CHAPTER 3

DEVELOPMENT OF NEW LAMBDA

PHAGE BASED DISPLAY VECTORS FOR

HIGH DENSITY DISPLAY

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3.1 INTRODUCTION

Filamentous phage M13/fd has been proven to be the vector of choice to generate small peptide libraries or display specialized repertoires where variability is confined to a few amino acids in the context of a fixed scaffold (i.e.

antibody libraries). One caveat of the M13 display is that fusion products must be secreted into the periplasm prior to assembly onto the phage capsid (Model & Russel, 1988). This requirement may introduce a bias during phage production presumably due to inefficient recombinant protein translocation (Malik et al., 1996) that in turn would lead to under representation, or even the absence, of many polypeptides in the library. The gIIIp of filamentous phage has been shown to tolerate insertion of foreign peptides of considerable size (Felici et al., 1995), but this is achieved at the expense of the multivalency of the display, which is an essential requisite when using weak or multispecific ligand for affinity selection (Folgori et al., 1994). Moreover, in the gIIIp based phage display systems only a fraction of phages carry the displayed fusion protein and most of the phages carry only the gIIIp protein encoded by the helper phage genome. Thus, while it is successful in applications where high affinity interactions are to be studied but for several applications like, identification of immunodominant epitopes using patient’s sera, high-density display of peptides or protein domains are required. Thus, as an alternative to filamentous phage, new display vectors based on lytic bacteriophage lambda have been reported wherein the encapsidation of the fusion protein is an intracellular event, thus making assembly of chimeric phage a less demanding process (Maruyama et al., 1994; Kuwabara et al., 1997). There have been several reports describing N- and C-terminal display of proteins or peptides on phage lambda as fusion to the capsid protein gpD and tail protein gpV of lambda, (Dunn et al., 1995; Sternberg and Hoess, 1995; Mikawa et al., 1996). Use of the lambda capsid protein gpD appears to be a particularly attractive option, since a variety of proteins or protein domains as large as β-galactosidase have been successfully displayed as fusions to its N or C termini (Sternberg and Hoess 1995; Mikawa et al., 1996).

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In this chapter we have described the construction and characterization of lambda phage vectors based on 2-gpD-gene system wherein the second copy of D gene with an amber mutation is present within the left arm of the vector derived from λDam. The other copy of recombinant gpD is located within the plasmid inserted between the terminator and initiator domains followed by the right arm of lambda derived from λZap. The left and right arm carried the information required for the production of an infective phage particle, whereas the inserted plasmid contains the DNA sequence encoding for the D-fusion products with desired N-terminus and C-terminus affinity tags. This would generate mosaic phage particles that contain both native and recombinant versions of gpD. Different lambda constructs were designed containing plasmids with variable sequences. The phages produced were characterized for the display density of the fusion protein using western blot. To further check the functional display, three mycobacterial proteins of variable sizes were cloned into the λZapDamDc106 vector to be displayed on the lambda surface as fusion to the C-terminus of the D protein and then characterized by western blot and affinity selection studies to show high density display of proteins. 3.2 MATERIALS AND METHODS 3.2.1 Materials

TG1 strain (F', traD36 proAB+ lacIq lacZΔM15) supE thi-1 Δ(lac-proAB)

Δ(mcrB-hsdSM)5, (rK-mK

-) were obtained from New England Biolabs (Beverly, MA). TG1 cells are most commonly used for phage display.

pVCDcDL1 (3431 bp) is a high copy number plasmid having backbone derived from pUC119 and carries under the control of the lac promoter operator (lacPO), a sequence encoding gpD of lambda followed by four amino acid GGSG spacer(s), a collagenase site, NheI site, a stuffer segment, MluI site and a c-myc tag. Cloning of DNA sequence as NheI - MluI inserts in place of stuffer allows expression of D-fusion protein with collagenase site between D and the foreign

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protein and c-myc tag at the C-terminus. The vector also contains the M13 phage origin of replication (f-ori) flanked by loxPwt and loxP51 recombination sequences.

pVCEPI13960 (6305 bp) is a high copy number plasmid having backbone derived from pUC119 and carries under the control of lac promoter operator between HindIII and EcoRI a cassette comprising XbaI site, followed by ribosome binding site (RBS), codons of Pectate lyase B (PelB) signal sequence, cloning sites and gene encoding full length gene III. The lac promoter operator is preceded by a transcriptional terminator from glutamine permease operon (glnHPQ) of E. coli.

The vector backbone further comprises of the intergenic region of phage M13 (f-ori), β-lactamase gene as selection marker, ColE1 origin of replication and T7 terminator sequence between EcoRI site and f-ori. In this chapter this vector has been used as HindIII – EcoRI vector.

pVCSDH1033 (3487 bp) is a high copy number vector that contains under the control of lac promoter operator a DNA cassette encoding Strep tag, gpD of lambda, four amino acid GGSG spacer(s), a collagenase site, NheI site, a stuffer

segment containing the MCS, Bsu36I site and a hexa-histidine tag followed by translational stop and EcoRI site. The vector backbone comprises of the β-lactamase gene as selection marker and ColE1 origin of replication. In this chapter, this vector has been used as a source for Strep tag-gpD-H6 fusion gene.

pVLSDH1013 (5353 bp) is a low copy T7 promoter based expression vector containing a sequence encoding N-terminus Strep tag - gpD of lambda followed by a small stuffer segment and a hexa-histidine tag at the C-terminus. The vector

backbone comprises of the intergenic region of phage M13 (f-ori), β-lactamase gene as selection marker, ColE1 origin of replication, rop gene, and lac repressor laci. This vector was modified by site directed mutagenesis to delete BglII site at

position 5342 bp by changing the base from ‘A’ to ‘C’ (shown as bold and underlined) and create a unique SacII restriction site (underlined) using an oligonucleotide pVLdelSacII (5’

GTCGTATTAATTTCGCGACCGCGGGATCTCGATCCTCTACGC 3’) to obtain a

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derivative named as pVLSDH1013 SacII plasmid. This vector was used to obtain the laci repressor and rop gene to convert a high copy number vector to low copy number.

Lambda ZAP vector (Short et al., 1988) that excises the pBluescript phagemid was constructed by separating two overlapping domains of the f1 origin of replication. The initiator domain contains the site for nicking of double stranded DNA by gene II protein and the initiator site of DNA synthesis for the plus strand. The terminator domain contains the site for cleavage of single stranded DNA by gene II protein and circularization of the single stranded DNA. The plasmid pBluescript SK(-) was inserted into the nonessential region of a lambda phage genome such that the plasmid sequences were flanked by the

initiator and terminator domains. The 3.0 Kb pBluescript phagemid in the Lambda ZAP vector conferred ampicillin resistance, contained a ColEl origin of replication, and, the alpha portion of the lacZ gene required for alpha-complementation and a pBluescript SK polylinker with 21 unique cloning sites

flanked by T3 and T7 RNA polymerase promoters. The right arm of lambda ZAP also contains the nin5 (2.5 Kb) deletion. The Lambda ZAP vector containing 40.8 Kb long DNA has six unique cloning sites that can accommodate DNA inserts from <1 to 10 Kb in length.

The Lambda ZAP vector was designed to allow simple, efficient in vivo excision and recircularization of any cloned insert contained within the lambda vector to form a phagemid containing the cloned insert. The lambda phage (target) is made accessible to the f1-derived proteins by simultaneously infecting a strain of E. coli with both the lambda vector and the f1 bacteriophage. Inside E.

coli, the "helper" protein gene II (i.e., proteins from f1 or M13 phage) recognize the initiator DNA and introduces nicks in one of the two DNA strands. At the site of this nick, unidirectional replication proceeds from the origin displacing the f1 plus strand to synthesize new DNA strand until a termination signal, positioned

3´ to the initiator signal, is encountered within the constructed lambda vector. The single-stranded DNA is circularized by the gene II product, forming a circular

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DNA molecule containing the DNA between the initiator and terminator. The plus strand DNA is converted to a double stranded RF molecule by synthesis of the minus strand origin utilizing host derived proteins. Signals for “packaging” the newly created phagemid are contained within the f1 terminator that allows the circularized DNA to be “packaged” and secreted from the E. coli.

3.2.2 Construction of Lambda phage based display vector 3.2.2.1 Construction of pVCDc01 plasmid vector DNA fragment encoding full-length 1-109 residues of gpD, spacer,

collagenase site, 30-nucleotide long stuffer and decapetide c-myc tag was obtained from pVCDcDL1 vector by digestion with HindIII - EcoRI restriction enzymes. Following treatment with phenol - chloroform and ethanol precipitation, the digested DNA was purified from 1.2 % low melting agarose (SeaKem) using QIAquick Gel Extraction kit (Qiagen, Hilden, Germany) and then cloned into the

similarly digested, dephosphorylated vector backbone of pVCEPI13960 to obtain recombinants named as pVCDc01. The recombinants were identified by colony

PCR using ori2 (5’ ACCTCTGACTTGAGCGTCGA 3’) and T7Tn (5’

GTCGGTTGAGTCGAAGGAAA 3’) primers followed by sequencing of plasmid

with ori2, M13R (5’ AGCGGATAACAATTTCACACAGGA 3’), L6 (5’

TTATTGTCTCATGAGCGGATA 3’), L7 (5’ GGAGGCTGCCAGCGACGAGAC 3’) and T7Tn primers using Big Dye terminator chemistry and automated DNA

sequencer (ABI Prism 3700). 3.2.2.2 Site directed mutagenesis to convert pVCDc01 to pVCDc05 vector pVCDc01 vector was subjected to site directed mutagenesis using uracil containing single stranded DNA template following the protocol described in Appendix III.16 (Kunkel et al., 1985). Mutagenesis was carried out in sequential steps to bring the following modifications

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a) Deletion of BsaI site located within the β-lactamase gene, b) Introduction of FseI site between the phage origin of replication (f-ori)

and β-lactamase gene, c) Insertion of SacI site upstream of lacPO,

d) Deletion of Bsu36I site located just upstream of the lacPO and e) To insert AvrII site just after the T7 terminator sequence.

Oligonucleotide pVLBsadel (5'

CAGTGCTGCAATGATACCGCGAGAACCACGCTCACCGGCTCCAGA 3') was used to delete BsaI site present within the β-lactamase gene (at position 2141 bp) of pVCDc01 vector and FseI-Amp (5’ TAGGGGTTCCGCGCACATTTGGCCGGCCAGTGCCACCTAAATTGTAAGCGT

TAATATT 3') was used to introduce a unique FseI restriction site (underlined) (at

position 1306 bp) between the phage origin of replication (f-ori) and β-lactamase gene. Oligonucleotide SacI-Cap (5' TGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCCGAGCTCTAGG

TGGGCTGCAAAACAAAAC 3’) was used to create SacI restriction site (underlined) upstream of the cap binding site of lac promoter operator (at position

3195 bp) and to delete Bsu36I site located (at position 3188 bp) upstream of the lac promoter operator. A unique AvrII (underlined) site was introduced just downstream of the T7 terminator sequence using an oligonucleotide AvrII-ApaI (5’ TACGCCAGAATTGATCTCGATGGCCACCTAGGTCCTCCTTTCAGCAAAAAA

CCCCTC 3’). The final mutant obtained was named as pVCDc05 and characterized by restriction enzyme digestion followed by DNA sequencing of plasmid with ori2,

M13R, L6, L7 and T7Tn primers. 3.2.2.3 Construction of pVCDc101 plasmid for insertion into lambda vector DNA encoding Strep tag, full length gpD protein, spacer, collagenase site

and polyhistidine (His6) tag was amplified from purified DNA of pVCSDH1033

as template using 5’ primer SacI-cap-1033 (5’

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GGAAAGCGGAGCTCGGGCGCAACGCAATTAATGTGAGTTAGCTCACTCAT

TAGG 3’) that annealed in the cap binding site upstream the lac promoter

operator with a tail carrying SacI restriction site (underlined) at the 5’ end and 3’

primer Dc101-EcoRI (5’ CGAAGTTATGAATTCTTAATGGTGGTGATG 3’) that annealed in the template region encompassing the EcoRI site (underlined) located immediately after the translational stop codon. PCR was set up in 200 µl reaction (4 x 50 µl) volume containing 20 ng of template, 100 pmoles of the each primer, 200 µM dNTPs and 1.2 U of Expand High Fidelity polymerase (Roche Molecular Biochemicals, Mannheim, Germany). The amplification steps involved initial

heating at 95oC for 4 min followed by 25 cycles of denaturation at 95oC for 30 sec,

annealing at 55oC for 30 sec and polymerization at 72oC for 50 sec with 2 sec extension in each cycle. The final polymerization was carried out at 72oC for 4 min. The PCR product was purified using QIAquick PCR purification kit (Qiagen, Hilden, Germany), digested with SacI - EcoRI restriction enzymes and finally ligated to similarly digested, dephosphorylated backbone of pVCDc05 vector. The recombinants pVCDc101 were confirmed by sequencing of plasmid

DNA using ori 2, M13R and L7 primers. 3.2.2.4 Construction of Dc101-Zap intermediate vector A segment of Lambda Zap (λZap) DNA encoding H, L, K, I, J, lom essential genes, BsiWI restriction site and terminator domain (Fter; T) of phage origin of replication was amplified from λZap DNA (Stratagene) as template

using 5’ primer λBsadelT4 (5’ CGCTAGAGACTTGGGCAGACAGGACTGCG 3’) that annealed within the H gene encompassing the BsaI site located at position

11418 bp position of wild type lambda sequence to delete the site and 3’ primer

λZapFse (5’ CCGCGCACATTTGGCCGGCCAGTGCCACCTGACGCGGCCT 3’) which annealed downstream of terminator sequence (T) and introduced a unique

FseI restriction site (underlined) at the 3’ end of the amplified product. In another PCR, the complete DNA sequence of pVCDc101 vector excluding the f-ori region was amplified with 5’ primer PspT4Dc101 (2) (5’

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GGCCACCTAGGTCCTCCTTTCAGC 3’) that annealed downstream of T7

terminator followed by AvrII restriction site and 3’ primer FseDc101 (5’

AGGTGGCACTGGCCGGCCAAATG 3’) that binds upstream of β-lactamase gene encompassing the FseI site (underlined). Each amplified product was purified

using QIAquick PCR purification kit and 3 µg of each purified PCR product in two separate reactions was digested with 10 U FseI restriction enzyme in reaction volume of 100 µl. The digested DNA samples were separated on 1.2 % low melting agarose gel to obtain desired bands and DNA was extracted using QIAquick gel extraction kit. These DNA fragments were ligated overnight in the presence of 1 U T4 DNA ligase (1 U/µl, Roche Molecular Biochemicals, Germany). The ligated DNA was employed as template for two separate PCR.

One PCR was set up using 5’ λ19223F (3) (5’

CGCGTCGAAAAAGAGCAGCACAG 3’) that anneals within the J gene

upstream of the BsiWI restriction site and 3’ L6-1 (4) (5’

TATTATTGAAGCATTTATCAGGGT 3’) primer that anneals within the β-lactamase gene of the plasmid pVCDc101 situated downstream of Fter domain of Lambda. Another PCR was set up with 5’ primer Fterseq (1) (5’

CTTGACGGGGAAAGCCGGCGA 3’) that binds within the Fter sequence of λ

Zap DNA and 3’ primer PspT4DC101 (2) that anneals downstream of T7 terminator. The two PCR products were purified using QIAquick PCR purification kit, mixed in an equimolar ratio and then employed as a template for

PCR using 5’ λ19223F primer (3) and 3’ DC101-Xho (5’

TGATCTCGATGTCGACCTAGGTCCTCCTTTCAGC 3’) primer that annealed at the end of the T7 terminator and introduced SalI restriction site (underlined) at

the 3’ end of the amplified product. The resultant PCR product of ~ 11.0 Kb was purified and then recircularized by self-ligation after blunting with Klenow fragment (5 U/µl, New England Biolabs, Beverly, MA) and kinasing in the presence of T4 polynucleotide kinase (10 U/µl, New England Biolabs, MA). The recircularized plasmid was sequenced with L7, L6-1 and ori 2 primers and finally named as named Dc101-Zap. The sequencing data showed that the gpD gene contained several mutations introduced during PCR. Thus, to replace the

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mutated gpD gene of Dc101-Zap vector, DNA cassette encoding strep tag, full-length gpD gene and hexa-histidine tag was obtained from pVCDc101 vector after digestion with BspLU11I - EcoRI restriction enzymes and then ligated to similarly digested vector backbone of Dc101-Zap to construct pVCDc101- Zap1 vector. 3.2.2.5 Construction of λZapDc101 phage vector

To construct λZapDC101, 5 µg of λZap DNA was digested with 10 U of BsiWI and 40 U of XhoI restriction enzymes in reaction volume of 50 µl with 2 hrs

incubation at 37oC, followed by additional 20 U BsiWI and incubation at 55oC for 2 hrs. To insert plasmid into λZap, 10 µg of pVCDc101-Zap1 DNA was digested

with 30 U BsiWI and 60 U of SalI restriction enzymes in a reaction volume of 300

µl with 3 hrs incubation at 37oC followed by 2 hrs at 55oC. Following digestion, the DNA was extracted using phenol-chloroform, precipitated with ethanol, resuspended in 0.1 x TE and purified on 1.0 % low melting agarose gel. The

desired bands of BsiWI - XhoI digested two arms of lambda (18.6 Kb and 19.3 Kb)

and BsiWI - SalI digested DNA fragment of pVCDc101-Zap (3.1 Kb) were ligated to reconstruct sequence as in λZap followed by Fter, FseI site, β-lactamase gene, ColE1 origin of replication, lac promoter operator, XbaI site, ribosome binding site (RBS), gpD of lambda preceded by Strep tag and followed by multiple cloning site (MCS), hexa-histidine tag, translational stop, EcoRI and T7 terminator. DNA was extracted using QIAquick gel extraction kit followed by estimation on 1.2 % -TAE agarose gel. 10 µl ligation reaction was set up with 50 ng of BsiWI - XhoI digested lambda arms and 15-fold molar excess i.e., 50 ng of BsiWI - SalI digested plasmid insert in the presence of 5 U T4 DNA ligase (5 U/µl, Roche Molecular Biochemicals, Mannheim, Germany). The ligation mix was then packaged in vitro using the Gigapack II Packaging Extract (Stratagene, La Jolla, CA, USA; packaging efficiency = 2 x 109 pfu/µg) following manufacturer’s protocol and plated on a lawn of E. coli TG1 cells to obtain isolated plaques. The plaques were

analyzed for recombinants by PCR using 5’ primer Fterseq and 3’ primer Fintseq

(5’ GAATTTTAACAAAATATTAACG 3’) and confirmed by sequencing with ori

2, M13R, T7Tn and L7 primers. The recombinant phage containing ~ 40 Kb

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genome was named λZapDc101. Selected clone was used for the preparation of small- scale phage lysate using the lysis protocol as described in Appendix III.17.1. Briefly, a well-isolated plaque was picked and used to infect 200 µl TG1

cells of OD600nm = 0.5 (~ multiplicity of infection MOI = 0.001) for 20 min at 37oC without shaking. The infected cells were diluted in 2 ml LB medium

supplemented with 10 mM MgCl2 followed by growth at 37oC with shaking at 220 rpm for 5 - 6 hrs till complete lysis was observed. The cell free supernatant was obtained by two times centrifugation at 14,000 rpm (SIGMA centrifuge, 1-15, Germany) for 20 min at 4oC. To further confirm the insertion of plasmid pVCDc101-Zap1 into the lambda DNA, in vivo excision of the plasmid was carried out using ExAssist helper phage protocol as described in Appendix III.17.6. The plasmid DNA was isolated and characterized by digestion of the excised phagemid DNA with XbaI and NgoMIV restriction enzymes. After characterization, the selected phage clone of λZapDc101 was used to extract genomic DNA from 500 ml culture using the Zinc chloride (ZnCl2) based phage precipitation method as described in Appendix III.17.5. 3.2.2.6 Construction of λZapDamDc101 phage display vector To construct λZapDamDc101, 2 µg genomic DNA of λZapDc101 was digested for 2 hrs at 55oC with 20 U of BsiWI restriction enzyme in 20 µl reaction volume to obtain two fragments. In another digestion reaction, 2 µg of λ Dam genomic DNA (gifted by Dr Ron Hoess) was similarly digested with BsiWI to obtain two fragments. For ligation, 7 µl of digested λZapDc101 was mixed in an equimolar ratio with 7 µl of digested λDam, heated at 80oC for 20 min followed by slow cooling to room temperature (25oC) and finally incubated overnight in the presence of 2 U T4 DNA ligase. The ligation mix was packaged in vitro and plated on lawn of TG1 cells to obtain plaques as described earlier in section 4.2.2.5. The recombinant plaques were screened by PCR using ori 2 and T7Tn primers. The selected recombinants were further screened in another PCR using

5’ primer L8 and 3’ primer L10 to confirm the presence of D gene, followed by sequencing with L9 primer that anneals upstream of D gene with amber mutation

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(Dam). Small-scale lysate was prepared from the selected recombinant clone as

described in section 3.2.2.5. For large-scale production of phages, 10 ml TG1 cells

(OD600nm= 0.7-0.8) were infected with λZapDamDc101 phages at MOI of 0.0003

followed by incubation at 37oC for 20 min without shaking. 5 ml of the infected cells were diluted 100-fold in 495 ml LB media supplemented with 10 mM MgCl2

and grown at 37oC, 275 rpm, till complete lysis (~ 6 - 7 hrs). Phage supernatant was obtained by centrifugation at 10,000 rpm (GSA rotor Sorvall RC5B) for 20 min at 4oC. Phages were concentrated using PEG - NaCl precipitation method

described in detail in Appendix III.17.3. The concentrated phage supernatant obtained after ultra centrifugation was used to infect the TG1 cells to determine plaque-forming unit (pfu) as described in Appendix III.17.4. The purified phage lysate was used to extract genomic DNA of λZapDamDc101 phage using the Zinc chloride (ZnCl2) based phage precipitation method. 3.2.2.7 Construction of series of λZapDam based phage vectors using different intermediate plasmids

3.2.2.7.1 Construction of intermediate vector pVCLDc102 and its corresponding lambda phage vector λZapDamDc102 To construct pVCLDc102, oligonucleotide duplex containing sequence encoding for ten histidine residues was synthesized by annealing 2 µg each of two complementary oligonucleotides H10BE-51 (5’

GCGCGCAGCACCACCATCACCACCATCACCATCACCATTAAG 3’) and

H10BE-31 (5’ AATTCTTAATGGTGATGGTGATGGTGGTGATGGTGGTGCTGC

3’) respectively, of 42 bases each in the presence of annealing buffer (10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl2, 1 mM Dithiothreitol) in a 100 µl reaction volume. The oligonucleotides were designed to create BssHII compatible 4-base overhangs at 5’ end of sense strand (bold and underlined) and EcoRI compatible

4-base overhangs at the 3’ end (bold and underlined) in the duplex synthesized after annealing. The annealed duplex was ligated to BssHII - EcoRI digested backbone of pVCDc101 vector to obtain recombinants named as pVCLDc102. The

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recombinants were characterized by sequencing of plasmid DNA with ori2 and L7 primers. To incorporate plasmid vector pVCLDc102 into λZap DNA, 10 µg purified DNA of pVCLDc102 was digested with 20 U of FseI and 50 U of EcoRI restriction

enzymes in a reaction volume of 300 µl with 3 hrs incubation at 37oC to obtain two fragments. 10 µg genomic DNA of λZapDamDc101 was similarly digested with FseI - EcoRI restriction enzyme to obtain two large fragments of nearly same size and a small fragment of inserted phagemid. The digested DNA samples were extracted with phenol-chloroform followed by ethanol precipitation. The precipitated DNA was resuspended and subjected to 1.2 % low melting agarose gel electrophoresis to excise desired bands of 2575 bp fragment from pVCLDc102 plasmid as insert and two fragments of almost same size 19.7 Kb and 18.7 Kb from λZapDamDc101 phage DNA as vector. Excised DNA were extracted using the QIAquick gel extraction kit followed by ligation of 15-fold excess of insert over the vector as described earlier. Further cloning steps of packaging of the ligation mix in vitro, plating on lawn cells to obtain plaques and phage

production has been described previously in section 3.2.2.6. The phages were

also characterized for plasmid rescue as described in section 3.2.2.5 and then one phage clone was grown for further work. 3.2.2.7.2 Construction of low copy number vector pVLLDc103 and its corresponding lambda phage vector λZapDamDc103 To convert the high copy number vector to low copy number, 10 µg

purified DNA of low copy number pVLSDH1013-SacII vector was digested with

SacII - ScaI restriction enzymes followed by dephosphorylation. The 3625 bp fragment containing ColE1 origin of replication, rop gene and laci repressor from

pVLSDH1013-SacII was ligated to similarly digested 1883 bp fragment from pVCLDc102 encoding Strep tag - gpD - His10 tag under the control of lac promoter

operator. The recombinant obtained was named as pVLLDc103 and was characterized by sequencing of plasmid with T7Tn primer.

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To insert into lambda display vector, 10 µg purified DNA of pVLLDc103 was digested with 20 U of FseI and 50 U of EcoRI restriction enzymes as described above in section 4.2.2.7.1 followed by ligation of 4818 bp DNA fragment to similarly digested two arms of λZapDamDc101 phage vector. The recombinant

phage vector was named as λZapDamDc103. . The phages were characterized for

plasmid rescue as described in section 3.2.2.5 and then one phage clone was grown for further work. 3.2.2.7.3 Construction of intermediate vector pVLTLDc103 and corresponding lambda phage vector λZapDamDc104

To insert the T7 promoter sequence upstream of the lac promoter operator, 2 µg each of complementary oligonucleotide pair T7lac-51 (5’ CTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCATGCA

TAGCT 3’) and T7lac-31 (5’ ATGCATGGAATTGTTATCCGCTCACAATTCCCCTATAGTGAGTCGTATTAG

AGCT 3’) containing sequence of T7 promoter followed by lac operator was

annealed as described above in section 3.2.2.7.1 to obtain a duplex bearing 4-base

SacI compatible overhang at 5’ and 3’ ends (shown in bold and underlined).

Purified DNA of pVLLDc103 vector was linearised by SacI restriction enzyme digestion and ligated to the annealed duplex to obtain recombinant vector named

as pVLTLDc103 vector which was characterized by sequencing of the plasmid

DNA with laci31 (5’ GATATAGGCGCCAGCAACCGCACCT 3’) primer that anneals 100 bp upstream of the laci repressor gene.

T7-lac promoter based low copy intermediate vector pVLTLDc103 was inserted into λZapDamDc101 phage vector as FseI - EcoRI insert as described above to get the recombinant phage vector named as λZapDamDc104. 3.2.2.7.4 Construction of intermediate vector pVTLDc105 and its corresponding lambda phage vector λZapDamDc106

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DNA fragment containing the 1.8 Kb stuffer sequence was amplified from

pVCEPI23961 DNA as template using 5’ primer DC105-51 (5’ CAGCCAGCGGCTAGCGGCAGCGGCCAAGGGGGCCAATGACGCCGATAA

AGGCCCCACGTACAACAAG 3’) and 3’ primer DC105-31 (5’ GGATCCAGCCTGAGGCGCCGCCACCGGCCCCACCGGCCGATCCTTAAAC

TCCCGACGACGTCGGTGCAGGA 3’). The primers were designed to

incorporate two non-compatible SfiI sites (GGCCN1N2N3N4N5CCGG) having

different recognition sequences at the 5’ end {SfiI(A)} and 3’ end {SfiI(B)} (bold

and underlined) of the amplified product. The 5’ and 3’ primers also inserted a

NheI site upstream of SfiI(A) and a Bsu36I site downstream of SfiI(B) restriction site respectively. PCR was carried out using Expand HF enzyme and involved initial heating at 95oC for 4 min followed by 25 cycles of denaturation at 95oC for

30 sec, annealing at 55oC for 30 sec and polymerization at 72oC for 90 sec with 2 sec extension in each cycle and final polymerization at 72oC for 4 min. The amplified product was purified using QIAquick PCR purification kit, digested

with NheI - Bsu36I restriction enzymes and ligated to similarly digested and

dephosphorylated vector backbone of pVLTLDc103. The recombinants were

screened by colony PCR using 5’ primer M13R and 3’ primer T7Tn followed by

sequencing of the plasmid DNA with M13R primer and Mtb81-57 internal primer within the stuffer. The recombinants were named as pVLTLDc105. 10 µg of pVLTLDc105 vector was digested with XbaI - EcoRI restriction

enzymes in 300 µl reaction with incubation at 37oC for 3 hrs to obtain two fragments. Similarly, 10 µg of genomic DNA of λZapDamDc104 phage was also digested with XbaI - EcoRI. The restriction enzyme digested DNA were extracted with phenol - chloroform and precipitated using ethanol. The DNA were subjected on 1.2 % agarose gel electrophoresis on low melting agar as described above to excise desired fragment encoding the strep tag, full length gpD, stuffer sequence flanked by SfiI(A) & Sfi(B) sites followed by deca-histidine tag from pVLTLDc105 vector and isolate two arms of almost same size from λZapDamDc104. DNA were extracted using QIAquick gel extraction kit and

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ligated as described earlier to construct a new phage display vector λZapDamDc106. The recombinant plaques were screened by plaque PCR using

5’ primer M13R and 3’ primer T7Tn and sequenced as described above. Small and large-scale phage lysate were produced from each construct

following the lysis protocol as described above in section 3.2.2.5. The large-scale lyaste was concentrated using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane (Amicon, Millipore, Chelmsford, MA) according to manufacturer’s protocol. The concentrated phage particles were finally purified on Sepharose CL-6B (GE-Amersham Health Sciences, Uppsala, Sweden) packed in disposable 5 ml polypropylene column (Pierce Biotechnology, Rockford, USA, Cat no. 29924). Before loading of phage, the column was washed with 2 CV of autoclaved ddH2O followed by column equilibration with 2 CV T50M10 buffer (50 mM Tris supplemented with 20 mM MgCl2). Following column equilibration, 1.5 ml phage supernatant was loaded and flow-through was discarded. Column was washed with 0.5 ml T50M10 buffer. Finally phages were eluted from the column using 1.5 ml T50M10 buffer. The column was regenerated with 4 CV T50M10 buffer. The eluted phages were titrated in TG1 cells using plaque forming unit assay (pfu) and stored as aliquots at - 20oC. 3.2.2.8 Characterization of λZapDamDc102/103/104/106 phage display vectors by western blot analysis For western blot analysis, 2 x 109 column purified phage particles of

different constructs λZapDamDc102/103/104/106 displaying the S-D-H fusion were prepared in 1 x Laemmli buffer (Laemmli et al., 1970) under reducing conditions and loaded on four different 0.1 % SDS - 15 % PAG. Following electrophoresis, the proteins from each gel were transferred overnight at 60 mA (Towbin et al., 1979) on 4 x 0.2 micron immobilon PVDF membranes (Cat No. ISEQ00010, Millipore, MA, USA). PVDF membranes, after electro-transfer, were blocked with 2 % SMPBST for 1 hr followed by three washes with PBST. The three membranes were incubated for 1 hr with different purified primary

130

monoclonal antibodies namely, anti-D (against gpD head protein) at 1 µg/ml, anti-His (against poly-histidine tag) at 100 ng/ml and anti-V (against gpV tail

protein) at 3 µg/ml concentration prepared in 1 % SMPBST. The membranes were washed thrice with PBST and then probed with 5000-fold dilution of HRP-conjugated goat anti-mouse IgG (H+L) (diluted in 1 % SMPBST) following 1 hr

incubation. The fourth membrane was first incubated for 30 min incubation with biotin blocking buffer (IBA, Germany) followed by incubation with 1:4000 dilution of HRP conjugated streptactin. All the incubations were carried out at room temperature (25 - 27oC). Finally, the membranes were washed thrice with PBST followed by three washes with PBS. The binding of HRP conjugated secondary antibodies and streptactin was revealed by incubating the membrane

in 1 mg/ml solution of DAB (3,3'-diaminobenzidine; Cat. No. D-5837, SIGMA,

St. Louis, MO) containing 1 µl/ml of 30 % H2O2 and 0.03 % NiCl2 in PBS. 3.2.2.9 Construction of Lambda phages displaying Ag85A (Rv3804c), Ag85B (Rv1886c) and MPT51 (Rv3803c) mycobacterial proteins

DNA encoding native mycobacterial proteins, Ag85A (Rv3804c), Ag85B

(Rv1886c) and MPT51 (Rv3803c) were amplified from purified DNA of

pVLExp85A4235, pVLExp85B4235 and pVLExpMPT514235 as template containing the respective genes using gene specific primers carrying additional sequence to introduce two non-compatible SfiI sites (A) & (B) at either ends (Table IV.1) to obtain amplified genes flanked by SfiI sites. Three individual PCR were set up in 200 µl reaction volume each containing 20 ng of respective template

DNA, 100 pmoles of specific 5’ and 3’ primers and 4 U of Expand HF enzyme.

The amplification steps involved initial heating at 95oC for 3 min followed by 25

cycles of denaturation at 95oC for 30 sec, annealing at 55oC for 30 sec and polymerization at 72oC for 70 sec with 2 sec extension in each cycle. The final

polymerization was carried out at 72oC for 3 min. PCR products were purified using QIAquick PCR purification kit, digested with 50 U of SfiI restriction enzyme in three separate reactions, purified on low melting agarose gel and

TABLE III.1 Sequences of primers used for the cloning of mycobacterial genes in λZapDamDc106 phage vector. Primers used for the amplification of mycobacterial genes (A) Ag85A (B) Ag85B and (C) MPT51 to clone into λZapDmaDc106 phage display vector. Underlined sequences represents the restriction site SfiI contained within the oligonucleotides. ‘+’ plus sign followed by a number shows the residue position of protein.

PRIMERS USED FOR CLONING OF MYCOBACTERIAL GENES IN λZapDamDc106

S.No. Plasmid (template)

Cloning

site

Primers used for cloning (Sequence between first and the last codon)

A

pVLE

xp85

A42

35

SfiI

DcAg85A-51 - 5' GGAGGTTCAGGCCAAGGGGGCCAGGGTTTTTCCCGGCCGGGCTTGCCGGTG 3' G Q G G Q G F S R P G L P V +1 of Ag85A DcAg85A-31 - 5' GCCACTAGTGGCCCCACCGGCCGATCCGGCGCCCTGGGGCGCGGGCCCGGT 3' A G G A S G A G Q P A P G

B

pVLE

xp85

B423

5

SfiI

DcAg85B-51 - 5' GGAGGTTCAGGCCAAGGGGGCCAGGGTTTCTCCCGGCCGGGGCTGCCGGTC 3' G Q G G Q G F S R P G L P V +1 of Ag85B DcAg85B-31 - 5' GCCACTAGTGGCCCCACCGGCCGATCCACCGGCGCCTAACGAACTCTGCAG 3' A G G A S G A G L S S Q L D

C

pVLE

xpM

PT51

4235

SfiI

DcMPT51-51 - 5' GGAGGTTCAGGCCAAGGGGGCCAGGGTGCCCCATACGAGACCCTGATGGTG 3' G Q G G Q G A P Y E N L M V +1 of MPT51 DcMPT51-31 - 5' GCCACTAGTGGCCCCACCGGCCGATCCGCGGATCGCACCGACGATATCGCC 3' A G G A S G R I A G V I D G

131

132

finally ligated in 5-fold molar excess with left and right arms of SfiI digested λZapDamDc106 phage vector. In vitro packaging, plating, screening of recombinant plaques and sequencing was carried out as described above. The recombinants obtained were named as ZapDamDc106-85A, ZapDamDc106-85B and ZapDamDc106-MPT51. Large-scale lysate produced from each construct was concentrated and then purified on Sepharose 6B-CL column as described in

section 3.2.2.7.1. 3.2.2.10 Characterization of λZapDamDc106Ag85A / Ag85B / MPT51 phage display vectors by western blot analysis For western blot analysis, 1 x 1010 column purified phage particles of three phage constructs displaying mycobacterial proteins as gpD fusion protein and λZapDamDc104 phages were made in 1 x Laemmli buffer under reducing conditions and loaded on four different 0.1 % SDS - 15.0 % PAG. Two

concentrations, 34 ng and 17 ng of purified SDH6 protein were also electrophoresed to serve as control. Following electrophoresis, the proteins were transferred onto 0.2 micron immobilon PVDF membrane, blocked and then incubated with three different primary monoclonal antibodies namely, anti-D, anti-His and anti-V and processed for visualization of bands as described in

section 3.2.2.8.

The other three membranes after blocking were incubated for 1 hr with purified rabbit polyclonal antibody against whole lambda particles (anti-λ), recombinant histidine tagged Antigen 85B (anti-Ag85B) and recombinant histidine tagged MPT51 (anti-MPT51). After washing with PBST, the membranes were incubated with HRP- conjugated goat anti-rabbit IgG followed by visualization of bands using DAB. 3.2.2.11 Affinity selection of the lambda phages displaying Ag85B with monoclonal antibody (mAb) recognizing the specific displayed protein

133

For carrying out affinity selection, the purified phages of λZapDamDc106 displaying Ag85B as D fusion with N-terminus Strep tag and C-terminus His tag were spiked in two different ratios of 1 in 105 and 1 in 106 into λZapDamDc104 phages that do not display any protein. For panning, eight wells of maxisorp microplate strips (Nunc. Inc) were coated with 2 µg of specific MAb Ag85AB04 and incubated overnight at 4oC. The wells were blocked with 2 % BSA in PBST

(PBS containing 0.1 % Tween 20) for 1 hr at 37oC. The blocked wells were washed three times with PBST and a total of 1 x 109 spiked phages in 100 µl PBSTB (PBST containing 1 % BSA) were added to each well followed by

incubation at 37oC for 1 hr. The unbound phages were removed by ten washes with PBST followed by three washes with PBS. All the washings were carried out in a microplate washer (Model Columbus, TECAN Austria GmbH, Grodig, Austria). The bound phages were eluted by adding one unit of collagenase in 100 µl of phosphate buffer (20mM, pH 7.4) to each well and incubating for 10 min at RT (25-27oC). A small fraction of the eluate was titrated on TG1 cells with appropriate phage dilutions. After titration, the eluted phages were mixed with 10 ml of TG1 cells (OD600nm= 0.5) to obtain MOI of 0.001 and incubated for 20 min

at 37oC without shaking. Following this, the infected cells were diluted with LB

medium supplemented with 10 mM MgCl2 to 50 ml and grown at 37oC with shaking at 220 rpm for 5 - 6 hrs till complete lysis. The phages were obtained by centrifugation to remove the cell debris and subjected to the second round of affinity selection on the same immobilized monoclonal antibody. Bound phages were eluted and titrated as described above. Phage plaque plates containing

approximately 200 – 300 well-isolated plaques were made from the initial phage mix used for Pan I and also from eluates of Pan I and Pan II for both the conditions i.e., 1 Ag85B phage per 105 or 106 non specific phages. The plaques were transferred onto a dry nitrocellulose filter (Schleicher & Schuell, Keene, NH) for 4 hrs at room temperature. The filters were blocked for 2 hrs at room temperature in 2 % skimmed milk (SM) prepared in 1 x PBST followed by washing with PBST. Immunoscreening was performed by incubating the washed filters with 5 µg/ml of mAb Ag85AB04 (prepared in SMPBST) for 2 hrs at room temperature. The filters were washed thrice with PBST and then probed with

134

5000-fold dilution of HRP-conjugated goat anti-mouse IgG (H+L) (diluted in 1 % SMPBST) following 1 hr incubation. Finally, the filters were washed three times with PBST followed by three washes with PBS. The spots were revealed by using

in 1 mg/ml solution of DAB containing 1 µl/ml of 30 % H2O2 and 0.03 % NiCl2 in PBS. 3.3 RESULTS

The main purpose of this work was to develop a phage lambda based

vector for the display of proteins at high density and after selection of desired clone from a library, there could be an easy process of expressing the proteins from the selected clone. For this purpose, a well-established lambda cloning vector, lambda Zap was employed. This vector carried after J gene a insertion of a plasmid pBluescript SK(-) comprising gene encoding β-lactamase, high copy ColE1 origin of replication and lacPO driving lacZα fragment required for alpha-complementation with multiple cloning sites. This plasmid sequence was flanked by terminator sequence (T) and initiator (I) sequence of the f-ori. Using these sequences the inserted plasmid can be excised and recircularised to obtain phagemid containing the cloned insert. In this section, effort has been made to replace this inserted plasmid with a plasmid to code for D protein of phage lambda under the control of lacPO with N-terminal and C-terminal tags and having cloning sites to insert foreign DNA and libraries to code for D-fusion proteins. Efforts have also been made to modify the tags, copy number and even to insert T7 promoter to drive high level of expression. Therefore, various lambda vectors have been constructed in two steps involving first the construction of plasmid and then its insertion into the lambda vector. The first vector has been constructed involving several steps. This vector allows cloning of a gene and its display as fusion with D protein carrying N-terminal Strep tag and a hexa-histidine tag at the C-terminus of the fusion protein. The lambda vector was modified to carry native D gene with amber mutation so that depending upon the host there would be native D and D fusion protein in the cytosol to compete for incorporation into the lambda head.

135

3.3.1. Construction of lambda phage display vector, λZapDamDc101

For the construction of λZapDamDc101, first a plasmid pVCDc101-Zap1 was assembled in several steps and then it was inserted in place of plasmid carried in λZap.

First, an intermediate plasmid pVCDc01 was constructed by taking HindIII-EcoRI segment from pVCDcDL1 and inserting into the high copy number

backbone derived from pVCEPI13960 as depicted in Fig.III.1a. The pVCDc01 was further modified by site-directed mutagenesis to create some desired restriction sites such as SacI just upstream of the lacPO, AvrII after the T7 terminator

sequence and FseI upstream of β-lactamase gene, and to delete Bsu361 upstream of lacPO and a BsaI site within the β-lactamase gene. The plasmid with these mutations was named pVCDc05 (Fig.III.1b). In the next step, SacI-EcoRI backbone of pVCDc05 was used and the remaining segment was replaced by

another gene segment that was obtained from pVCSDH1033 by PCR to create

SacI site upstream of lacPO and EcoRI at the 3’ end as shown in the Fig.III.2. The resultant plasmid, pVCDc101 carries under lacPO, a gene segment coding for D-protein of phage lambda with N-terminal octa-peptide strep tag (WSHPQFEK), and at its C-terminus a collagenase cleavage recognition site (GPVG), NheI and

Bsu36I unique restriction sites for cloning followed by hexa-histidine tag. In between each of these functional tag / site there are spacer sequences of glycine and serine residues. The backbone further comprises of tHP transcription terminator upstream of lacPO with SacII and SacI unique sites; high copy number ColE1 origin of replication, β-lactamase gene without internal BsaI site, a unique FseI site just upstream of β-lactamase, f-ori, a unique AvrII site and T7 terminator (T7Tn).

To insert into λZap, pVCDc101 was further modified to include few restriction sites and terminator sequence (T) of f-ori in several steps. As depicted

in Fig.III.3, a segment of λZap extending from BsaI site within H gene to the terminator sequence (T) was amplified using 5’ primer Bsadel1-T4 that anneals

Fig.III.1. Schematic representation of construction of vectors pVCDc01 and pVCDc05. Only relevant genes and restriction sites are shown. The maps are not to scale. lacP/O, lac promoter-operator; D, segment encoding amino acid residues 1–109 of gpD of lambda; Stuffer, a 30 nucleotide long sequence; c-myc, decapeptide; F+ origin of replication of filamentous phage ; Ampr, β-lactamase gene; Ori, ColE1 origin of replication; lox Pwt, wild-type lox site; lox P511, lox site with mutation 511; pelB, pectate lyase signal sequence; gene III, gIII protein of filamentous phage and tHP, transcriptional terminator. a. Plasmid pVCDcDL1 was digested with HindIII and EcoRI restriction enzymes to obtain a fragment encoding gpD, stuffer and c-myc tag followed by its ligation to similarly digested vector backbone of pVCEPI13960 phagemid vector to yield recombinants named as pVCDc01. Dashed arrows mark the location of various sequencing primers; 1, M13R; 2, L7; 3, T7 terminator; 4, L6; 5, ori2. b. pVCDc01 was then subjected to site directed mutagenesis to bring about various modifications like deletion of BsaI present within β-lactamase gene, to insert FseI site upstream of β-lactamase gene, to delete Bsu36I between tHP and lacP/O, to introduce SacI just downstream of lacP/O and to insert AvrII restriction site just below the T7 terminator sequence (modifications are depicted in bold).

CONSTRUCTION OF pVCDc01 AND pVCDc05 INTERMEDIATE VECTORS FOR INSERTION INTO LAMBDA ZAP

HindIII, EcoRI

Digested Insert (475 bp)

EcoRIHindIII NheIXbaI BglIIMluI

D Stuffer cmycpVCDcDL1Ori

SacI

HindIII NheIXbaI

lacP/O

BglIIEcoRI

MluI

D Stuffer cmyc

loxP511

1

loxPwtF+Ampr

T7Tn

Site Directed Mutagenesis using I. pVLBsadel II. FseI-Amp III. SacI-cap IV. AvrII-ApaI oligonucleotides aaa

b.

FseI

F+tHP

EcoRI

Ampr

HindIII NheIXbaI BglIIMluI

D Stuffer cmyclacP/OpVCDc05

SacI

Ori

SacII AvrIIT7Tn

a. SacII

BsaI

F+OritHP

EcoRI

Bsu36IHindIII NheIXbaI BglIIMluI

D Stuffer cmyc T7TnlacP/OpVCDc01

1 2 3

4 5

Ampr

HindIII, EcoRI

HindIIIXbaI

F+ pVCEPI13960

OritHP

BsaI

EcoRI

lacP/O gene IIIpelB

Ampr

Bsu36ISacII

T7Tn

Digested Vector BsaI

F+OritHP

HindIII EcoRI

Ampr

Bsu36ISacII

T7Tn

136

Fig.III.2. Schematic representation of construction of plasmid pVCDc101. Only relevant genes and restriction sites are shown. The maps are not to scale. lacP/O, lac promoter-operator; D, segment encoding amino acid residues 1–109 of gpD of lambda; Stuffer, a 30 nucleotide long sequence; c-myc, decapeptide; F+ origin of replication of filamentous phage ; Ampr, β-lactamase gene; Ori, ColE1 origin of replication; tHP, transcriptional terminator; ST, strep-tag; MCS, multiple cloning sites; H6, sequence encoding for hexa-histidine tag; T7tn, T7 terminator. Fragment encoding ST, D, stuffer containing MCS and hexa-histidine tag was amplified from pVCSDH1033 plasmid using 5’ SacI-cap-1033 and 3’ Dc101-EcoRI primers to obtain an amplified product of 702 bp which was further subjected to digestion with SacI - EcoRI to obtain a 680 bp digested product and ligated into SacI and EcoRI digested vector backbone of pVCDc05 to yield recombinants named as pVCDc101.

CONSTRUCTION OF pVCDc101 PLASMID TO CLONE INTO LAMBDA ZAP

SacI, EcoRI

Digested Vector FseI

F+OritHP

SacI EcoRI

AmprF+tHP pVCDc05

FseI

EcoRI

Ampr

HindIII NheIXbaI MluI

D Stuffer cmyclacP/O

SacI

Ori

SacII

PCR with 5ʼ SacI-cap-1033 3ʼ Dc101-EcoRI

Sca I

AmprOri

ST D H6 MCS

XbaI EcoRI RI

pVCSDH1033

5ʼ Sac-CAP-1033

3ʼ Dc101-EcoRI lacP/O

Bsu36INheI

SacI, EcoRI

Bsu36INheI

lacP/O ST D H6 MCS

XbaI EcoRI RI

SacI

702 bp

Digested Insert (680 bp)

lacP/O ST D H6 MCS

XbaI EcoRI RI

SacI Bsu36INheI

CATATGGCTAGTTGGAGCCACCCGCAGTTTGAAAAAGGCGGATCCGGTGGCACTTCGAAA--ATTGTTGGCGGATCCGGACCGGTAGGGCCCGGTGGCAGCGGAGCTAGCGGC--MCS--GGCGCCTCAGGCACTAGTGGCGCGCAGCACCACCATCACCACCATTAAGAATTC M A S W S H P Q F E K G G S G G T S K I V G G S G P V G P G G S G A S G G A S G T S G A Q H H H H H H *

NheI His(6) tag Spacer Collagenase site Bsu36I

gpD

EcoRI NdeI Strep tag Spacer

gpD

SacII

pVCDc101F+

AmprOri

lacP/O T7tnST D H6 MCS

XbaI EcoRI RI

Fse I

SacI

tHP

Bsu36INheIAvrII

T7tnAvrII AvrII

T7tn

137

Fig.III.3. Schematic representation of incorporation of pVCDc101 plasmid DNA into Lambda Zap vector. Only relevant genes and restriction sites are shown. The map is not to scale. Ampr, β-lactamase gene; Ori, ColE1 origin of replication; F+, phage M13 origin of replication; H6, sequence encoding for hexa-histidine tag; lacP/O, lac promoter and lac operator; T7tn, T7 terminator; ST, strep-tag; D, recombinant lamda head protein; MCS, multiple cloning sites; tHP, transcriptional terminator. Only some of the lambda genes are shown. Only the important unique restriction sites in the lambda genome have been shown. T, terminator domain of the phage origin of replication; I, initiator domain of the phage origin of replication derived from Lambda genome; lacZ, lacZ-alpha fragment for alpha complementation with MCS containing several unique restriction sites. (a) A 8.3 Kb segment of Lambda Zap genome was amplified with 5’ primer Bsadel1-T4 and 3’ primer ZapFse to incorporate FseI site. (b) pVCDc101 plasmid vector was amplified with 5’ primer FseDc101 and 3’ primer PspT4Dc101 to obtain a band of ~ 2.7 Kb. Both the PCR products were purified, digested with FseI and then ligated to obtain a product of 11 Kb.

CLONING OF pVCDC101 INTO λZAP PHAGE VECTOR

Ligate

BsiWI Ligated product of 11 Kb

PspOMI

T7tnST D H6MCSlacP/OAmpr OriTJ H

FseI SacI SacII

Digest with FseI

PCR with 5ʼ-Bsadel1-T4 3ʼ-ZapFse primers

BsiWI

TJ H

FseI

BsiWI

TJ H

FseI

8.3 Kb

λ ZAP

SacI

XhoI

Bam

HI

EcoR

I

Hind

III

XbaI

ApaI

KpnI

MCSlacP/OAmprT Ori I lacZ

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro Bsadel1-T4

ZapFse

BsaI

BsiWI

T I

PCR with 5ʼ-FseDc101 3ʼ- PspT4Dc101 primers

Digest with FseI

XbaI

T7tnST D H6MCSlacP/OOriFseI

Ampr

PspOMI

2.7 Kb

SacI

SacI

pVCDc101F+

AmprOri

lacP/O T7tnST D H6MCS

XbaI EcoRI RI

Fse IFseDc101

PspT4Dc101

SacII

tHP

XbaI PspOMI SacI

T7tnST D H6MCSlacP/OOriFseI

Ampr

a. b.

138

139

around position 11418 bp (numbering as in wild type) and 3’ primer, Zap-Fse that anneals just after the terminator sequence (T) and creates a unique FseI site to

obtain an amplified product of 8.3 Kb (Fig.III.3a). In another PCR, complete plasmid pVCDc101 excluding the f-ori was amplified using 5’ primer, FseDc101

that annealed encompassing FseI site, and the 3’ primer PspT4Dc101 that anneals downstream of T7Tn but upstream of f-ori, to get an amplified product of 2.7 Kb

(Fig.III.3b). After digestion with FseI, the two fragments were mixed in an equimolar ratio and ligated. Attempts were made to amplify 11 Kb resultant

product from the ligation mixture using a 5’ primer, Bsadel1-T4 and 3’ primer PspT4Dc101 but PCR did not work. Hence, another strategy was followed. The ligated product was used as a template in two separate PCR (Fig.III.4). One with

5’ primer, λ19223F (Primer 3 in Fig.III.4) that anneals within the lom gene and 3’ primer, L6-1 (Primer 4 in Fig.III.4) which anneals in the β-lactamase gene to give a

product of 635 bp that contains terminator sequence of f-ori flanked by BsiWI and FseI sites. In another PCR, the same template was used with 5’ primer Fterseq

(Primer 1 in Fig.III.4) that anneals within the terminator domain and 3’ primer PspT4Dc101 (Primer 2 in Fig.III.4) that anneals within the T7Tn. This PCR

produced a 2.8 Kb fragment. The 3’ end of 635 bp product and the 5’ end of 2.8 Kb product have overlap of 47 bases. The two fragments were spliced in another

PCR using 5’ primer, 19223F (Primer 3) and a new 3’ primer, DC101-Xho (Primer

5 in Fig.III.4) that annealed in T7 terminator region and created SalI site. The 3.4 Kb amplified product was end-repaired, kinased and recircularised by ligase to produce plasmid Dc101-Zap. However, sequencing of candidate recombinants showed that there were several mutations in the segment encoding D, which probably took place due to multiple rounds of PCR. To fix this problem, a segment between BspLU11I and EcoRI of pVCDc101 was ligated into the backbone of pVCDc101-Zap, to create the final intermediate vector, pVCDc101-Zap1 (Fig.III.4). This vector carries under the lacPO a DNA segment encoding D protein with strep tag at N-terminus and collagenase cleavage site followed by hexa-histidine tag at the C-terminus. The backbone contains a unique SacI and BspLU11I sites upstream of lacPO, ColE1 origin of replication, β-lactamase gene

Fig.III.4. Schematic representation of the construction of intermediate vector pVCDc101-Zap1. Only relevant genes and restriction sites are shown. The map is not to scale. Ampr, β-lactamase gene; Ori, ColE1 origin of replication; F+, phage M13 origin of replication; H6, sequence encoding for hexa-histidine tag; lacP/O, lac promoter and lac operator; T7tn, T7 terminator; ST, strep-tag; D, recombinant lambda head protein; D*, recombinant lambda head protein with mutations generated during PCR; MCS, multiple cloning sites; T, terminator domain of the phage origin of replication. Only some of the lambda genes are shown. 11 Kb ligated product obtained after incorporation of pVCDc101 into Lambda Zap genome was used as template for PCR. A 635 bp fragment was amplified with 5’ - λ19223F primer and 3’- L6-1 primer. Same template was also used for amplification with 5’ - Fterseq and 3’ - PspT4Dc101 to obtain a band of ~ 2.7 Kb. The two PCR products were mixed in appropriate ratio and then amplified with 5’- λ19223F primer and 3’- Dc101-Xho primer carrying XhoI site to give an amplified product of 3.4 Kb. Linear product of 3.4 Kb was blunt ended, kinased and then recircularized to obtain Dc101-Zap intermediate vector. The mutated D* of Dc101-Zap was replaced by obtaining the S-D-H DNA cassette from pVCDc101 vector as BspLU1I-EcoRI insert and ligated into the backbone of Dc101-Zap to obtain pVCDc101-Zap1.

CLONING OF pVCDC101-ZAP1 INTERMEDIATE VECTOR

PCR with primer 3 & 5 3. λ19223F 5. Dc101-Xho

Blunt Ended, Kinased & Recircularized

PCR with primer 3 & 4 3. λ19223F 4. L6-1

PCR with primer 1 & 2 1. Fterseq 2. PspT4Dc101

SacI BsiWI FseI 3

635 bp

T

PspOMI

T7tn ST D H6 MCS lacP/O Ori FseI

5 2.8 Kb

Ampr

1

T J H

FseI 3 BsiWI

T7tn ST D H6 MCS lacP/O Ori Ampr

PspOMI

2 4

SacI

BsiWI Ori Ampr T

FseI PspOMI SalI

EcoRI

T7tn ST D H6 MCS lacP/O 3.4 Kb

SacI

Digested Vector

BspLU1I, EcoRI

SalI BsiWI

Dc101-Zap Ampr Ori

lacP/O T7tn ST D* H6 MCS

XbaI EcoRI RI

FseI T

SacI BspLU11I

tHP

SalI BsiWI

Ampr Ori

T7tn

FseI T

EcoRI RI

BspLU11I

BspLU1I, EcoRI

ST D H6 MCS lacP/O

BspLU11I SacI EcoRI RI

Digested Insert

SacI

pVCDc101 F+

Ampr Ori

lacP/O T7tn ST D H6 MCS

XbaI EcoRI RI

Fse I

BspLU11I

tHP

SalI BsiWI

pVCDc101-Zap1 Ampr

Ori

lacP/O T7tn ST D H6 MCS

XbaI EcoRI RI

FseI T

SacI BspLU11I

tHP

tHP

140

141

and terminator sequence (T) of the f-ori from λZap flanked by FseI and BsiWI restriction sites, a unique SalI site and T7 terminator.

To insert pVCDc101-Zap1 into λZap, λZap DNA was digested with BsiWI

and XhoI to obtain 19.3 Kb left and 18.6 Kb right arm and these two arms were

ligated to 3.1 Kb fragment of BsiWI and SalI digested pVCDc101-Zap1 (Fig.III.5). Ligation of BsiWI end restored the lambda sequence and inserts segment from pVCDc101-Zap1 comprising of terminator sequence of f-ori (T), unique FseI site, high copy ColE1 origin of replication, lacPO, unique XbaI site, gene segment encoding D protein of phage lambda with N-terminal strep tag, C-terminal hexa-histidine tag with cloning sites in between a translational stop and T7Tn. The ligation of SalI and XhoI ends destroyed either site and the sequence is followed by initiator sequence of f-ori (I). Thus, the recombinant λZapDc101 is similar to λZap but contains a different plasmid in between terminator (T) and initiator (I) sequences of f-ori. The correct recombinants were identified by amplification of DNA from plaques followed by sequencing.

The λZapDc101 vector contains two copies of gpD or gpD like genes. One copy of gpD is encoded by the native gpD gene located within the left arm of lambda genome and the other as gpD fusion protein encoded by lacPO driven D fusion. During the assembly of phages both types of gpD can get incorporated into the lambda particles. In order to control the incorporation of native D so that high density display of D-fusion takes place, the left arm of λZapDc101 was changed with that of a lambda derivative λDam that carries an amber mutation in native D gene hence its expression can vary depending upon the suppressor strains of the E. coli host. This was achieved by digesting λZapDc101 and λDam genomic DNA with unique BsiWI and mixing the left and right arms (~20 Kb each) from both lambda DNA samples in a ligation reaction (Fig.III.6). Following packaging and plating there would be 4 different types of plaques produced.

Upon analysis of 40 plaques by PCR, 13 clones were found to contain pVCDc101

plasmid. These 13 clones were further screened by PCR using L8 and L10 primers to amplify the wild type gpD gene in the left arm and then sequenced

Fig.III.5. Schematic representation of the construction of λZapDc101 phage vector. Only relevant genes and restriction sites are shown. Only some of the lambda genes are shown. The map is not to scale. T, terminator domain of the phage origin of replication derived from Lambda genome; Ampr, β-lactamase gene; Ori, ColE1 origin of replication; lacP/O, lac promoter and operator; ST, strep-tag; D, recombinant lambda head protein; Dam, wild type D protein with amber mutation; MCS, multiple cloning sites; H6, sequence encoding for hexa-histidine tag; T7tn, T7 terminator; I, initiator domain of the phage origin of replication derived from Lambda genome. 40.5 Kb Lambda Zap genome was digested with BsiWI and XhoI restriction enzymes to obtain three fragments of 19.3 Kb, 18.6 Kb and 2.6 Kb. The two bands of 19.3 Kb left arm and 18.6 Kb right arm were extracted on prep gel and then ligated with 3.1 Kb fragment of pVCDc101-Zap1 intermediate vector to obtain final recombinant λZapDc101. Dashed arrows represent the location of various primers used for sequencing; 1, Fterseq; 2, ori2; 3, M13R; 4, L7; 5, T7 terminator; 6, Fint.

INSERTION OF pVCDC101-ZAP1 PLASMID INTO LAMBDA ZAP

3.1 Kb

Digest with BsiWI and SalI

Gel Extraction

FseIBsiWI EcoRI

T7tnST D H6MCSlacP/OOriAmprT

SalI

3.1 Kb

SalI BsiWI

pVCDc101-Zap1AmprOri

lacP/O T7tnST D H6MCS

XbaI EcoRI RI

FseI T

SacI BspLU1I

tHP

Gel Extraction

Digest with BsiWI and XhoI

19.3 Kb + 18.6 Kb

SacI

XhoI

Bam

HI

EcoR

I

Hind

III

XbaI

ApaI

KpnI

MCSlacP/OAmprT Ori I lacZ

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro

BsiWI

T I

A B K L H Z E C I J lom G BsiWI

int ex P O cII rex ral bet Q S Rz cro XhoI

18.6 Kb

I

19.3 Kb 2.6 Kb lacP/OAmprT

XhoI BsiWI

lacZOri

λZap

Ligate

λZapDc101

6 5 1 2 3 4 FseIlacP/OAmpr Ori T

EcoRI XbaI

T7tnST H6DMCS I

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom

G int exo

P O cII rexB

ral bet Q S Rz cro

BsiWI

I I

142

CONSTRUCTION OF PHAGE DISPLAY VECTOR λZapDamDc101

Fig.III.6. Schematic representation of the construction of phage display vector λZapDamDc101. Only relevant genes and restriction sites are shown. Only some of the lambda genes are shown. The map is not to scale. T, terminator domain of the phage origin of replication derived from Lambda genome; Ampr, β-lactamase gene; Ori, ColE1 origin of replication; lacP/O, lac promoter and operator; ST, strep-tag; D, recombinant lambda head protein; Dam, wild type D protein with amber mutation; MCS, multiple cloning sites; H6, sequence encoding for hexa-histidine tag; T7tn, T7 terminator; I, initiator domain of the phage origin of replication derived from Lambda genome. λ Dam DNA was digested with BsiWI restriction enzyme to obtain the left arm of 19.3 Kb (shown in bold) carrying the D protein with amber mutation (Dam). This was ligated to BsiWI digested right arm of 21.7 Kb (shown in bold) of λZapDc101 phage vector containing the recombinant lambda head D protein (D) to obtain a final recombinant phage display vector λZapDamDc101. Dashed arrows L8 and L10 are the oligonucleotide primers used for the PCR based analysis of the recombinants and L9 is sequencing primer used to sequence the D with amber mutation (Dam) in the left arm of λZapDamDc101.

Ligate

λ ZapDamDc101

5K 10K 40K 35K 30K 25K 20K 15K BsiWI

BsaI A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro Dam

FseIlacP/OAmpr Ori T

L9 L8 L10

EcoRI XbaI

T7tnST H6DMCS I

Ori2

T7tn

I

λ Dam

5K 10K 40K 35K 30K 25K 20K 15K

int exo

P O cII rexB

ral bet Q S Rz cro A B K L H Z E C I J lom

G Dam

SacI

XhoI

SalI

SpeI

Ba

mH

I EcoR

I Hi

ndIII

XbaI

No

tI Ap

aI

KpnI

BsaI

BsiWI

λZapDc101

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom

G int exo

P O cII rexB

ral bet Q S Rz cro

BsiWI

FseIlacP/OAmpr Ori T

EcoRI XbaI

T7tnST H6DMCS I

I

Digest with BsiWI

19.3 Kb (Left Arm) + 21.7 Kb (Right Arm)

19.3 Kb (Left Arm) + 26.7 Kb (Right Arm)

Digest with BsiWI

143

144

with L9 primer to check for the presence of amber mutation in the gpD gene. Two clones were found to contain the Dam gene. This resulting lambda phage of ~ 41 Kb genome size was named as λZapDamDc101 (λ-101) consisting of a left arm from lambda Dam and right arm from lambda Zap that contained the essential genes responsible for the phage production and infectivity. In this

vector the initiator and terminator sequences were positioned on two ends of 3.1 Kb plasmid DNA that confers ampicillin resistance, had ColE1 origin of replication and fragment encoding Strep tag - D - small stuffer segment with multiple cloning sites - hexa-histidine tag (His6) under the control of lac promoter. Replacement of the stuffer segment with any gene of interest would allow its display as D fusion protein on the phage head. Thus, mosaic phage particles would be generated displaying both native and recombinant versions of gpD. A collagenase recognition sequence included between D protein and the stuffer sequence allowed the rescue of bound phages after affinity selection. The phagemid was excised and then characterized by digestion with NgoMIV and XbaI unique restriction enzymes. The presence of unique restriction sites in the plasmid allowed a convenient and restriction digestion based method to modify and derive a series of phage display vectors. To carry out modifications downstream of the lac promoter operator, unique XbaI and EcoRI restriction sites could be used whereas to change the promoter sequence and selection marker downstream of terminator (T) sequence, unique FseI and EcoRI restriction sites could be used. Small-scale phage lysate was produced from a well-isolated single plaque and then concentrated using the PEG - NaCl precipitation method

as described in the Appendix III.17.3. The lysate was used to extract the genomic DNA as described previously to carry out the cloning of series of lambda vectors.

3.3.2. Construction of other λZapDam vectors with modifications in the inserted phagemid

As described above the λZapDam series vectors were derived by the insertion of various phagemids with different features between the T and I sequences of f-ori. For this, first the plasmid to be inserted, pVCDc101 is

145

modified and then the segment carrying modification is inserted in the λZapDamDc101 using unique sites such as Fse-EcoRI or Xba-EcoRI.

First, the C-terminal tag of the D-fusion protein was changed to code for a

deca-histidine. For this, a synthetic oligonucleotide duplex carrying sequence for ten codons of histidine was cloned into BssHII-EcoRI digested pVCDc101 to obtain pVCDc102 (Fig.III.7A). Next, FseI-EcoRI fragment carrying the change in D-fusion was cloned into left and right arms of FseI-EcoRI digested λZapDamDc101 to yield λZapDamDc102 (λ-102) (Fig.III.7B). This vector displays D fusion proteins with C-terminal deca-histidine tag.

Since both λ-101 and λ-102 carry plasmid with backbone of high copy number plasmid without lac repressor, if the excised plasmids were to be employed for expression, there would be leaky expression. To address this, the D fusion was first cloned into a low copy vector backbone carrying laci repressor

gene. For this, the backbone was derived from pVLSDH1013SacII as SacII-ScaI fragment and ligated with D-fusion containing SacII-SacI fragment from

pVCDc102 to yield pVLLDc103. In this case also, FseI-EcoRI fragment containing almost entire plasmid was ligated with left and right arms of FseI-EcoRI digested

λ-101 to obtain λZapDamDc103 (λ-103) (Fig.III.8).

The plasmid pVLLDc103 was further modified by inserting a synthetic oligonucleotide duplex carrying sequence for T7 promoter-lac operator using the SacI site located upstream of lacPO. The SacI compatible ends of the duplex were designed in a manner such that SacI is recreated at the end distal to the lacPO and

the new plasmid was named pVLTLDc103 (Fig.III.9). To construct λZapDamDc104 (λ-104), Fse-EcoRI fragment carrying almost entire plasmid was ligated with left and right arms of FseI-EcoRI digested λ-101. In this display vector the rescued plasmid could be used to express D fusion protein in host

strains carrying T7 RNA polymerase gene such as BL21 (λDE3).

Fig.III.7A. Schematic representation of construction of plasmid vector pVCLDc102. Only relevant genes and restriction sites are shown. The map is not to scale. Ampr, β-lactamase gene; Ori, ColE1 origin of replication; F+, phage M13 origin of replication; H6, sequence encoding for hexa-histidine tag H10, sequence encoding for deca-histidine tag; lac P/O, lac promoter and lac operator; T7tn, T7 terminator; ST, strep-tag; D, recombinant lambda head protein; MCS, multiple cloning sites. pVCDc101 bearing the hexa-histidine tag (H6) was digested with BssHII and EcoRI restriction enzyme to obtain a fragment of 3224 bp vector backbone. Insert was prepared by annealing two complementary oligonucleotides H10BE-51 and H10BE-31 of 42 mer, encoding the deca-histidine tag with 4 – base overhang at 5’ and 3’ ends (shown in bold) compatible to BssHII and EcoRI digested ends respectively of the vector pVCDc101. Ligation of vector and insert produced recombinants named as pVCLDc102 that consisted of deca-hisitidine tag in place of hexa-histidine tag at the C-terminus of the fusion protein.

5’GCGCGCAGCACCACCATCACCACCATCACCATCACCATTAAG 3’

3’CGTCGTGGTGGTAGTGGTGGTAGTGGTAGTGGTAATTCTTAA 5’

His (10)

Annealed Oligonucleotide H10BE-51 (42 mer) H10BE-31 (42 mer)

Insert

BssHII, EcoRI

NheIXbaI BssHII

Sca I

pVCDc101F+

AmprOri

lacP/O T7tnST D H6MCS

EcoRI RI

SacIIBsu36I

FseI

tHP

Digested Vector

SacII

ScaI

F+AmprOri

lacP/O

BssHIIXbaI NheI

ST D MCS T7tn

EcoRI RI

Bsu36I

FseI

tHP

His (10)

GGCGGCGCCTCAGGCACTAGTGGCGCGCAGCACCACCATCACCACCATCACCATCACCATTAAGAATTC

Bsu36I BssHII EcoRI

BssHIIBsu36I

NheI

pVCLDc102

XbaI

Sca I

F+AmprOri

lacP/O

EcoRI RI

SacII

T7tnST D H10MCS

FseI

tHP

CONSTRUCTION OF PLASMID pVCLDc102 FOR THE LAMBDA VECTOR λZapDamDc102

146

CONSTRUCTION OF PHAGE DISPLAY VECTOR λZapDamDc102

Fig.III.7B. Schematic representation of the construction of phage display vector λZapDamDc102. Only relevant genes and restriction sites are shown. Only some of the lambda genes are shown. The map is not to scale. T, terminator domain of the phage origin of replication derived from Lambda genome; Ampr, β-lactamase gene; Ori, ColE1 origin of replication; lacP/O, lac promoter and operator; ST, strep-tag; D, recombinant lambda head protein; Dam, wild type D protein with amber mutation; MCS, multiple cloning sites; H10, sequence encoding for deca-histidine tag; T7tn, T7 terminator, I, initiator domain of the phage origin of replication derived from Lambda genome. λZapDamDc101 DNA was digested with FseI-EcoRI restriction enzyme to obtain the left arm of 19.7 Kb, right arm of 18.7 Kb (shown in bold) and intermediate plasmid sequence of 2575 bp. pVCLDc102 plasmid was digested similarly to obtain 2575 bp fragment and ligated with left and right arm of λZapDamDc101 to obtain a final recombinant phage display vector λZapDamDc101.

Digest with FseI, EcoRI Digest with FseI, EcoRI

Ligate

19.7 Kb (Left Arm) + 2575 bp + 18.7 Kb (Right Arm)

EcoRI BssHII

Bsu36I FseIlacP/OAmpr Ori

XbaI

ST H10D MCS

NheI EcoRI FseI

T7tn F+

λ ZapDamDc101

5K 10K 40K 35K 30K 25K 20K 15K BsiWI

BsaI A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro Dam

FseIlacP/OAmpr Ori T

EcoRI XbaI

T7tnST H6DMCS I

I

λ ZapDamDc102

BssHII

NheI Bsu36I

5K 10K 40K 35K 30K 25K 20K 15K BsiWI

BsaI A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro Dam

FseIlacP/OAmpr Ori T

EcoRI XbaI

T7tnST H10D MCS I

I

BssHIIBsu36I

NheI

pVCLDc102

XbaI

Sca I

F+AmprOri

lacP/O

EcoRI RI

SacII

T7tnST D H10MCS

FseI

tHP

147

SacII, ScaI

ScaI

roppVLSDH1013SacII

F+AmprOri

T7P T7tnST D H6 MCS

XbaI EcoRI RI

laci

SacII

AmprOrila

cI

SacII

ScaI

rop

SacII, ScaI

ScaI

pVCLDc102F+

AmprOri

lacP/O T7tnST D H10 MCS

XbaI EcoRI RI

SacII

FseI

lacP/OXbaI

DST T7tnH10

MCSO

SacII

ScaI FseI

tHP

Fig.III.8. Schematic representation of construction of plasmid vector pVLLDc103 and corresponding phage display vector λZapDamDc103. Only relevant genes and restriction sites are shown. The map is not to scale. Ampr, b-lactamase gene; Ori, ColE1 origin of replication; F+, phage M13 origin of replication; H10, sequence encoding for deca-histidine tag; lacP/O, lac promoter and lac operator; T7tn, T7 terminator; ST, strep-tag; D, recombinant lambda head protein; MCS, multiple cloning sites. Only some of the lambda genes are shown. T, terminator domain of the phage origin of replication derived from Lambda genome; I, initiator domain of the phage origin of replication derived from Lambda genome; Dam, wild type D protein with amber mutation. Fragment containing laci and rop gene was obtained as a 3625 bp SacII - ScaI digested fragment from pVLSDH1013SacII vector. This fragment was ligated to similarly digested 1883 bp pVCLDc102 vector to replace the backbone and obtain a recombinant with low copy number named as pVLLDc103. The low copy intermediate vector was inserted into λZapDamDc101 as FseI - EcoRI insert to obtain phage display vector λZapDamDc103.

CONSTRUCTION OF PLASMID pVLLDc103 FOR THE LAMBDA VECTOR λZapDamDc103

FseI, EcoRI

ScaI

pVLLDc 103F+

AmprOri

lacP/O T7tnST D H10 MCS

XbaI EcoRI RI

SacII

laci

rop

FseIlaci-31

λZapDamDc103

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro

BsiWI

T I

FseI

lacPOAmpr Ori T laci rop

EcoRI Bsu36I NheI XbaI

T7tn H10 MCSST D I

148

CONSTRUCTION OF PLASMID pVLTLDc103 FOR THE LAMBDA VECTOR λZapDamDc104

Fig.III.9. Schematic representation of construction of intermediate vector pVLTLDc103 and the corresponding λZapDamDc104. Only relevant genes and restriction sites are shown. The map is not to scale. lacP/O, lac promoter operator; ST, strep-tag; D, recombinant lamda head protein; MCS, multiple cloning sites; H10, sequence encoding for deca-histidine tag; T7tn, T7 terminator; F+, phage M13 origin of replication; Ampr, β-lactamase gene; rop, rop gene to regulate the copy number of plasmid; Ori, ColE1 origin of replication and laci, lac repressor gene. Only some of the lambda genes are shown. T, terminator domain of the phage origin of replication derived from Lambda genome; I, initiator domain of the phage origin of replication derived from Lambda genome; Dam, wild type D protein with amber mutation. T7 promoter was incorporated upstream of lacPO by annealing two complementary oligonucleotides T7Lac51 and T7Lac31 of 55 mer each, encoding the T7 promoter followed by lac operator with 4 – base overhang at either 3’ ends (shown in bold) compatible to SacI linearised ends of the vector pVLLDc103 to produce recombinants named as pVLTLDc103 that consisted of T7 promoter upstream of the lac promoter operator. The intermediate vector pVLTLDc103 was inserted into λZapDamDc101 as FseI - EcoRI insert to obtain λZapDamDc104 phage display vector.

SacI

pVLLDc103F+

AmprOri

lacP/O T7tnST D H10MCS

XbaI EcoRI RI

SacI

FseI

laci

\\

GAGCTCGGCGC- (N)40-AGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCA

Lac Operator -10 Region -35 Region

Sac I

rop

T7tnST D H10MCSlacP/O OriEcoRI

RIXbaI

Ampr laciSacI

F+SacI

Linearised vector

FseI

5’CTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCATGCATAGCT 3’

3’TCGAGATTATGCTGAGTGATATCCCCTTAACACTCGCCTATTGTTAAGGTACGTA 5’

T7 Promoter Lac Operator Annealed Oligonucleotide

T7Lac51 (55 mer) T7Lac31 (55 mer)

pVLTLDc103F+

AmprOri

lacP/O T7tnST D H10MCS

XbaI EcoRI RI

FseI

laci

T7P

GAGCTCTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCA CACGAGATTATGCTGAGTGATATCCCCTTAACACTCGCCTATTGTTAAGGT T7 Promoter Lac Operator

Sac I

rop

FseI, EcoRI

λZapDamDc104

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom G int exo P O cII rexB

ral bet Q S Rz cro

BsiWI

T I

T7P

FseIlacPOAmp

rOri T laci rop

XbaI

T7tn H10 MCSST DNheI EcoRI Bsu36

I I

149

Fig.III.10A. Schematic representation of construction of phagemid vector pVLTLDc105. Only relevant genes and restriction sites are shown. The map is not to scale. Ampr, β-lactamase gene; Ori, ColE1 origin of replication; lacP/O, lac promoter operator; pelB, pectate lyase signal sequence; 1.8 Kb stuffer, 1.8 Kb stuffer sequence; Tryp, trypsin protease cleavage site; S, glycine and serine rich spacer, geneIII, geneIII of filamentous phage; F+, phage M13 origin of replication; ST, strep-tag; D, recombinant lamda head protein; MCS, multiple cloning sites; H10, sequence encoding for deca-histidine tag; T7tn, T7 terminator. 1.8 kb stuffer was amplified from phagemid vector pVCEPI23961 using 5’ primers DC105-51 and 3’ primerDC105-31 that incorporated two non compatible SfiI restriction sites at either ends of the amplified product of 1963 bp. The PCR product obtained was digested with NheI - Bsu36I to give a fragment of 1837 bp that was ligated to digested vector backbone of pVLTLDc103. The recombinants obtained were named as pVLTLDc105.

CONSTRUCTION OF PLASMID pVLTLDc105 FOR THE LAMBDA VECTOR λZapDamDc106

PCR with DC105-51, DC105-31 1963 bp

1.8 Kb stuffer

NheISfiI(A)

Bsu36ISfiI(B)

NheI, Bsu36I

Digested Insert 1837 bp

1.8 Kb stuffer

SfiI(A) SfiI(B)

NheI, Bsu36I

EcoRI RI

Bsu36INheI

pVLTLDc103F+

AmprOri

lacP/O T7tnST D H10MCS

XbaI

ScaI

laci

T7P

rop

F+AmprOri

laci

Bsu36I EcoRI RI

T7tnH10ST D

XbaI

lacP/OT7PNheI

ScaIDigested Vector

rop

Bsu36I

F+Ampr

XbaI

pVCEPI 23961Ori

tHPlacP/O geneIIIpelB TrypS1.8 Kb Stuffer

EcoRINheI

pVLTLDc105F+

AmprOri

ScaI

laci

XbaI

STlacP/O T7P

EcoRI RI

Bsu36INheID 1.8 Kb StufferH10T7tn

SfiI(A) SfiI(B)

GCTAGCGGCAGCGGCCAAGGGGGCCAATGA-(N)-GGAGTTTAAGGATCGGCCGGTGGGGCCGGTGGCGGCGCCTCAGGCACTAGTGGCGCGCAG(CAC)10

CGATCGCCGTCGCCGGTTCCCCCGGTTACT-(N)-CCTCAAATTCCTAGCCGGCCACCCCGGCCACCGCCGCGGAGTCCGTGATCACCGCGCGTC(GTG)10

A S G S G Q G G Q * G V * G S A G G A G G G A S G T S G A Q H10

SfiI(B) SfiI(A)

stuffer

NheI Bsu36I

rop

150

CONSTRUCTION OF PHAGE DISPLAY VECTOR λZapDamDc106

Fig.III.10B. Schematic representation of the construction of phage display vector λZapDamDc106. Only relevant genes and restriction sites are shown. Only some of the lambda genes are shown. The map is not to scale. T, terminal domain of the phage origin of replication derived from Lambda genome; I, initiator domain of the phage origin of replication; Ampr, β-lactamase gene; Ori, ColE1 origin of replication; T7P, T7 promoter; lacP/O, lac promoter and operator; ST, strep-tag; D, recombinant lambda head protein; Dam, wild type D protein with amber mutation; 1.8 Kb stuffer, stuffer segment of 1.8 Kb in size; MCS, multiple cloning sites; H10, sequence encoding for deca-histidine tag; T7tn, T7 terminator. λZapDamDc104 DNA was digested with XbaI-EcoRI restriction enzyme to obtain the left arm of 19.7 Kb, right arm of 20.7 Kb (shown in bold) and intermediate plasmid sequence of 536 bp. pVTLDc105 plasmid was digested similarly to obtain 2254 bp fragment and ligated with left and right arm of λZapDamDc104 to obtain a final recombinant phage display vector λZapDamDc106.

Ligate

Digest with XbaI, EcoRI

19.7 Kb (Left Arm) + 536 bp + 20.7 Kb (Right Arm)

Digest with XbaI, EcoRI

ScaI

XbaI

STlacP/O T7P

EcoRI RI

Bsu36INheID 1.8 Kb StufferH10T7tn

SfiI(A) SfiI(B)

pVLTLDc105F+

AmprOri

laci

rop

SfiI(A)

XbaI

STEcoRI

RIBsu36INheI

D 1.8 kb Stuffer H10

SfiI(B)

rop

ScaI

XbaI EcoRI RI

T7tn

F+AmprOri

laci

lacP/O T7PT7P

λ ZapDamDc104

5K 10K 40K 35K 30K 25K 20K 15K BsiWI

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro Dam

BsaI

FseI

Ampr Ori T lacP/OT7P

XbaI EcoRI RI

T7tnST D H10 MCS I

I

λ ZapDamDc106

XbaI

STEcoRI

RIBsu36INheI

D 1.8 kb StufferH10T7tn

SfiI(A) SfiI(B)

I

5K 10K 40K 35K 30K 25K 20K 15K BsiWI

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro Dam

FseIAmpr Ori T lacP/O T7P

BsaI

I

laci

151

OUTLINE FIGURE OF VARIOUS λZapDam DERIVED PHAGE DISPLAY VECTORS

Fig.III.11. Outline figure of different lambda constructs. A. λZapDamDc102; B. λZapDamDc103; C. λZapDamDc104; D. λZapDamDc106. The different lambda constructs have been shown with various plasmids positioned between the terminator domain (T) and initiator domain (I) of the phage origin of replication. The key features of each construct have been shown in bold.

λZapDamDc103

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro

BsiWI

T I

FseIlacPOAmpr Ori T laci rop

EcoRI Bsu36I NheI XbaI

T7tn H10 MCSST D I B.

λZapDamDc104

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro

BsiWI

T I

T7P

FseIlacPOAmpr Ori T laci rop

XbaI

T7tnH10 MCSST D

NheI EcoRI Bsu36I

I C.

D.

λZapDamDc106

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro

BsiWI

T I

I

NheI Bsu36I XbaI

1.8 Kb StufferST D T7tn H10

EcoRI

SfiI(A) SfiI(B)

T7P

FseIlacPOAmpr Ori T laci rop

5K 10K 40K 35K 30K 25K 20K 15K

A B K L H Z E C I J lom G int exo P O cII rexB ral bet Q S Rz cro

BsiWI

T I

λZapDamDc102

EcoRI XbaI FseIlacPOAmpr Ori T T7tnST H10 MCSD I A.

152

153

The next modification was to produce a lambda display vector where cloning could be carried out directly into the lambda vector. For this, the

pVLTLDc103 was modified by inserting a 1.8 Kb DNA segment flanked by two

SfiI sites and inserted into the NheI and Bsu36I sites (Fig.III.10A). This new plasmid pVLTLDc105 carries D fusion with the two SfiI sites which produce non-

compatible 3-base over hangs and can be used for directional cloning with no chances of vector self ligation. This feature was brought into λZapDam by inserting XbaI-EcoRI fragment from pVLTLDc105 into left and right arms of XbaI-EcoRI digested λ-104 to yield λZapDamDc106 (λ-106) (Fig.III.10B). In this vector, the two SfiI sites are unique and any DNA can be inserted with ease. 3.3.3. Characterization of various Lambda display vectors by western blot analysis

All the λZapDam (Fig.III.11) vectors were tested for plasmid rescue using helper phage as described in Appendix III.17.6 and were found to be equally efficient indicating the full functionality of initiator and terminator sequences of f-ori which flank the inserted plasmids.

Further, phages were prepared and purified by PEG precipitation and chromatography on Sepharose 6B-CL to isolate concentrated phage particles free of any soluble contaminating protein. The intactness of the phage particles, display of D fusion proteins and display density was checked by western blot analysis using different reagents as described below. To estimate the number of D fusion proteins, known quantities of purified recombinant S-D-H (Strep tag-D-H6 fusion protein) were also loaded on gel. (A) RD113 (anti-gpD MAb)

This antibody binds to D protein of phage lambda and requires almost

complete D protein for binding. The western blot with RD113 revealed detectable

bands of D fusion protein around 17 kDa in case of λ-102 and λ-103. But no

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Fig.III.12. Western Blot analysis of different constructs of Lambda phage displaying D - fusion. For Western blot: 2 x 109 lambda phages of different constructs, Lane 1, λZapDamDc102; Lane 2, λZapDamDc103; Lane 3, λZapDamDc104; Lane 4, λZapDamDc106; Lane 5 - 7, 50 ng, 25 ng and 12.5 ng purified SDH6 protein respectively were electrophoresed on 0.1% SDS-15% PAG then transferred onto PVDF membrane and probed with (A) anti-gpD MAb, RD113, (B) anti-His MAb AE-26 (C) anti-gpV MAb, P043 followed by HRP-conjugated goat anti-mouse IgG and (D) anti-Strep MAb conjugated with HRP; Lane M, molecular mass markers with molecular mass in kDa.

WESTERN BLOT ANALYSIS OF DIFFERENT CONSTRUCTS OF LAMBDA DISPLAYING D - FUSION

D.

206

54

37

29

6

17

4 M 1 3 2 5 6 7

S-gpD-H S-gpD

206

54

37 29

6

17

4 M 1 3 2 5 6 7 B.

S-gpD-H

A.

206

54

37

29

17

6

116

4 M 1 3 2 5 6 7

S-gpD-H

gpD

4 M 1 3 2 206

54

37

29

17

6

97

C.

gpV

155

detectable protein was observed in the case of λ-104 and 106 (Fig.III.12A). Since, these phages carry native D protein also, a strong band at ~ 10 kDa was also seen in all phages. Although previous experiments using another type of lambda display vector had suggested that in lambda system very large proportion of D

could be incorporated as D fusion protein, the reactivity of RD113 shows presence of D and only a small fraction is the D fusion protein. This unexpected result was

probably due to the poor reactivity of RD113 to D fusion protein expressed by lambda vectors and incorporated in the phage particles. The reactivity of this antibody to recombinant SDH was good but this SDH was not the same as found

in the phage particles. Indeed, reactivity of RD113 was an issue that was confirmed by the reactivity with other reagents that bind to D-fusion proteins.

(B) AE26 (Anti-His MAb) and HRP- conjugated Streptactin

This MAb is capable of binding to poly histidine sequence containing six or ten histidine residues placed at either termini of the protein. The western blot

analysis showed that λ-102, λ-103 and λ-104 are reactive, of which λ-103 showed the strongest reactivity followed by λ-102 and λ-104 (Fig.III.12B). This result showed that different lambda display vectors incorporate large amount of D fusion protein. The densitometric scanning in comparison to the standard

recombinant S-D-H protein revealed that λ-102 and λ-103 display more than 100 copies of fusion protein per phage particle. λ-106 did not show reactivity to anti-His MAb because in this construct D-fusion protein was interrupted by a stuffer, that contained several translational stops. In fact, this construct would express D with N-terminal strep tag but would terminate after adding just six amino acids beyond 109 residues of D, thus making a D fusion without C-terminus histidine tag that would be smaller in size. Indeed, this was further proved by western blot using HRP conjugated streptactin (Fig.III.12D), which binds to N-terminal strep tag. Here, λ-106 showed a reactive band of size smaller than S-D-H. The western

blot analysis showed that, λ-103 has the strongest reactivity with HRP -

streptactin suggesting that the plasmid inserted in λ-103 was most suitable for high-density display.

156

The western blot analysis using MAb P043 (Fig.III.12C), an antibody against V tail protein of lambda, showed that all types of phage preparations contain equal number of intact phages having equal amount of tails.

The above characterization clearly demonstrated that the λZapDamDc103 carrying low copy number vector is the best for high-density display. 3.3.4. Evaluation of λZapDam display system for the efficient display of proteins

The display capability of the new λZapDam was evaluated by cloning three different proteins as SfiI inserts in λZapDamDc106 that carries a low copy

number vector. These three proteins Ag85A (Rv3804c), Ag85B (Rv1886c) and

MPT51 (Rv3803c) are produced by M. tuberculosis and differ in molecular weight and also solubility when produced as recombinant proteins in E. coli using T7 promoter based expression system where MPT51 is most soluble followed by Ag85B and Ag85A. The coding regions of these three proteins were amplified

using primers to create SfiI site with appropriate sequences at the 5’ and 3’ ends and cloned in SfiI digested λZapDamDc106. After characterization of phage plaques by PCR and sequencing, phages from one clone each were prepared and purified. The phages from the lysate were concentrated using Amicon-100 filters followed by gel filtration on Sepharose 6B-CL to obtain concentrated phages free of any soluble contaminant. It was found that the purity of phages prepared by this procedure is almost same as that produced by PEG precipitation followed by ultracentrifugation.

The purified phages λZapDamDc106-85A (displaying 32 kDa Ag85A, λ-85A), λZapDamDc106-85B (displaying 28 kDa Ag85B, λ-85B), λZapDamDc106-MPT51 (displaying 24 kDa MPT51, λ-MPT51) and a control phage λZapDamDc104 (displaying Strep tag-D-H10 fusion protein of 17 kDa, λ-104) were analyzed by western blot analysis using different monoclonal and

4 M 1 3 2 5 6 8 7 206

6

17

29

37

54 97

D.

Pd. Ag85B

gpD-Ag85B

206

6

17

29

37

54

97

4 M 1 3 2 5 6 8 7 E.

Pd. MPT51

gpD-MPT51

Fig.III.13. Western Blot analysis of Lambda phages displaying mycobacterial proteins Ag85A, Ag85B and MPT51. For Western blot A-C: 1 x 109 lambda phages of different constructs, Lane 1, λZapDamDc104 Lambda phage displaying Strep-gpD-H10; Lane 2, λZapDamDc106-85A Lambda phage displaying Ag85A as gpD fusion; Lane 3, λZapDamDc106-85B Lambda phage displaying Ag85B as gpD fusion; Lane 4, λZapDamDc106-MPT51 Lambda phage displaying MPT51 as gpD fusion; Lane 5 and 6, 34.6 ng and 17.3 ng of purified SDH6 protein respectively; Lane M, molecular mass markers with molecular mass in kDa were electrophoresed on 0.1% SDS-12.5% PAG, transferred onto PVDF membrane and probed with (A) anti-gpD MAb, RD113, (B) anti-His MAb AE-26 followed by HRP-conjugated goat anti-mouse IgG, (C) anti-Lambda phage proteins Rabbit anti Lambda followed by HRP-conjugated goat anti-rabbit IgG. For Western blots D-E: Lane 1-4, 1 x 109, 5 x 108, 2.5 x 108 and 1.25 x 108 lambda phage displaying Ag85B and MPT51; Lane 5-8, 20 ng, 10 ng, 5 ng and 2.5 ng of purified Ag85B and MPT51 purified protein were electrophoresed on 0.1% SDS-12.5% PAG transferred onto PVDF membrane and probed with (D) Rabbit anti polysera against Ag85B and (E) Rabbit anti polysera against MPT51 followed by HRP-conjugated goat anti-rabbit IgG.

4 M 1 3 2 M 5 6 206

54

37

29

17

6

116 97

A.

6

116

206

97

54

37

29

17

4 M 1 3 2 5 6 M B.

WESTERN BLOT ANALYSIS OF LAMBDA PHAGES DISPLAYING MYCOBACTERIAL PROTEINS

116 206

97

54

37 29 17 6

4 M 1 3 2 C.

gpE gpV

gpD

SDH

D-Fusion

157

158

polyclonal antibodies and other reagents. The western blot analysis with

RD113 (Fig.III.13A) revealed that λ-104, λ-85A, λ-85B and λ-MPT51 showed two reactive bands, one corresponding to the fusion protein of 17 kDa, 48 kDa, 44 kDa and 40 kDa in respective phage lane of varying intensity and a band corresponding to the native D protein of 10 - 11 kDa. Although it was difficult to make judgment about the display density by comparing the reactivity of D fusion protein with that of standard recombinant SDH, it was very clear that MPT51 was best displayed. Further, there was no degradation of incorporated D fusion proteins.

The western blot using MAb AE26 (Fig.III.13B), which reacts with C-terminal deca-histidine tag showed almost equal intensity bands with all four phage preparations suggesting that display of proteins was equal and might not be affected by size and nature of the protein. The comparison of reactivity of fusion proteins with standard recombinant SDH showed that nearly 150 - 200 fusion proteins were present on each phage particle which correspond to about 50 % of the D being the fusion protein. Further, number of molecules displayed, as fusion with D did not seem to be affected by the size and nature of the protein.

However, the reactivity with RD113 did not truly reflect the amount of displayed protein, as its reactivity seemed to be affected by nature of the fusion protein.

The western blot using a rabbit polyclonal antibody against whole lambda

(Fig.III.13C) also showed that D fusion proteins of calculated sizes were present in

almost same intensity with MPT51 fusion showing better reactivity (Fig.III.13C, lane 4). The fusion protein carried by λ-104 phages could not be seen clearly

(Fig.III.13C, lane 1). This western blot also showed that all the phage preparations contained equal amount of intact phages as revealed by equal reactivity to gpE (head protein) and gpV (tail protein). This was important as some of the earlier preparation when probed with this polyclonal sera showed that different preparations of phages contained different ratios of gpE and gpV indicating preponderance of either unassembled phage heads or phage tails

159

which may arise due to faulty assembly or disruption of phages during preparation, concentration and purification.

To more accurately calculate the display density and for functional characterization, λ-85B and λ-MPT51 phages were probed with polyclonal sera

against recombinant Ag85B and MPT51 proteins (Fig.III.13D & E). The phages showed specific reaction to respective polyclonal antibodies. Further, estimation of display density revealed that there were approximately 200 copies of D-Ag85B fusion and 150 copies of D-MPT51 fusion on each phage. These results corroborated with the display density estimations using anti-His MAb AE26.

This analysis clearly demonstrated that the new lambda display system was compatible with protein of any size and composition and is suitable for high-density display.

3.3.5. The new lambda phage display system results in rapid selection during panning

The power of any phage display system resides in the fact that through affinity selection (panning) phages displaying desired protein can be selected from a milieu of millions of other phages. The efficacy of the panning could be dependent on the display density. To examine the potential of this new lambda phage displaying Ag85B fused to D protein with N-terminal strep tag and C-terminal His tag (λ-85B, λZapDamDc106-85B) were mixed with λZapDamDc104 phages displaying only D protein with N-terminal strep tag and C-terminal His tag (λ-104, λZapDamDc104) in ratio of 1:104 (105 λ-85B + 109 λ-104/ml phages; mix A) and 1:105 (104 λ-85B + 109 λ-104/ml phages; mix B). These mixtures of phages were subjected to affinity selection (panning) on MAb Ag85AB04, coated on well surface of microtitre plate. After binding followed by washing of unbound phages, the bound phages were eluted using collagenase. The eluates were titrated by infecting TG1 cells and plated for pfu. The plaques from plates were transferred onto nitrocellulose membrane. The membrane was blocked and

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A. λZapDamDc104:λZapDamDc106-85B: 109:105 (i) (ii) (iii)

B. λZapDamDc104:λZapDamDc106-85B: 109:104 (iii) (ii) (i)

Fig.III.14. Affinity selection of λZapDamDc106-85B phages by plaque filter lift assay. Lambda ZapDamDc106 phages displaying Ag85B protein as gpD – fusion were spiked in two different ratios with the non-specific λZapDamDc104 phages displaying S-D-H fusion (A) 105:109 (B) 104:109. Two rounds of affinity selection were carried out with the spiked phages on purified MAbAg85AB04. Filters containing plaques from the (i) initial mix (ii) enriched phages after the first cycle of selection Pan I (iii) enriched phages after the second cycle of selection Pan II, were immunostained with MAbAg85AB04, followed by HRP conjugated goat anti-mouse IgG antibody. (C) Table showing the fold enrichment of λZapDamDc106 phages displaying Ag85B after (i) initial (ii) Pan I and (iii) Pan II as determined by the number of positive plaques obtained after immunostaining with MAbAg85AB04.

S.No. Spike Ratio Sample Plaques

obtained Positive plaques

Fold Enrichmenta

A.

109:105

i) Initial ii) Pan I iii) Pan II

250 350 200

- 50

150

- ~ 1400 fold ~ 7500 fold

B.

109:104

i) Initial ii) Pan I iii) Pan II

300 300 200

- 3-4 100

- ~ 1000 fold ~ 50,000 fold

a (Positive plaques/Plaques obtained) x Spike ratio

C.

AFFINITY SELECTION OF λZapDamDc106-85B PHAGES BY PLAQUE FILTER LIFT ASSAY

161

plaques from λ-85B phages were visualized by immunostaining by incubating the nitrocellulose membrane with MAb Ag85AB04 followed by HRP conjugated goat anti-mouse IgG and finally the reactivity was seen using DAB as a substrate. The major portion of eluate from panning I was amplified by infecting TG1 cells to prepare phage lysate followed by titration for pfu. A solution containing 109 phages/ml was subjected to second round of affinity selection on MAb Ag85AB04 as described above and bound phages were eluted and titrated. Here also, plaques were transferred onto nitrocellulose and presence of λ-85B was detected by immunostaining using MAb Ag85AB04. An appropriate dilution of each unselected phage mix before panning I was also titrated and phages were transferred onto nitrocellulose membrane to estimate the presence of λ-85B by immunostaining.

The results (Fig.III.14A & B) show that immunostaining of initial mix did

not show any MAb Ag85AB04 reactive phage plaque as the ratio of λ-85B phage

was 1/104 or 1/105 while the plated phages were only ~ 200 - 300. However, after

first round of panning of mix A (1 λ-85B/104 λ-104) 50 out of 350 phages were λ-85B, which is equal to 1400-fold enrichment. This affinity selection further improved to 150 λ-85B phages out of 200 total plaques after II round of panning with total enrichment of ~ 7500 fold. In the mix B, that had much lower number of λ-85B (1 λ-85B / 105 λ-104), first round of panning resulted in 1000-fold

enrichment, as 3 - 4 λ-85B phages were present among 300 plaques. This enrichment improved to ~ 50,000 folds as 100 λ-85B phage plaques were present in 200 total plaques after second round of panning (Fig.III.14C).

These results clearly established the efficacy of the new lambda display system that even a few specific phages in a million of non-specific phages can be efficiently selected in two rounds of panning.

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3.4. DISCUSSION

In this chapter, we have described development of a new lambda phage based vector for high-density display of peptides and proteins fused to the ‘D’ head protein of the bacteriophage lambda. The vector is based on a well-known cloning vector lambda Zap, which is not a display vector. The new phage vector contains a plasmid carrying sequence of ColE1 origin of replication, gene encoding β-lactamase and lacPO driving the expression of D fusion protein. The plasmid sequence is flanked by initiator (I) and terminator sequence (T) of f-ori of replication and therefore, the plasmid can be rescued by coinfecting a strain of E.

coli with the lambda phage and a M13 helper phage and using the lysate to transduce rescued plasmid present as phagemid particle, as after rescue from lambda, the f-ori is reconstituted. Thus, inserted plasmid produces ampicillin resistant colony and can be used for expression of D fusion protein under lacPO.

Several variants of the lambda display vectors have been constructed.

These variants differ in carrying plasmid with low and high copy number derivatives of ColE1 origin, hexa-histidine or deca-histidine as C-terminal tag in the expressed D-fusion protein and the cloning sites. However, it seems that lambda vector carrying low copy number ColE1 origin of replication shows good display. One important feature of this lambda display is that proteins of different sizes and solubility could be displayed with equal efficiency and there was no degradation product. Similar observation was made while developing another lambda display system, which involves insertion of plasmid at a different location in lambda DNA but the inserted plasmid cannot be rescued for expression of D-

fusion protein (Gupta et al., 2003). In this series of lambda display vector, a cloning vector with two SfiI sites has been constructed. Since these sites are unique and produce non-compatible ends, any gene with compatible ends can be directly cloned in lambda DNA and displayed on the surface of encoded phage particles.

163

M13 phage based display vectors have been developed and used extensively for a variety of applications including antibody cloning, epitope mapping, for constructing gene fragment and random peptide libraries.

However, in the M13 based system generally only one protein molecule is displayed per phage particle. Therefore, this system is very useful where panning can be adversely affected by avidity such as during the affinity maturation/ improvement of antibodies. However, there are several applications such as epitope mapping using random peptide library or gene fragment library, which can be greatly benefited by avidity due to high-density display. Moreover, the high density can lead to rapid selection of specific clones. This was seen in panning experiment where in two rounds of panning, specific phage displaying Ag85B protein could be enriched 50,000 times from a mixture having 105 times more non-specific phages. The system needs to be tested in comparison to more

popular M13 display vector to truly establish its superiority.

It is also clear that lambda D protein based display is not affected by size or properties of the displayed protein and there is no degradation of the D fusion protein. This feature could be due to cytosolic assembly of the phage and thus avoiding the complex secretion apparatus where many of the secretion incompetent protein may get stuck or only their degraded secretion competent

derivations get preferentially incorporated in the M13 phage particles. Another important application of lambda phage based system would be in cDNA cloning

where M13 system has not been widely utilized due to the presence of

translational stop and poly-A tail at the 3’ end of the cDNA which would not allow production of gIII fusion protein. In lambda phage the display is due to the

fusion of foreign gene at the 3’ end of D at the C-terminus.

The system has been designed in such a way that inserted plasmid can be easily modified using FseI, XbaI or EcoRI sites to meet any need of cloning within the inserted plasmid. The lambda system produces two types of D proteins, one from the lambda DNA itself and the other from the cloned DNA as D fusion. The relative concentration of the two decides the final display density.

164

Beside lambda, other cytoplasmic lytic virus such as T4 and T7 has been employed for display. Especially, T7 system has been used extensively as the system is commercially available from M/s Novagen, USA. But that system also does not support high-density display of proteins.

The high-density display might also find use in in vivo targeting to either select tissues targeting peptides or for delivering the cargo. Due to high density, the panning condition can be made more stringent so that non-specific phages are completely removed. This is evident from the panning experiment where despite very low amount (1 in 105), specific phages could be enriched in two rounds of panning so that more than 50 % phages after the second round will be specific desired phages.