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

• 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.
Illustration and verification of the CoFi
blot procedure

Nature Methods 2, 507 - 509 (2005)


Proteins Vol.65, 2 Pages: 266-273
Copyright © 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: 266-273


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

(9) 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)

(11) 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