4:00 – 6:00 Main Conference Pre-Registration

MONDAY, APRIL 6

New or Improved Expression Systems

7:00am   Registration and Morning Coffee

8:30 Chairpersons Opening Remarks

9:10 Improving Integral Membrane Protein Production in the  Baculovirus Expressions System

Suzanne M. Thiem, Ph.D., Department of Biochemistry and Molecular Biology, Michigan State University

We are using model membrane proteins fused with GFP as tools for investigating why integral membrane proteins are difficult to express in the baculovirus expression system, and for identifying and developing new cell lines better suited for membrane protein production. A new insect cell line and progress in developing baculovirus vectors designed for enhanced membrane protein production will be presented.

Sponsored by

9:40 
Template-Directed-Assembly of RTK TK Domains Restores Biologically-Relevant Function
Stephen Zolnay, Vice President of Business Development, P.A. Technologies
Receptor tyrosine kinases and other membrane associated kinases are important targets in drug discovery. Use of recombinant kinase domains, from targets which are normally membrane-associated, in early stage HTS efforts often provide unreliable results including false positives, missed leads, and poor selectivity data (IGF-1R, TrkB, Tie2, and ErbB4, as examples). Because these target proteins normally reside on the cell membrane and utilize the membrane as a scaffold for assembly of complex signaling networks, we have developed a simple, homogenous, in vitro self-assembly approach to create more meaningful assays. Our template-directed self-assembly approach promotes the assembly of functioning protein complexes on a soluble nanometer scale template, so that the recombinant fragments function more like they do inside the cell. Our results indicate that these proteins are more active, more faithfully represent their cellular function, and have altered substrate selectivity when properly assembled, and therefore provide more meaningful results. 

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9:55 Rapid Recombinant Protein Expression Screening and Production in Insect Cells

Barbara Morris, Senior Scientist, Product Applications, EMD Chemicals, Novagen Brand

The pIEX/Bac plasmids are for plasmid or baculovirus- mediated expression in insect cells. An immediate early promoter allows expression directly in Sf9 cells without viral infection, providing an ideal format for screening multiple constructs. For large scale protein production, the pIEx/Bac plasmids are easily recombined into a baculovirus genome.

10:10 Grand Opening Coffee Break in the Exhibit Hall

11:10 Controlling Transgene Expression via Intragenic CpG Dinucleotides

Asli Bauer, Ph.D., Senior Scientist, Molecular Microbiology and Gene Therapy, University of Regensburg

Here we provide evidence that the intragenic content of CpG dinucleotides significantly influences the level of transient and stable protein expression in mammalian cells such as 293T or CHO. Whereas a maximal increase of CpG numbers in the open reading frame of several reporter transgenes resulted in the highest expression yields albeit codon quality was reduced, the elimination of CpGs clearly diminished transgene expression both in vitro and in vivo. Recent results suggest an influence of intragenic CpG dinucleotides on nucleosome assembly potentially affecting chromatin structure. Deeper insights into CpG-mediated epigenetic mechanisms might serve to develop novel optimization strategies for improved protein production.

11:40 Novel Expression and Production Methods for Difficult to Express Proteins in Tetrahymena thermophila

Paul Colussi, Ph.D, Director of Protein Expression, Tetragenetics

Tetrahymena shares many of the attractive production features of other microbial systems and is uniquely suited to the production of difficult to express proteins, most notably highly hydrophobic membrane proteins. Key to this feature is a significant metabolic commitment to membrane protein production and a large membrane surface area that is greatly exaggerated by hundreds of cilia. At the core of our expression system is a ribosomal DNA based vector that is targeted to a unique chromosome encoding ribosomal DNA that is formed at 9000 copies per cell. The palindromic nature of the rDNA chromosome allows stable maintenance of up to 18,000 copies of the transgene per cell. Expression is driven from a choice of powerful but tightly regulated metallothionein promoters. In this presentation we will be detailing case studies of recombinant membrane protein production validating the versatility of Tetrahymena as an expression host.

12:10pm A Novel and Cost-Effective Method to Express Antimicrobial Peptides for Therapeutic Use in Escherichia coli

Bettina Bommarius, Ph.D., Research Associate, Pathology, Emory University

The successful recombinant expression of antimicrobial peptides in bacteria with sumo as a protective group is a useful tool to overcome critical shortfalls in the therapeutic use of antimicrobial peptides, since existing methods for peptide synthesis are too expensive to provide the amounts of AMPs needed in clinical use. Potentially, bacterial overproduction with the subsequent 3 step purification using Ni-NTA and RPC could be scaled up for industrial use and make antimicrobials readily available in clinical trials as a alternative to antibiotics in treatment of MDR pathogens.

12:40 Luncheon Presentation I                                                                                          Sponsored by  

Shuffle™, A Novel E. Coli Strain That Can Promote the Formation of Correct Disulfide Bonds in the Cytoplasm
Mehmet Berkmen, Ph.D., Research Scientist, Gene Expression, New England Biolabs
Disulfide bond formation is a post-translational modification essential for the folding of many proteins. However, Escherichia coli is ill equipped to correctly fold multi-disulfide bond proteins to high yields. Here we introduce a novel Escherichia coli strain called Shuffle™ that can correctly oxidize protein within the cytoplasm. Proteins within the cytoplasm of Escherichia coli are maintained in their reduced non-disulfide bonded state by thioredoxin and glutaredoxin reductases. Proteins which require disulfide bonds for their folding are therefore restricted to those that are secreted to extracytoplasmic compartments. In gram negative bacteria such as E. coli, a set of Dsb proteins catalyze the formation of disulfide bonds in the periplasmic compartment. DsbA catalyzes the formation of disulfide bonds and the chaperone DsbC isomerizes mis-oxidizes substrates into their correctly folded state. Mutant E.coli lacking the two reductases (trxB and gor), with an additional suppressor mutation (ahpC*) which restores viability, allow the formation of stable disulfide bonds in the cytoplasm. Under these conditions thioredoxins are in their oxidized state, converting them from reductases to oxidases. Proteins which require disulfide bonds for their folding thus can be oxidized and form stable disulfide bonds within the cytoplasm. In this presentation we describe a strain which, in addition, expresses the disulfide bond isomerase DsbC within its cytoplasm. This feature greatly enhances the fidelity of disulfide bond formation in the cytoplasm, and proteins with multiple disulfide bonds are correctly oxidized to significantly higher yields. This strain is now commercially available from New England Biolabs as Shuffle™ and is constructed in E. coli K12 and E. coli B backgrounds. Further developments were engineering for the expression of T7 derived protein expression and toxic proteins. We will present detailed characterization of Shuffle™ along with yields of protein obtained.

1:10 Luncheon Presentation II (Opportunity Available)

1:40 Break

Protein Engineering Approaches

2:00 Chairpersons Remarks

Deb Chatterjee, Ph.D., NCI

2:05 Innovative Low Human Homology Affinity Tags

Fons  Bosman, Ph.D., Formulation Expert, Biologicals, Innogenetics NV

We designed flexible small (multifunctional) low human homology affinity tags (LHH-tag) that maintain the benefit of single-step purification on commercially available resin, and that can resolve these general immunology-related disadvantages. The selection strategy of these LHH-tags, as well as purification- and immunology-related results will be presented and discussed.

2:35 Fusion Proteins in Mammalian Expression

Christopher Mehlin, Ph.D., Senior Scientist, Protein Science, Amgen

I will present a small study we have done with six difficult-to-express proteins.  We made a set of vectors which appended three different solubility and expression enhancing tags and screened the resultant fusions via transient transfection in a HT, parallel system. Some of these tags were clear winners, and others didn’t do so well.  I will also discuss issues concerning scale-up and cleavage of these tags.

3:05 Refreshment Break in the Exhibit Hall

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3:45 Comparative Study on Autologous Expression Improvement in Human Cells by Gene Optimization: Results and Applications

Stephan Fath, Ph.D., Scientist, Research & Development, GENEART AG

We report the largest gene expression study on synthetic optimized genes in mammalian cells to date. Fifty human genes from the NCBI Entrez database representing different protein classes such as protein kinases, cytokines, membrane proteins and transcription factors, were optimized for increased mRNA half-life and protein expression in human cells. Expressed protein levels in HEK‑293T cells were quantified and compared. The results clearly indicate a significant improvement of expression yield with optimized constructs compared to respective wildtype versions. Therefore, gene synthesis is not only a versatile manner to obtain individualized genes but also allows for autologous expression increase in most cases.

4:15 E. Coli Mutants Selected for Improved Protein Expression

James Bowie, Ph.D., Professor, Chemistry and Biochemistry, University of California at Los Angeles

We have developed a simple genetic selection for improved protein expression.  The selection is powerful enough to isolate host mutations that increase expression.  I will describe our progress in obtaining E. coli strains with improved abilities to produce membrane proteins.

4:45 Problem Solving Break-Out Sessions

Table 9 Expression of G Protein-Coupled Receptors for Structural Studies

Moderator: Alexei Yeliseev, Ph.D., National Institutes of Health

• o How to choose the expression strategy
  o Stabilization of the recombinant receptors in detergent micelles
  o Site-specific and uniformed labeling for spectroscopic studies

Table 10 Protein Production in Alternative Microbial Systems- Harvesting High-Hanging Fruit

Moderator: Paul Colussi, Ph.D., Tetragenics, Inc.

• o Protists (ciliated or otherwise) as expression Moderators
  o Codon Optimization in alternative expression systems
  o High-throughput screening applications for alternative expression Moderators

Table 11 Solutions for Difficult Proteins: What to Try First?

Moderator: Christopher Mehlin, Ph.D., Amgen

• o Refolding from E. coli?
  o Expression in alternate Moderator?
  o Genetic optimization?
  o Fusion partners?

Table 12 The Dirty Little Secrets of Production Cell Lines-Rearrangements and Their In-Depth Analysis

Moderator: Dayou Liu, M.S., Amgen

• o How rearrangements happened.
  o Why rearrangements are important for production cell lines
  o Methodology for rearrangement analysis
  o Assessment of impact of rearrangements

Table 13 The Quest to Increase Protein Expression: Navigating a Battlefield

Moderator: Mouna Guerfal, University of Ghent

• This discussion will focus on different approaches used to manipulate organisms in order to increase the production of heterologous proteins.
  o  Techniques as co-expression of molecular chaperones, altering expression parameters such as growth temperature, supplementing culture medium with chemicals, etc.
  o  Even though these manipulations can lead to increased expression levels, most of them cannot be used as a general technique to improve the yield of heterologous proteins. Different proteins encounter problems at different levels when expressed in heterologous host strains.
  o  What more can we do to increase the expression levels of proteins? Which organism is best for which protein? Are we capable to develop routine techniques to increase protein yield? Can the battle for efficient protein expression be won?

Table 14 Will High Through put protein production solve our problems?

Moderator: Marco Casteleijn, Ph.D., University of Oulu

• o  Introduction - yourself and "agenda"
  o  Proper Protein Folding - pilot versus parallel approaches
  o  High Yield versus Low Volume
  o  The Major bottlenecks in Protein Engineering (and Metagenomics)
  o  Future outlook - what to solve next?

5:45 Networking Cocktail Reception in the Exhibit Hall