

75

Appendix DvEHuni and vECuni Baculovirus Reagent Sets

Contents of the vEHuni Baculovirus Reagent Set (Cat. No. 21523K)


vEHuni Baculovirus DNA (Cat. No. 21524P) 5 µg in 50 µl

vEHuni Baculovirus DNA is a modified AcNPV DNA in which an hsp70 promoter from Drosphilamelanogaster and a multiple cloning site (MCS) containing two Bsu36I restriction sites were insertedinto the nonessential Ecdysteroid UDP-glucosyltransferase (egt) gene of the wild type AcNPVgenome. vEHuni Baculovirus DNA is provided undigested and untreated. Store at –20°C.

vEHuni High Titer Virus Stock (Cat. No. 21525E) 1 ml

vEHuni High Titer Viral Stock contains 1 × 108 pfu/ml. vEHuni virus is provided to allow for pro-duction of vEHuni DNA. For isolating AcNPV particles and DNA see Section 4.4. Store at 4°C.

Contents of the vECuni Baculovirus Reagent Set (Cat. No. 21526K)


vECuni Baculovirus DNA (Cat. No. 21527P) 5 µg in 50 µl

vECuni Baculovirus DNA is a modified AcNPV DNA in which a PcapminXIV hybrid late/very late poly-hedrin promoter and a multiple cloning site (MCS) containing two Bsu36I restriction sites wereinserted into the nonessential Ecdysteroid UDP-glucosyltransferase (egt) gene of the wild typeAcNPV genome. vECuni Baculovirus DNA is provided undigested and untreated. Store at –20°C.

vECuni High Titer Virus Stock (Cat. No. 21528E) 1 ml

vECuni High Titer Viral Stock contains 1 × 108 pfu/ml. vECuni virus is provided to allow for pro-duction of vECuni DNA. For isolating AcNPV particles and DNA see Section 4.4. Store at 4°C.


21527P vECuni Baculovirus DNA 5 µg21528E vECuni High Titer Virus Stock 1 ml


21524P vEHuni Baculovirus DNA 5 µg21525E vEHuni High Titer Virus Stock 1 ml

77

Appendix EBaculovirus Transfer Vectors

PharMingen sells a diverse line of BV vectors ranging from polyhedrin-derived singlepromoter to p10 derived multiple promoter vectors. Table 3, Chapter 4 provides anoverview to help in your vector selection. Contact PharMingen Technical Serviceat 800-TALK-TEC for vector sequences that are available free of charge ondisk (Macintosh or IBM compatible) or by e-mail.

I. Polyhedrin Locus-based Transfer VectorsA. Single Promoter Transfer VectorspVL1392, pVL1393 Baculovirus Transfer Vector Set

Catalog No. 21201P Set Individual: 21485P, 21486P

Description: The pVL1392 and pVL1393 Baculovirus Transfer Vectors are derivatives of theplasmid pVL941. They contain the complete polyhedrin gene locus including flanking regions ofAcNPV cloned into the pUC8 vector, but they lack part of the polyhedrin gene coding region. AMCS region has been inserted 37 nucleotides downstream of the ATG polyhedrin start codon,which has been changed into an ATT. pVL1392 and pVL1393 contain the MCS in opposite ori-

HindIII (1)

SacII (868)

PvuII (1307)ApaI (1395)

StyI (1501)

XhoI (1901)

SphI (2131)BclI (2232)

NsiI (2669)

SalI (2947)

NsiI (3169)SalI (3232)

NaeI (3770)EcoRV (3998)

MCSHindIII (4253)

KpnI (4634)HindIII (5181)

SalI (6175)HindIII (6217)

PvuII (6752)

PvuII (7183)

AlwNI (7772)

ScaI (8732)

NdeI (9424)

PvuII (9547)

R

ColEori

polyhedrinpromoter

Amp

polyhedrin promoter

polyhedrin promoter

Unique sites

BglII (4134)

AGATCTGCAGCGGCCGCTCCAGAATTCTAGAAGGTACCCGGGATCCTCTAGACGTCGCCGGCGAGGTCTTAAGATCTTCCATGGGCCCTAGG

PstI (4138)

EagI (4144)

EcoRI (4155)

XbaI (4159)

SmaI (4170)

BamHI (4174)

pVL1392

MCS

CGGATCCCGGGTACCTTCTAGAATTCCGGAGCGGCCGCTGCAGATCTGCCTAGGGCCCATGGAAGATCTTAAGGCCTCGCCGGCGACGTCTAGA

BamHI (4129)

SmaI (4133)

XbaI (4144)

EcoRI (4148)

NotI (4143)

NotI (4158)

EagI (4159)

PstI (4165)

BglII (4169)

Unique sites

MCSpVL1393

pVL1392/13939639 bp

unique sites underlined

78

entation to one another. The MCS regions reads: BglII, PstI, NotI, EcoRI, XbaI, SmaI/XmaIand BamHI (from 5’ to 3’ for pVL1392 and from 3’ to 5’ for pVL1393). The insert of choice mustprovide its own ATG start signal at the 5’ end of the gene. The distance between the cloning siteand the ATG start of the gene should not be longer than 100 nucleotides, otherwise the proteinexpression will be poor. These vectors may be used for high level expression of non-fused foreignproteins under the strong polyhedrin promoter of AcNPV. These vectors are recommended foruse in conjunction with PharMingen’s BaculoGold™ Baculovirus DNA (Cat. No. 21100D) toachieve virtually 100% recombination efficiencies.

Note: The plasmid pVL941 (PharMingen) was a predecessor form of pVL1392 andpVL1393. Instead of the MCS of the latter two it had a single BamHI cloning site.

79

pAcSG2 Baculovirus Transfer Vector

Catalog No. 21410P

Description: The pAcSG2 Baculovirus Transfer Vector is a newly developed, streamlinedderivative of pVL941. It contains the essential parts of the polyhedrin gene locus of theAcNPV, cloned into a derivative of the pUC8 vector. To create a vector small in size, severalnon-essential parts of the polyhedrin locus have been deleted, including out-of-frame por-tions of the polyhedrin gene and portions of ORF 603. The MCS immediately follows the endof the polyhedrin promoter for improved expression levels. The MCS region of pAcSG2 reads(from 5’ to 3’): XhoI, EcoRI, StuI, NcoI/StyI, SacI, NotI, EagI, PstI, KpnI, SmaI/XmaI andBglII. The pAcSG2 has an ATG inside the NcoI site, thus sequences cloned downstream ofthe NcoI must NOT contain their own start codon or they must be in-frame with the ATG ofthe NcoI site. Sequences cloned upstream of the NcoI site must provide their own ATG andwill be expressed as a non-fusion protein. This vector may be used to produce high levelexpression of foreign proteins under the strong polyhedrin promoter of AcNPV. Because ofits small size, it may be used to accommodate inserts as large as 8 kb. The pAcSG2 vector canbe used in conjunction with PharMingen’s BaculoGold™ Transfection Kit (Cat. No. 21100K)to achieve virtually 100% recombination efficiencies.

polyhedrin promoter

NaeI (361)

EcoRV (589)

MCS

SnaBI (933)

HindIII (1297)

AgeI (1783)

ClaI (1906)

SalI (2292)

HindIII (2334)MscI (2513)

PvuII (2869)BstBI (2947)

BspE1 (3042)HpaI (3098)

PvuII (3300)SapI (3355)

AlwNI (3889)

BsaI (4438)GsuI (4456)BglI (4485)

PvuI (4738)ScaI (4849)

EcoO109I (5346)

AmpR

ColE ori

pAcSG25544 bp


KpnI (734)

EagI (721)

polyhedrin promoter

Unique sites

BglII (746)

PstI (728)StuI (702)

EcoRI (696)

BanII (714)

SmaI (740)

pAcSG2MCS

NotI (720)XhoI (690)

NcoI (708)StyI (708)DsaI (708)

CTCGAGGAATTCAGGCCTCC ATG GGA GCT CGC GGC CGC CTG CAG GGT ACC CCC GGG AGA TCT

SacI (714)

Sse8387I (727)

met gly ala arg gly arg leu asn gly thr pro gly arg ser

80

pAcMP2, pAcMP3 Baculovirus Transfer Vector Set

Catalog No. 21209P Set Individual: 21210P, 21211P

Description: The pAcMP2/pAcMP3 Baculovirus Transfer Vectors are derivatives of the afore-mentioned pVL1392/1393 vectors. The pAcMP2/3 plasmids contain a copy of the AcNPVbasic protein promoter instead of the AcNPV polyhedrin promoter. They will recombine withthe polyhedrin locus of the AcNPV virus since the vectors contain residual out-of-frame poly-hedrin gene coding sequences and their flanking regions. The pAcMP2/pAcMP3 vectors havea MCS inserted downstream of the basic protein promoter. This MCS reads BamHI, XbaI,EcoRI, NotI, EagI, PstI and BglII (from 5’ to 3’ for pAcMP3 and from 3’ to 5’ for pAcMP2).These vectors permit foreign gene expression in the late phase of virus infection, i.e., prior tothe very late phase when polyhedrin and p10 promoter-driven genes are expressed. Whileexpression levels may be somewhat reduced in comparison to polyhedrin and p10 promoter-driven expression, post-translational modifications (e.g., glycosylation and phosphorylation)are more readily accomplished.41 These vectors are recommended for use in conjunction withPharMingen’s BaculoGold™ Baculovirus DNA (Cat. No. 21100D) to achieve virtually 100%recombination efficiencies.

Note: The plasmid pAcMP1 (PharMingen) was a predecessor form of pAcMP2 and pAcMP3.Instead of the MCS of the latter two it had a single BamHI cloning site.

XcmI (739)

SacII (868)

BanII (1395)

SphI (2131)

NaeI (3770)

SnaBI (5029)

SapI (7451)

AlwNI (7985)

ScaI (8945)

NdeI (9637)

ori

Basic ProteinPromoter

GGATCCCGGGTACCTTCTAGAATTCCGGAGCGGCCGCTGCAGATCTCCTAGGGCCCATGGAAGATCTTAAGGCCTCGCCGGCGACGTCTAGA

GGATCTGCAGCGGCCGCTCCAGAATTCTAGAAGGTACCCGGGATCCCCTAGACGTCGCCGGCGAGGTCTTAAGATCTTCCATGGGCCCTAGG

Unique sites

XbaI (4357)NotI (4371)

PstI (4378)BglII (4382)

XbaI (4367)NotI (4351)

PstI (4346)

BamHI (4382)

Unique sites

pAcMP2

pAcMP3

MCS

BamHI (4342)

MCS

EcoR1 (4363)

EcoR1 (4361)

EagI (4352)

EagI (4352)

basic protein promoter

basic protein promoter

pAcMP29847 bp


9852 bppAcMP3

MCS

pAcMP3:pAcMP2: SnaBI (5024)

pAcMP3:

pAcMP3:

pAcMP3:

pAcMP3:

pAcMP2: SapI (7446)

pAcMP2: AlwNI (7980)

pAcMP2: ScaI (8940)

pAcMP2: NdeI (9632)

81

pAcUW21 Baculovirus Transfer Vector

Catalog No. 21206P

Description: The pAcUW21 Baculovirus Transfer Vector is an AcNPV polyhedrin locus-based vector that contains the AcNPV p10 promoter and SV40 transcription terminationsignals inserted upstream of the complete AcNPV polyhedrin gene. Foreign genes may becloned into the BglII or EcoRI site located downstream of the p10 promoter. The recom-binant virus will be occlusion body-positive. This vector will be of use to those researchersinterested in producing recombinant protein in insect larvae. pAcUW21 contains the f1 ori-gin of replication and can produce, by helper phage mediation, single strand DNA, usefulin sequencing and mutagenesis. pAcUW21 is best used in conjunction with BaculoGold™

DNA (Cat. No. 21100D).

polyhedrin gene

SphI (230)

BstXI (1248)

NaeI (1869)

XcmI (2255)

EcoRI (2548)BglII (2554)

PacI (2597)

AflII (2790)

BamHI (3079)PpuMI (3091)HindIII (3158)

KpnI (3539)

SnaBI (3721)HindIII (4086)AgeI (4571)HindIII (5132)

MscI (5311)

EagI (5920)

NaeI (6515)BanII (6545)

DraIII (6618)

GsuI (7929)

AlwNI (8493)

SapI (9029)

ColE ori

AmpR pAcUW21

9267 bpunique sites underlined

polyhedrinpromoter

f1 ori

p10promoter

82

Fusion VectorspAcGHLT-A, -B, -C Baculovirus Transfer Vector Set

Catalog No. 21463P Set Individual: 21460P, 21461P, 21462P

Description: The pAcGHLT-A, -B and -C Baculovirus Transfer Vectors are derivatives of thepAcG1 vector. They contain a 6xHis tag and a glutathione S-transferase (GST) tag upstreamof the MCS. The recombinant protein will be expressed as a 6xHis-containing GST fusionprotein. 6xHis fusion proteins bind with high affinity to Ni-NTA Agarose and GST fusion pro-teins have a high affinity for reduced glutathione.40 Therefore, a highly efficient single stepaffinity purification can be done on GST-6xHis-tagged proteins using either Ni-NTA Agarose(a metal chelating agent)30 or Glutathione Agarose Beads (Cat. No. 21427B). Purifiedrecombinant proteins can be phosphorylated at a protein kinase A site which follows the6xHis sequence. This phosphorylation should not alter the binding affinity of the recombi-nant proteins to any of its ligands. After purification, the GST and 6xHis tags can beremoved by incubating the fusion protein in the presence of thrombin. All foreign insertsmust be in frame with the GST open reading frame (ORF). These vectors are recom-mended for use in conjunction with PharMingen’s BaculoGold™ DNA (Cat. No. 21100D) toachieve virtually 100% recombination efficiencies.

Amp

glutathione S-transferase

SphI (230)

BstXI (1248)

NaeI (1869)

EcoRV (2099)

EcoNI (2212)

BamHI (2862)

6xHis TagProtein Kinase AThrombin Cleavage

MCS

SnaBI (3211)HindIII (3576)

AgeI (4061)HindIII (4622)

PvuII (5157)

PvuII (5735)

NaeI (6005)

DraIII (6108)

GsuI (7419)

AlwNI (7983)

PvuII (8575)

R

ColE ori

pAcGHLT-A, -B, -C8757 bp


polyhedrinpromoter

83

Multiple Cloning Regions of pAcGHLT-A, -B and -C

2851

GST protein

StuI (2980)

PstI (3006)

GCG GGA ATT TTG GTC CCT CGT GGA AGC CCA GGA CTC GAT GGC ATA TGC TCG ATC GAG GAA TTC AGGAla Gly Ile Leu Val Pro Arg Gly Ser Pro Gly Leu Asp Gly Ile Cys Ser Ile Glu Glu Phe Arg

EcoRI (2974)

DsaI (2986)

CCA CCA AAA TCG GAT CCG ATG GGA CAT CAT CAT CAT CAT CAC GGA AGG AGA AGG GCC AGT GTT GCGPro Pro Lys Ser Asp Pro Met Gly His His His His His His Gly Arg Arg Arg Ala Ser Val Ala

Thrombin cleavage site

Thrombin cut

6xHis tag Protein kinase A site

NdeI (2958)

CCT CCA TGG GAG CTC GCG GCC GCC TGC AGG GTA CCC CCG GGA GAT CTG TAC CGA CTC TGC TGAPro Pro Trp Glu Leu Ala Ala Ala Cys Arg Val Pro Pro Gly Asp Leu Tyr Arg Leu Cys Stop

NcoI (2986)StyI (2986)

SacI (2992)

NotI (2998) Sse8387I (3005)Kpn I (3012)

SmaI (3018)XmaI (3018) BglII (3024)

BamHI (2862)

GST protein

StuI (2978)

PstI (3004)

GCG GGA ATT TTG GTC CCT CGT GGA AGC CCA GGA CTC GAT GGC ATA TAT GCT CGA GGA ATT CAG GCCAla Gly Ile Leu Val Pro Arg Gly Ser Pro Gly Leu Asp Gly Ile Tyr Ala Arg Gly Ile Gln Ala

EcoRI (2972)

DsaI (2984)



Thrombin cut

6xHis tag

XhoI (2966)

TCC ATG GGA GCT CGC GGC CGC CTG CAG GGT ACC CCC GGG AGA TCT GTA CCG ACT CTG CTG AAG ...Ser Met Gly Ala Arg Gly Arg Leu Gln Gly Thr Pro Gly Arg Ser Val Pro Thr Leu Leu Lys

NcoI (2984)StyI (2984)

SacI (2990)

NotI (2996) Sse8387I (3003) KpnI (3010)

SmaI (3016)XmaI (3016)

BglII (3022)

Protein kinase A site

2851 BamHI (2862)

GST protein

StuI (2976)

PstI (3002)

GCG GGA ATT TTG GTC CCT CGT GGA AGC CCA GGA CTC GAT GGC ATA TGC TCG AGG AAT TCA GGC CTCAla Gly Ile Leu Val Pro Arg Gly Ser Pro Gly Leu Asp Gly Ile Cys Ser Arg Asn Ser Gly Leu

EcoRI (2970)



Thrombin cut

6xHis tag

XhoI (2964)

CAT GGG AGC TCG CGG CCG CCT GCA GGG TAC CCC CGG GAG ATC TGT ACC GAC TCT GCT GAA GAG ...His Gly Ser Ser Arg Pro Pro Ala Gly Tyr Pro Arg Glu Ile Cys Thr Asp Ser Ala Glu Glu

SacI (2988)NotI (2994)

Sse8387I (3001) KpnI (3008)

SmaI (3014)XmaI (3014)

BglII (3020)


2851

NdeI (2958) DsaI (2982)

StyI (2982)NcoI (2982)

BamHI (2862)

pAcGHLT-B 8755 bp

pAcGHLT-A 8757 bp

pAcGHLT-C 8753 bp

84

pAcHLT-A, -B and -C Baculovirus Transfer Vector Set


Description: The pAcHLT-A, -B and -C Baculovirus Transfer Vectors are derivatives ofthe pAcG1 vector. They contain a 6xHis tag upstream of the MCS and the recombinantprotein will be expressed as a 6xHis-containing fusion protein. The presence of a 6xHistag substantially eases the purification of the recombinant proteins since 6xHis fusionproteins bind with high affinity to Ni-NTA Agarose (a metal chelating agent).30 Mosthost cell proteins do not bind to such a matrix. Therefore, a highly efficient single-stepaffinity purification can be done with 6xHis-tagged proteins. Purified recombinant pro-teins can be phosphorylated at a protein kinase A site which follows the 6xHissequence. This phosphorylation should not alter the binding affinity of the recombi-nant protein to any of its ligands. If desired, the 6xHis tag can be removed by incu-bating the fusion protein in the presence of thrombin. Additional features of thesevectors include their expanded MCS. All foreign inserts must be in frame with the 6xHisORF. These vectors are recommended for use in conjunction with PharMingen’s Baculo-Gold™ DNA (Cat. No. 21100D) to achieve virtually 100% recombination efficiencies.

SphI (230)BclI (331)

BstXI (1248)

NaeI (1869)

EcoRV (2097)


MCS

SnaBI (2566)

HindIII (2931)

AgeI (3416)

HindIII (3977)

MscI (4156)PvuII (4512)

PvuII (5090)

NaeI (5360)

DraIII (5463)

EcoO109I (5883)

ScaI (6381)

GsuI (6774)

AlwNI (7338)

SapI (7874)

PvuII (7930)

pAcHLT-A, -B, -C8112 bp


RAmp

ColE ori

polyhedrinpromoter

85

Multiple Cloning Regions of pAcHLT-A, -B and -C

pAcHLT-B 8110 bp

pAcHLT-A 8112 bp

pAcHLT-C 8108 bp

2206

polyhedrinpromoter

Stu I (2335)

PstI (2361)

GCG GGA ATT TTG GTC CCT CGT GGA AGC CCA GGA CTC GAT GGC ATA TGC TCG ATC GAG GAA TTC AGGAla Gly Ile Leu Val Pro Arg Gly Ser Pro Gly Leu Asp Gly Ile Cys Ser Ile Glu Glu Phe Arg

EcoR I (2329)

DsaI (2341)

ATG TCC CCT ATA GAT CCG ATG GGA CAT CAT CAT CAT CAT CAC GGA AGG AGA AGG GCC AGT GTT GCGMet Ser Pro Ile Asp Pro Met Gly His His His His His His Gly Arg Arg Arg Ala Ser Val Ala


Thrombin cut

6xHis tag Protein kinase A site

Nde I (2313)

CCT CCA TGG GAG CTC GCG GCC GCC TGC AGG GTA CCC CCG GGA GAT CTG TAC CGA CTC TGC TGAPro Pro Trp Glu Leu Ala Ala Ala Cys Arg Val Pro Pro Gly Asp Leu Tyr Arg Leu Cys Stop

NcoI (2341)StyI (2341)

SacI (2347)

NotI (2353) Sse8387I (2360)KpnI (2367)

SmaI (2373)XmaI (2373) BglII (2379)

Stu I (2333)

PstI (2359)

GCG GGA ATT TTG GTC CCT CGT GGA AGC CCA GGA CTC GAT GGC ATA TAT GCT CGA GGA ATT CAG GCCAla Gly Ile Leu Val Pro Arg Gly Ser Pro Gly Leu Asp Gly Ile Tyr Ala Arg Gly Ile Gln Ala

EcoR I (2327)

DsaI (2339)



Thrombin cut

6xHis tag

Xho I (2321)

TCC ATG GGA GCT CGC GGC CGC CTG CAG GGT ACC CCC GGG AGA TCT GTA CCG ACT CTG CTG AAG ...Ser Met Gly Ala Arg Gly Arg Leu Gln Gly Thr Pro Gly Arg Ser Val Pro Thr Leu Leu Lys

NcoI (2339)StyI (2339)

SacI (2345)

NotI (2351) Sse8387I (2358) KpnI (2365)

SmaI (2371)XmaI (2371)

BglII (2377)


2206

polyhedrinpromoter

polyhedrinpromoter

StuI (2331)

PstI (2357)

GCG GGA ATT TTG GTC CCT CGT GGA AGC CCA GGA CTC GAT GGC ATA TGC TCG AGG AAT TCA GGC CTCAla Gly Ile Leu Val Pro Arg Gly Ser Pro Gly Leu Asp Gly Ile Cys Ser Arg Asn Ser Gly Leu

EcoRI (2325)



Thrombin cut

6xHis tag

XhoI (2319)

CAT GGG AGC TCG CGG CCG CCT GCA GGG TAC CCC CGG GAG ATC TGT ACC GAC TCT GCT GAA GAG ...His Gly Ser Ser Arg Pro Pro Ala Gly Tyr Pro Arg Glu Ile Cys Thr Asp Ser Ala Glu Glu

SacI (2343)NotI (2349) Sse8387I (2356) KpnI (2363)

SmaI (2369)XmaI (2369)

BglII (2373)


2206

NdeI (2313) DsaI (2337)

StyI (2337)NcoI (2337)

86

pAcG1 Baculovirus Transfer Vector

Catalog No. 21413P

Description: The pAcG1 Baculovirus Transfer Vector is a derivative of the pAcCL29 vector.42

Foreign genes are expressed as GST fusion proteins when cloned into one of the availablerestriction enzyme sites (BamHI, SmaI or EcoRI). All foreign inserts must be in frame withthe GST ORF. The GST fusion protein expression is under the control of the strong AcNPVpolyhedrin promoter. Because GST fusion proteins have a high affinity for reduced glu-tathione, they can be purified in a single step using glutathione agarose beads.40 Thisvector is recommended for use in conjunction with PharMingen’s BaculoGold™ DNA(Cat. No. 21100D) to achieve virtually 100% recombination efficiencies.


SphI (230)

BstXI (1248)

EcoRV (2097)

EcoNI (2212)

BamHI (2862)

XmaI (2867)SmaI (2867)

EcoRI (2872)SnaBI (2968)

HindIII (3333)


PvuII (4914)

EagI (5167)

PvuII (5492)

BanII (5792)DraIII (5865)

GsuI (7176)

AlwNI (7740)

PvuII (8332)

AmpR

polyhedrinpromoter

ColE ori


8514 bppAcG1

GST protein

EcoRI (2872)

SmaI (2867)

BamHI (2862)

CCA AAA TCG GAT CCC CGG GAA TTC ATC GTG ACT GAC TGAPro Lys Ser Asp Pro Arg Glu Phe Ile Val Thr Asp Stop

87

pAcG2T Baculovirus Transfer Vector

Catalog No. 21414P

Description: The pAcG2T Baculovirus Transfer Vector is a derivative of the pAcCL29vector.43 Foreign genes are expressed as GST fusion proteins when cloned into one of theavailable restriction enzyme sites (BamHI, SmaI or EcoRI). All foreign inserts must be inframe with the GST ORF. The GST fusion protein expression is under the control of thestrong AcNPV polyhedrin promoter. Because GST fusion proteins have a high affinity forreduced glutathione, they can be purified in a single step using glutathione agarosebeads.40 After purification, the GST affinity tag can be removed by incubating the fusionprotein in the presence of the site-specific protease, thrombin. This vector is recommendedfor use in conjunction with PharMingen’s BaculoGold™ DNA (Cat. No. 21100D) to achievevirtually 100% recombination efficiencies.


SphI (230)

BstXI (1248)

EcoRV (2097)EcoNI (2212)

BamHI (2878)

XmaI (2883)SmaI (2883)


HindIII (3349)


PvuII (4930)

EagI (5183)

PvuII (5508)

BanII (5808)

DraIII (5881)

GsuI (7192)

AlwNI (7756)

PvuII (8348)

pAcG2T8530 bp

unique sites underlinedpolyhedrinpromoter

AmpR

ColE ori

GST protein

EcoRI (2888)

SmaI (2883)

BamHI (2878)

CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA


Leu Val Pro Arg Gly Ser Pro Gly Ile His Arg Asp Stop

Thrombin cut

88

pAcG3X Baculovirus Transfer Vector

Catalog No. 21415P

Description: The pAcG3X Baculovirus Transfer Vector is a derivative of the pAcCL29 vec-tor.42 Foreign genes are expressed as GST fusion proteins when cloned into one of the avail-able restriction enzyme sites (BamHI, SmaI or EcoRI). All foreign inserts must be in framewith the GST ORF. The GST fusion protein expression is under the control of the strongAcNPV polyhedrin promoter. Because GST fusion proteins have a high affinity forreduced glutathione, they can be purified in a single step using glutathione agarosebeads.40 After purification, the GST affinity tag can be removed by incubating the fusion pro-tein in the presence of the site-specific protease, factor Xa. This vector is recommended foruse in conjunction with PharMingen’s BaculoGold™ DNA (Cat. No. 21100D) to achieve vir-tually 100% recombination efficiencies.

SphI (230)

BstXI (1248)

EcoRV (2097)

EcoNI (2212)

Factor Xa siteBamHI (2882)SmaI (2887)EcoRI (2892)

SnaBI (2988)

HindIII (3353)


PvuII (4934)

EagI (5187)

PvuII (5512)

BanII (5812)

DraIII (5885)

GsuI (7196)

AlwNI (7760)

PvuII (8352)


pAcG3X8534 bp


polyhedrinpromoter

AmpR

ColE ori

GST protein

EcoRI (2892)

SmaI (2887)

BamHI (2882)

ATC GAA GGT CGT GGG ATC CCC GGG AAT TCA TCG TGA

Factor Xa

Ile Glu Gly Arg Gly Ile Pro Gly Asn Ser Ser Stop

Factor Xa cleavage site

recognition sequence

89

pAcSecG2T Baculovirus Transfer Vector

Catalog No. 21469P

Description: The pAcSecG2T Baculovirus GST-fusion expression is a derivative of thepAcCL29 vector.42 Foreign genes are inserted downstream of the GST coding region intoone of the available restriction enzyme sites (BamHI, SmaI or EcoRI). All foreign insertsmust be in frame with the GST ORF. The GST gene is preceded by an in-frame gp67 signalsequence to allow secretion of the GST-fusion protein. The AcNPV polyhedrin-promoter-driven synthesis generates a fusion protein composed of the gp67 signal sequence, GST,and the foreign sequence. The gp67 signal sequence is cleaved from the fusion protein dur-ing its transport across the cell membrane. The GST fusion protein is purified from the infec-tion supernatant. Because GST fusion proteins have a high affinity for reduced glu-tathione, they can be purified in a single step using glutathione agarose beads.40 Afterpurification, the GST affinity tag can be removed by incubating the fusion protein in thepresence of the site-specific protease, thrombin. This vector is recommended for use in con-junction with PharMingen’s BaculoGold™ DNA (Cat. No. 21100D) to achieve virtually 100%recombination efficiencies.


AmpR

SphI (230)

BstXI (1248)

NaeI (1869)

EcoRV (2097)SpeI (2205)

StyI (2223)EcoNI (2326)

BamHI (2992)SmaI (2997)

EcoRI (3002)

HindIII (3460)


PvuII (5041)

EagI (5294)

PvuII (5619)

NaeI (5889)BanII (5919)DraIII (5992)

GsuI (7303)

AlwNI (7867)

PvuII (8459)

polyhedrinpromoter


8641 bppAcSecG2T

ColE ori

gp67 leader sequence

2200

polyhedrinpromoter

EcoRI (3002)

ATT GTT TTA TAT GTG CTT TTG GCG GCG GCG GCG CAT TCT GCC TTT GCG GAT CTG ATG TCC CCT ...Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His Ser Ala Phe Ala Asp Leu Met Ser Pro

ATG CTA CTA GTA AAT CAG TCA CAC CAA GGC TTC AAT AAG GAA CAC ACA AGC AAG ATG GTA AGC GCT Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr Ser Lys Met Val Ser Ala

gp67 secretion signal sequence (underlined)

glutathione S-transferase gene ... CTG GTT CCG CGT GGA TCC CCG GGA ATT CAT CGT GAC TGA Leu Val Pro Arg Gly Ser Pro Gly Ile His Arg Asp Stop

SmaI (2997)XmaI (2997)

BamHI (2992)


Thrombin cut


Signal peptide cleavage site

90

pAcGP67-A, -B and -C Baculovirus Transfer Vector Set


Description: The acidic glycoprotein gp67 (syn.: gp64) is the most abundant envelopesurface glycoprotein of the AcNPV, and is essential for the entry of Baculovirus particles intosusceptible insect cells.43 Since large amounts of this protein are secreted and anchored tothe virus peplomer, its gene contains one of the most effective Baculovirus-encoded signalsequences for protein secretion.44 We have constructed Baculovirus Transfer Vectors(pAcGP67-A, -B and -C)15 that contain the gp67 signal sequence upstream of a MCS (5’-BamHI, SmaI, XbaI or NcoI, EcoRI, NotI, EagI, PstI and BglII-3’). A gene of choicecan be inserted in one of these cloning sites and the protein of interest will be expressed asa gp67 signal peptide fusion protein under the control of the strong Baculovirus polyhedrinpromoter. The signal peptide mediates the forced secretion of the recombinant protein,even if it is normally not secreted. During transport across the cell membrane, the signalpeptide is cleaved and the native protein is easily purified from the infection supernatantwhen protein-free insect culture medium (Cat. No. 22128M) is used. This vector is recom-mended for use in conjunction with PharMingen’s BaculoGold™ DNA (Cat. No. 21100D) toachieve virtually 100% recombination efficiencies.

HindIII (1) PacI (579)XcmI (739)

SacII (868)BstEII (923)

PvuII (1307)ApaI (1395)

XhoI (1901)

SphI (2131)

BclI (2232)

NaeI (3770)

EcoRV (3998)gp67 Secretion Signal

MCS

HindIII (4375)SnaBI (4938)

HindIII (5303)

HindIII (6339)

PvuII (6874)

PvuII (7305)SapI (7360)

AlwNI (7894)

GsuI (8461)

ScaI (8854)NdeI (9546)

PvuII (9669)

ColE ori

AmpR

pAcGP67-A, -B, -C9761 bp


polyhedrinpromoter

91

Multiple Cloning Regions of pAcGP67-A, -B and -C

pAcGP67-B 9765 bp

pAcGP67-A 9761 bp

pAcGP67-C 9766 bp

4135

polyhedrinpromoter

XbaI (4266)

PstI (4287)

ATT GTT TTA TAT GTG CTT TTG GCG GCG GCG GCG CAT TCT GCC TTT GCG GCG GAT CCC GGG TAC CTTIle Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His Ser Ala Phe Ala Ala Asp Pro Gly Tyr Leu

EcoRI (4270) EagI (4281)



CTA GAA TTC CGG AGC GGC CGC TGC AGA TCT GAT CCT TTC CTG GGA CCC GGC AAG AAC CAA AAA ...Leu Glu Phe Arg Ser Gly Arg Cys Arg Ser Asp Pro Phe Leu Gly Pro Gly Lys Asn Gln Lys

NotI (4280)

SmaI (4255)XmaI (4255)

BglII (4291)

BamHI (4251)


PpuMI (4308)

SpeI (4140)

EagI (4285)

BamHI (4258)

4135

polyhedrinpromoter

PstI (4291)

ATT GTT TTA TAT GTG CTT TTG GCG GCG GCG GCG CAT TCT GCC TTT GCG GCG GAT CTT GGA TCC CGG Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His Ser Ala Phe Ala Ala Asp Leu Gly Ser Arg

EcoRI (4274)



GCC ATG GGA ATT CCG GAG CGG CCG CTG CAG ATC TGAAla Met Gly Ile Pro Glu Arg Pro Leu Gln Ile Stop

NotI (4284)

SmaI (4262)XmaI (4262)

BglII (4295)


NcoI (4268)

SpeI (4140)

BglII (4296)

BamHI (4259)

4135

polyhedrinpromoter

NcoI (4269)PstI (4292)

ATT GTT TTA TAT GTG CTT TTG GCG GCG GCG GCG CAT TCT GCC TTT GCG GCG GAT CTA TGG ATC CCG Ile Val Leu Tyr Val Leu Leu Ala Ala Ala Ala His Ser Ala Phe Ala Ala Asp Leu Trp Ile Pro

EcoRI (4275) EagI (4286)



GGC CAT GGG AAT TCC GGA GCG GCC GCT GCA GAT CTG ATC CTT TCC TGG GAC CCG GCA AGA ACC ...Gly His Gly Asn Ser Gly Ala Ala Ala Ala Asp Leu Ile Leu Ser Trp Asp Pro Als Arg Thr

NotI (4285)

SmaI (4263)XmaI (4263)


PpuMI (4313)

SpeI (4140)

signal peptide cleavage site



92

BioColors™ Baculovirus VectorsPharMingen introduces two new vector sets containing BioColors™ Genes, from the jel-lyfish Aquorea victoria. Each vector generates a fusion protein, consisting of the clonedgene product and the BioColors™ protein, which can be used for monitoring geneexpression and protein localization, in vivo and in vitro. The Baculovirus vector sets nowavailable with the BioColors™ Genes are: BioColors™ BV Control and BioColors™-His.

The BioColors™ Genes are:

• BioGreen™: Green Fluorescent Protein (GFP) absorbs UV light (max 395 nm,minor peak at 470 nm) and emits green light at 509 nm.45 Because the chro-mophore in GFP is intrinsic to the primary structure of the protein, the GFPsystem does not require exogenously added substrates. Purified GFP has spec-tral properties similar to the protein expressed in vivo: it absorbs blue light andemits green light which is detectable using a fluorescence microscope, fluo-rescence activated cell sorting (FACS), or visually, by UV light box (Fig. 18A).51

• BioBlue™: Blue Fluorescent Protein (BFP) absorbs UV light (max 382 nm) andemits blue light at 448 nm that can be detected using a fluorescence micro-scope, FACS, or visually, by UV light box.46, 47 Like the GFP system, the BFPsystem does not require exogenously added substrates (Fig. 18B).

• BioYellow™: Yellow Protein (YP) absorbs blue light (max 495 nm) and emitsa green light, at 509 nm, which can be detected visually by daylight.46,48

Since part of the emitted green light is reabsorbed by the protein due to over-lapping absorbancy and emission spectra, the net light emitted is yellow(Fig. 18C). Like the GFP system, the YP system does not require exogenouslyadded substrates.

Figure 16. BioColors™ in Sf9 cells. Sf9 cells expressing BioGreen™ (A), BioBlue™

(B) and BioYellow™ (C) were viewed under UV excitation filter (380–425 nm), a dichroicmirror (430 nm), and a barrier filter (450 nm).

BioGreen™ BioBlue™ BioYellow™

A B C

93

BioColors™ genes are useful tools for monitoring protein expression without theuse of antibodies. PharMingen’s BioColors™ genes differ in their excitation and/oremission spectra, suggesting that they may be useful in studying the interaction ofseveral proteins simultaneously using FACS (Fig. 19).

Figure 17. Separation of Baculovirus-expression GFP and BFP using fluorescence-activated cell sorting. Sf9 cells expressing a mixed population of BioBlue™ and BioGreen™

protein were viewed under UV excitation filter (380–425 nm), a dichroic mirror (430 nm), anda barrier filter (450 nm) (A), and as a histogram showing two distinguishable cell popu-lations (B).49 After FACS, Sf9 cells were evaluated again by fluorescence microscopy. TheR3-gated population (BioGreen™) is shown in (C), and the R2-gated population(BioBlue™) is shown in (D).

10 10 10 10 100 1 2 3 41010

1010

100

12

34

Green Fluorescence

Blu

e F

luo

resc

ence

R2

R3

B

C

A

D

94

BioColors™ Baculovirus Control Vector Set

Catalog No. 21518P

Description: The BioGreen™, BioBlue™ and BioYellow™ Baculovirus Control Vectors arederivatives of the pVL1393 plasmid. They are intended for use as a source for the BioColors™

gene or as a positive color control vector. A C-terminal BioColors™ fusion protein can be gen-erated if the foreign gene is cloned, in-frame, into the BamHI site. The BioColors™ codingregion is followed by a stop codon and does not allow for the expression of foreign insertscloned 3′ of the BioColors™ ORF. The BioColors™ gene is flanked by a number of uniquerestriction enzyme sites, which can be used for its removal. These vectors are recommendedfor use in conjunction with PharMingen’s BaculoGold™ Baculovirus DNA (Cat. No. 21100D)to achieve virtually 100% recombination efficiencies.

PacI (579) XcmI (739)

SacII (868)

BstEII (923)

ApaI (1395)

XhoI (1901)

SphI (2131)

BclI (2232)

NaeI (3770)

EcoRV (3998)

BamHI (4129)SmaI (4134)

NcoI (4319)XcaI (4601)


SapI (7969)

AlwNI (8503)

GsuI (9070)

PvuI (9352)

ScaI (9463)

BioGreen™ Control


BioColors™ ORF

polyhedrin promoter

ColE ori

RAmp

NotI (4889)EagI (4890)PstI (4896)BglII (4900)PpuMI (4917)

BioBlue™ Control BioYellow™ Control

95

BioColors™-His Baculovirus Transfer Vector Set

Catalog No. 21522P

Description: The BioGreen™, BioBlue™ and BioYellow™-His Baculovirus Vectors are deriva-tives of the pAcHLT-A plasmid.50 They contain a BioColors™ region with C-terminal 6xHistag downstream of the BioColors™ region and upstream of the MCS. Foreign genes may beexpressed as BioColors™-6xHis fusion proteins when cloned into one of the available restric-tion enzyme sites (EcoRI, StuI, SacI, NotI, PstI, KpnI, SmaI, or BglII). The presence ofthe BioColors™ coding sequence allows the visualization of protein expression (Fig. 18).The presence of a 6xHis tag substantially eases the purification of the recombinant proteinssince 6xHis fusion proteins bind with high affinity to Ni-NTA Agarose (a metal chelatingagent). Most host cell proteins do not bind to such a matrix. Therefore, a highly efficientsingle-step affinity purification can be done with 6xHis-tagged proteins. A protein kinase Asite follows the 6xHis sequence in the plasmid. Purified recombinant proteins can be phos-phorylated at a protein kinase A site which follows the 6xHis sequence. This phosphoryla-tion should not alter the binding affinity of the recombinant protein to any of its ligands. Ifdesired, the BioColors™-6xHis tag can be removed by incubating the fusion protein in thepresence of thrombin. All foreign inserts must be in frame with BioColors™-6xHis ORF. Thesevectors are recommended for use in conjunction with PharMingen’s BaculoGold™

Baculovirus DNA (Cat. No. 21100D) to achieve virtually 100% recombination efficiencies.

polyhedrinpromoter

BioColors™

ORF

BioGreen ™ His


AmpR

ColE ori

MCS

BioBlue ™ HisBioYellow ™ His

SphI (230)

BclI (331)

BstXI (1248)

NaeI (1869)

EcoRV (2099)

GsuI (2248)

MscI (2391)


SnaBI (3285)

HindIII (3650)


MscI (4875)

NaeI (6079)

DraIII (6182)

EcoO109I (6602)

ScaI (7100)

GsuI (7493)

AlwNI (8057)

SapI (8593)

96

B. Multiple Promoter Transfer VectorspAcUW51 Baculovirus Transfer Vector

Catalog No. 21205P

Description: The pAcUW51 vector contains a copy of the AcNPV p10 promoter and SV40transcription termination signal inserted in tandem, upstream of the AcNPV polyhedringene promoter, but in opposite orientation. One foreign gene coding region at the BamHIsite is under the control of the polyhedrin promoter and a second one at a BglII or EcoRIsite is under the control of the p10 gene promoter. Recombinant viruses will express twoforeign proteins. pAcUW51 contains the f1 origin of replication and can produce single-stranded DNA. These vectors are recommended for use in conjunction with PharMingen’sBaculoGold™ DNA (Cat. No. 21100D) to achieve virtually 100% recombination efficiencies.

p10 promoter

polyhedrinpromoter

NaeI (538)

BsmI (817)

XcmI (924)

PvuII (1211)

EcoRI (1217)BglII (1223)NsiI (1354)XbaI (1462)BclI (1486)

BamHI (1582)

AatII (1929)HindIII (2015)

AgeI (2500)

PvuII (3151)

NaeI (3351)

ScaI (4107)

GsuI (4500)

AlwNI (5064)

SapI (5600)

PvuII (5656)

ColE ori

Amp

F1 ori

R

pAcUW515863 bp


97

pAcDB3 Baculovirus Transfer Vector

Catalog No. 21532P

Description: The pAcDB3 vector is a 6 kb AcNPV polyhedrin locus-based vector that con-tains a copy of the AcNPV polyhedrin promoter and two AcNPV p10 promoters. Down-stream of the first p10 promoter are a SmaI and a BamHI cloning site, followed by poly-hedrin locus-derived terminator sequences. Upstream of this, an inverted polyhedrinpromoter has been inserted containing XbaI and StuI as single cloning sites, followed by asecond p10 promoter, EcoRI and BglII insertion sites and an SV40 terminator. The two p10promoters are in opposite orientations. This vector allows simultaneous expression of threeforeign genes during the very late phase of the Baculovirus infection cycle. The transfer vec-tor contains the F1 ori for production of single-stranded DNA. These vectors are recom-mended for use in conjunction with PharMingen’s BaculoGold™ DNA (Cat. No. 21100D) toachieve virtually 100% recombination efficiencies.

p10 promoter

polyhedrinpromoter

EcoRI (1217)BglII (1223)

StuI (1473)XbaI (1478)

SmaI(1724)

BamHI (1729)

SnaBI (1797)

AgeI (2647)DraIII (3601)

ScaI (4254)

AlwNI (5211)

SapI (5747)

ColE ori

Amp

F1 ori

R

pAcDB36010 bp


p10 promoter

98

pAcAB3 Baculovirus Transfer Vector

Catalog No. 21216P

Description: The pAcAB3 is a 10.0 kb AcNPV polyhedrin locus-based vector that con-tains a copy of the AcNPV polyhedrin promoter and two AcNPV p10 promoters.51 Down-stream of the first p10 promoter are a SmaI and a BamHI cloning site, followed by poly-hedrin locus-derived terminator sequences. Upstream of this, an inverted polyhedrinpromoter has been inserted containing an XbaI and a StuI cloning site, followed by asecond p10 promoter, a BglII insertion site and an SV40 terminator. The two p10 pro-moters are in opposite orientations. Using pAcAB, three foreign genes can be simultane-ously expressed during the very late phase of the Baculovirus infection cycle. This vectoris recommended for use in conjunction with PharMingen’s BaculoGold™ Baculovirus DNA(Cat. No. 21100D) to achieve virtually 100% recombination efficiencies.

AgeI (450)


StyI (1500)

XhoI (1900)

SphI (2130)BclI (2231)

NaeI (3769)

EspI (4439)EcoRI (4448)

BglII (4454)StuI (4704)

XbaI (4709)SmaI (4955)BamHI (4960)

SnaBI (5028)HindIII (5393)

AgeI (5878)

HindIII (6439)

EagI (7227)

EcoRI (7455)

NdeI (7678)

ScaI (8369)

GsuI (8762)

AlwNI (9326)

SapI (9862)

EcoRI (10096)

AmpR

ori

Promoters:polyhedrin

p10p10

pAcAB310096 bp


99

pAcAB4 Baculovirus Transfer Vector

Catalog No. 21412P

Description: pAcAB4 is a 10.0 kb AcNPV polyhedrin locus-based vector that containstwo copies of the AcNPV polyhedrin promoter and two AcNPV p10 promoters.56 Down-stream of the first p10 promoter are a SmaI and a BamHI cloning site, followed bypolyhedrin locus-derived terminator sequences. Upstream of this, an inverted poly-hedrin promoter has been inserted containing an XbaI and a StuI cloning site, fol-lowed by a second p10 promoter, a BglII insertion site and an SV40 terminator. Thereis an additional polyhedrin promoter in opposite orientation to the first copy, and is injuxtaposition to the first p10 promoter. The second polyhedrin promoter is followed bya BamHI cloning site. The two p10 promoters are in opposite orientations. UsingpAcAB, four foreign genes can be simultaneously expressed during the very late phase ofthe Baculovirus infection cycle. This vector is recommended for use in conjunction withPharMingen’s BaculoGold™ Baculovirus DNA (Cat. No. 21100D) to achieve virtually100% recombination efficiencies.

AgeI (450)


StyI (1500)

XhoI (1900)

SphI (2130)BclI (2231)

NaeI (3769)

EspI (4439)EcoRI (4448)

BglII (4454)StuI (4704)

XbaI (4709)SmaI (4960)

BamHI (5094)SnaBI (5162)

HindIII (5527)

AgeI (6012)

HindIII (6573)

EagI (7361)

EcoRI (7589)

NdeI (7812)

ScaI (8503)

GsuI (8896)

AlwNI (9460)

SapI (9996)

EcoRI (10230)

AmpR

ori

pAcAB410230 bp


Promoters: polyhedrin

p10p10

polyhedrin

100

I I. p10 Locus-based Transfer VectorsA. Single Promoter Transfer VectorspAcUW1 Baculovirus Transfer Vector

Catalog No. 21203P

Description: pAcUW1 is an AcNPV p10 locus-based vector that contains a copy of theAcNPV p10 promoter, but lacks part of the amino terminal p10 gene coding region. A BglIIcloning site, for foreign gene insertion, is located downstream of the p10 promoter.pAcUW1 is the plasmid of choice for the construction of a recombinant Baculovirus whichcan be fed to T.ni larva. Alternatively, this vector can be used for independent co-expressionof two proteins, one under polyhedrin expression, and one under p10 expression, codedfor by separate viruses. pAcUW1 is a non-polyhedrin-based vector which must be used inconjunction with linearized AcUW1.lacZ Baculovirus DNA (Cat. No. 21102D).

p10promoter

SalI (20)MscI (130)

BstXI (258)EagI (356)

SacII (400)PmlI (417)PflMI (461)

EspI (619)BsaB1 (699)

HpaI (735)

BclI (1332)PshAI (1436)HindIII (1460)BspE1 (1501)

BglII (1541)PacI (1585)

NsiI (1675)MluI (1753)XhoI (1778)

SphI (1785)XcmI (1917)

BglI (2067)NarI (2082)

AatII (2386)BsrBI (2465)

XmnI (2705)

ScaI (2826)

BglI (3185)GsuI (3219)

BsaI (3237)

AlwNI (3783)

BsrBI (4266)SapI (4319)

BsrBI (4507)

pAcUW14552 bp

ColE ori

AmpR


101

B. Multiple Promoter Transfer VectorspAcUW42, pAcUW43 Baculovirus Transfer Vector Set

Catalog No. 21208P

Description: pAcUW42, pAcUW43 are AcNPV, p10 locus-based vectors that are derivativesof the pAcUW41 transfer vector. Each plasmid has a copy of the polyhedrin gene promoterinserted downstream and in tandem with the p10 gene promoter. Between the two pro-moters, a copy of the SV40 transcription termination sequences has been inserted to pre-vent read-through into the polyhedrin gene promoter. A MCS has been inserted down-stream of the p10 promoter. The MCS reads: BglII, PstI, NotI, XbaI, KpnI, SmaI (from 5’to 3’ for pAcUW42 and 3’ to 5’ for pAcUW43). The insert of choice must provide its ownATG start signal at the 5’ end of the gene. The distance between the cloning site and theATG start of the gene should not be longer than 100 nucleotides, otherwise protein expres-sion may be poor. These vectors contain the f1 origin of replication and can produce, byhelper phage mediation, single-strand DNA, useful in sequencing and mutagenesis. This setof transfer vectors must be used in conjunction with linearized AcUW1.lacZ, BaculovirusDNA (Cat. No. 21102D).

R

ColE ori

p10 promoter

HindIII (1)SalI (33)

Spe I (465)

EcoRI (609)EcoRI (699)EcoRI (801)

EcoRV (853)EcoRI (908)

EcoRI (981)EcoRI (1088)

SalI (1222)SacI (1505)NaeI (1519)EcoRI (1595)PvuII (1612)NdeI (1624)SphI (1721)XhoI (1728)NsiI (1831)

MCS

BsmI (2409)BclI (2465)

BamHI (2562)

BclI (2696)

HpaI (3293)SacII (3628)SalI (4008)

EcoRI (4027)

PvuII (4205)Sap I (4260)

AlwNI (4794)

GsuI (5361)

ScaI (5754)

NaeI (6775)NarI (6974)

PvuII (7045)

AGATCCCGGGTACCTTCTAGAATTCTGAGCGGCCGCTGCAGATCTTCTAGGGCCCATGGAAGATCTTAAGACTCGCCGGCGACGTCTAGA

AGATCTGCAGCGGCCGCTCCAGAATTCTAGAAGGTACCCGGGATCTTCTAGACGTCGCCGGCGAGGTCTTAAGATCTTCCATGGGCCCTAGA

Unique sites

SmaI (1967) XbaI (1978)NotI (1992)

PstI (1999)

BglII (2003)

SmaI (1999)

XbaI (1988)NotI (1972)

PstI (1967)

BglII (1963)

Unique sites

pAcUW42

pAcUW43

MCS KpnI (1995)

KpnI (1971)

p10 promoter

p10 promoter

pAcUW42/437135 bp


polyhedrinpromoter

Amp

F1 ori

103

AAcNPV, Autographa californica

nuclear polyhedrosis viruscycle of infection of

in cell culture, 1, 2in host, 1, 2

AcUW1-lacZ DNA, 7-8,transfer-vector, 100-101

Amplification of viral stocks, 24, 29Autographa californica nuclear

polyhedrosis virus, 1, 2

Bβ-galactosidase, viii, 7 (see also X-gal)Baculoviruses

AcNPV, 1, 2amplification of, 24, 29basic protein, 11-12, 81budded virus, 2DNA genome, 1expression vectors, 7-9, 49-51gene expression, 14, 32host-range, 1isolation, 31nucleocapsid, 1, 2production of, 22polyhedrin, 1promoters, 2, 11, 12replication in vitro, 2replication in vivo, 2titration of, 24, 25, 26transfer vectors, 2, 10-12, 77-101

(see also Transfer vectors)virus particle, 1-2virus structure, 2

Bacterial transformation, 17Baculovirus expression system advantages, 3-5Baculovirus expression vector system (BEVS), 1Basic protein gene

promoter, 9, 11, 12transfer vector, 80

BioColors™, 92-95

Index

104

BioBlue™, 92-95BioGreen™, 92-95BioYellow™, 92-95Budded virus, 1-2

concentration and purification of, 30

DNA from, 31infection using, 29, 30storage of, 30structure of, 1

CCalcium phosphate-mediated

transfection, 22Catechol, 22-23, 68, 72Cell culture media, 65, 66Cell lines, 17-21

(see also Spodoptera frugiperda)Cell lysis buffer, 36, 67, 69, 71, 73Cell scrapers, 20Cloning virus isolates

by end-point dilution, 24by plaque purification, 26

Construction of recombinant transfer vectors, 2

(see also Transfer vectors)Co-transfection of insect cells

as a means of inserting foreigngenes, 23

calcium phosphate co-precipitation, 23

DDirect cloning vectors, 48-50DNA

extraction of, 31linearized, 7-9purification of, 30quality of, 22replication of, 29

DTE, viii, 57DTT, viii, 57Dual-expression vectors, 12, 96-101

EEarly promoters, 12Electron microscopy, AcMNPV-

infected cells, 22End-point dilution assay, 24-26Expressing recombinant protein, 31-32

105

FFactor Xa, 47-48Fall army worm cells, 66

(see also Spodoptera frugiperda)Fetal Bovine Serum (FBS), viii, 54-66Fluorescence (see BioColors™)Freezing of cells, 22

GGlutathione agarose beads, 40, 47Glutathione S-transferase

tag, 5, 39purification, 39, 43-47(see also GST)

Glycosylation in insect cells, 3gp64 gene (see gp67)gp67, 90-91Grace’s medium (see TNM-FH media)Green Fluorescent Protein (GFP), 4, 92

(see also BioColors™)GST, 5, 39, 43-47, 58-59, 71-73, 82-83, 86-89

(see also glutathione S-transferase)

H6xHis, 5, 9, 38-42, 46, 57-58, 67-69, 82-85Host gene expression in infection, 1Histidine tag (see 6xHis)

IImmediate early genes (AcNPV)

promoters, 12Infection of insect cells with

AcMNPV, 22, 32-33Insect cell culture

cell scrapers, 20contamination, 19freezing in, 22monolayer cultures, 20scale-up, 19seeding densities for

experimental work, 18spinner cultures, 20subculturing monolayer

cultures, 20-21suspension cultures, 21viability, 19

Insect cell culture media, 19-21, 66Insect cell lines, 17-21Insect cell lysis buffer, 34, 69, 73

106

Isolating virus particles, 30Isolating virus DNA, 31

LlacZ gene, 5-8Late promoters, 12, Ligations, 16Linearized DNA, 1, 8-10, 65

MMini-prep, DNA isolation method, 17Monolayer cell cultures, 18-20Monolayer insect cell cultures

infection with AcMNPV, 22-24, 32-33plaque-assay, 22scale-up, 17-21 (see also Insect cell culture)

Multiplicity of infection (MOI), 25, 32

NNi-NTA agarose, 5, 38-41, 61-62, 72, 82, 84

PPhenol-chloroform, 15, 49Phosphorylation, 3, 34, 39Plasmid (see Transfer vector)Plaque assay of virus isolates, 26Plaque-assay determination of virus stock titer, 24-28Plaque-picking, 28-29Plaque purification of virus isolates, 28Plaque-purification, 28Plasmid DNA isolation, 17Polyhedrin promoter, 9, 11, 12, 35, 47Poly(A) (polyadenylation signal), 13Polyhedrin-based transfer vectors, 7, 8, 12, 77-89p10-based transfer vectors, 9, 12, 91, 100-101Post-translational processing, 3, 32, 34Promoters,

choice of, 11,12early, 11,12late, 11,12very late, 11,12

Protein-free media, 20, 21Protein production,

harvesting of, 33, 34N-glycosylation of, 3, 32phosphorylation of, 3, 34

Purification of recombinant proteins, 36-48Purification of virus particles, 30Purification of virus DNA, 31

107

32P-labeled proteins, 46, 47

RRadiolabelling proteins in virus-infected cells, 45Recombinant viruses,

amplification of, 24, 29end-point dilution cloning of, 24identification of, 22phenotypes of, 22plaque purification of, 28screening of, 22selection of, 27-29

SScale-up of insect cell cultures, 19Scale-up of protein production

in cell culture, 32-33Seed stock of virus, 29Selection of recombinant transfer

vectors, 11-13Selection of recombinant virus,

polyhedrin-negative phenotype, 22polyhedrin-positive phenotype, 22

Serial passaging, effect on virus of, 29Serum-free media for cell culture

(see Protein-free media)Serum-supplemented media for cell

cultures (see TNM-FH media)Sf cells, 17-21, 66

(see also Spodoptera frugiperda cells)Spodoptera frugiperda (Sf) cells, 66

culture of, 17-21protein production in, 32storage of, 21

Suspension cell cultures, 21, 33

TThawing of cells, 22Thrombin,

cleavage, 38, 42, 45, 46-47, 59consensus site, 46powder, 67, 68, 71, 72

TNM-FH medium, 18-21, 66Transfection, 22, 54Transfer vectors,

basic protein promoter, 11, 12, 80BioColors™ BV Control (set), 92-94BioColors™ His (set), 92-95maps of, 77-101

108

multiple expression, 96-99, 101p10 locus-based, 100-101polyhedrin locus-based, 77-99polyhedrin promoter, 11, 12pVL1392/3 (set), 65-66, 77-78pAcSG2, 79pAcMP2/3 (set), 11, 80pAcUW21, 81pAcGHLT-A, -B, -C (set), 75, 76, 82-83pAcHLT-A, -B, -C (set), 67-68, 84-85pAcG1, 86pAcG2T, 87pAcG3X, 88pAcGP67-A, -B, -C (set), 90-91pAcSecG2T, 89pAcUW51, 96pAcAB3, 98pAcAB4, 99pAcDB3, 97p10 locus-based, 100-101pAcUW1, 100pAcUW42/43 (pair), 101

Troubleshooting guide, 53-59

VvECuni Baculovirus DNA, 48-50, 75Vectors, 2, 7-9

(see also Transfer vectors)vEHuni Baculovirus DNA, 48-50, 75Very-late genes,

p10, 12polyhedrin, 12promoters, 12

Very-late promoters, 12Viability of cells, 17-21Virus particle, 1Virus DNA purification, 30Virus purification, 31Virus stocks, 29Virus titration, 24-28

XX-gal, viii, 6, 28XylE, 22-25, 65, 67, 71

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Baculovirus Expression Vector System Manual

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