Antibody Engineering and Protein Expression Conferences Summary

Ge Wei

March 13, 2007

Some Interesting Presentations• TIPS: Titerless Infected-cells Preservation and Scale-up 

S. Edward Lee, Ph.D., Research Fellow, PGRD, Pfizer Inc. (Talk)

• The Colony Filtration Blot Method and Its Applications Sue-Li Dahlroth; Audur Magnusdottir; Maria Törnkvist; Victoria Lieu; Monica Ekberg; Pär Nordlund; and Tobias Cornvik (Poster)Karolinska Institute, Schlees vag 2, Stockholm, S-171 77 Sweden

• Translation Pauses are Conserved in Protein Families Joseph Kittle, CODA Genomics (Talks and Poster)

• Screening for soluble expression constructs using cell-free protein synthesis T. Lamla , S. Hoerer and M.M.T. BauerDepartment of Lead Discovery, Boehringer Ingelheim Pharma GmbH and Co. KG, Birkendorfer Strasse 65, D-88397 Biberach/Riss, Germany

Standard Baculovirus Protocol

• Multiple rounds of virus amplification, titering, and expression confirmation/optimization

• Inconsistency and variability in virus assays/titers and protein expression

• Large volume and instability of virus stocks

• Repeating virus amplification, titering, and expression confirmation/optimization

TIPS

• Keep the virus in the cells!

- Reduces the storage volume by nearly a 100-1000 fold.

- Increases the virus titers by the same fold.

• Can now stably cryo-preserve small aliquots of infected cells.

How to Create Infected-cell Stocks?

• Infect 100-200 ml sf9 cells (0.5-1x106 cells/ml) with 1-2 ml of virus

• When cell size increases by ~2μm and viability is still high (>90% preferred)

(1) Spin down cells, (2) re-suspend cell pellets in a freezing media, (3) aliquot, and (4) freeze (10%DMSO)

The TIPS Advantages

• The infected cell stocks are stable up to 60 months

• Easy to scale up. Reliable, flexible and amenable for automation

• No carry-over of degenerated, spent media

• Still need to do titration before doing large scale production.

The Colony Filtration Blot Method and Its Applications

• Incremental random N-terminal deletions to increase the success rate of soluble protein expression in E. coli.

• In vitro evolution (random mutagenesis) to improve protein solubility expressed in E. coli.

Sue-Li Dahlroth; Audur Magnusdottir; Maria Törnkvist; Victoria Lieu; Monica Ekberg; Pär Nordlund; and Tobias Cornvik

Illustration and verification of the CoFi blot procedure

Nature Methods  2, 507 - 509 (2005)

Proteins Vol.65, 2 Pages: 266-273Copyright © 2006 Wiley-Liss, Inc., A Wiley Company

A schematic representation of the Erase-A-Base process

Nature Methods  2, 507 - 509 (2005)

Random Mutagenesis

• Error-Prone PCR (↑ Mg2+ )

• Mutazyme

• The bacterial mutator strain method

• error-prone RCA (Rolling circle amplification)

Nucleic Acids Res. 2004; 32(19): e145

Figure 3. Examples of CoFi blots of N-terminal deletion libraries from six different proteins, (a) HP02, (b) HP03, (c) HP10, (d) HP11, (e)

HP25, and (f) HP26. The CoFi blots of HP03, HP10, HP11, and HP26 contain colonies expressing soluble protein (seen as black spots) while HP02 and HP25 were the two targets with no positives in the CoFi blot.

Proteins Vol.65, 2 Pages: 266-273Copyright © 2006 Wiley-Liss, Inc., A Wiley Company

Proteins Vol.65, 2 Pages: 266-273Copyright © 2006 Wiley-Liss, Inc., A Wiley Company

Proteins Vol.65, 2 Pages: 266-273Copyright © 2006 Wiley-Liss, Inc., A Wiley Company

CODA- Translational Pause and Pairs of Codon

Overview

• CODA's Translation Engineering TM technology is based on the observation by Hatfield G.W. (UCI) that living organisms from bacteria to fungi to higher plants and animals all use translational pause signals to regulate the amount and quality of protein translated from a given transcript. These explicit translation signals are encoded as pairs of codons. This phenomenon is independent of “codon usage” related to tRNA levels.

• The presence of these pause signals is universal. The actual codon pairs used to encode pauses vary widely from organism to organism, so moving an ORF into a heterologous host scrambles these signals, and can result in random pause signals. This is why many attempts to improve protein yield and expression with synthetic genes fail.

• Using CODA’s massive computing power running proprietary computer algorithms, they can:

(1) Detect and eliminate the unnecessary translational pauses of a foreign cDNA and re-introduction of codon pairs for optimal protein expression and functional equivalence when substituting non-native expression machinery for the native one. (Hot Rod GenesTM)

(2) Control the domain folding when introducing the correct pauses. (Planned Pause GenesTM)

Hot Rod Genes

Planned Pause Genes

Screening for soluble expression constructs

using cell-free protein synthesis T. Lamla , S. Hoerer and M.M.T. Bauer

Department of Lead Discovery, Boehringer Ingelheim Pharma GmbH and Co. KG, Birkendorfer

Strasse 65, D-88397 Biberach/Riss, Germany

•E. coli expression system: fast growth, easy handling, and low cost. However, recombinant proteins produced in E. coli often accumulate as insoluble aggregates.

•Traditional ways to express proteins in E. coli: Clone the target cDNA into an expression vector and try to change parameters such as temperature, additives, induction conditions, or the addition of affinity tags to improve the behavior of the recombinant proteins. However, only limited clones can by handled by this way.

•A faster and more convenient approach to screen a large number of expression constructs as well as expression conditions of recombinant proteins compared to classical in vivo systems is the cell-free protein expression.

•A main advantage of this method is the fact that linear templates generated by PCR can directly be used for in vitro expression of the encoded protein and thereby prevent time-consuming cloning steps. One open question is still the correlation of the in vitro expression based on an E. coli lysate and the expression in E. coli.

Comparison of in vitro and in vivo expressed constructs.

(I) E. coli expression; (II) and (III) in vitro expression with the cell-free systems from Roche and Qiagen

International Journal of Biological Macromolecules Volume 39, Issues 1-3 , 15 August 2006, Pages 111-121 Challenging Proteins

20 Structurally-Known SH2 were Used to Define the Boundary of SH2 of STAT6

International Journal of Biological Macromolecules Volume 39, Issues 1-3 , 15 August 2006, Pages 111-121 Challenging Proteins

70 PCR fragments of the STAT6 SH2 Domain were Screened and Two Partially Soluble Constructs were Identified

International Journal of Biological Macromolecules Volume 39, Issues 1-3 , 15 August 2006, Pages 111-121 Challenging Proteins

Many Constructs with Single and Double Mutations were Screened

International Journal of Biological Macromolecules Volume 39, Issues 1-3 , 15 August 2006, Pages 111-121 Challenging Proteins