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Despite decades of research and advances, some areas of protein science remain extremely challenging and complex.  Antibodies, vaccines, human proteins, and other difficult-to-express proteins have fueled new research and expression methodologies and technologies.  High throughput purification and tags promise great rewards, but still pose important questions for researchers.  Cell free expression methods hold great potential, but pose new challenges.  This meeting addresses the newest developments in these protein science fields and provides insights and solutions for conquering these issues.

1:00 pm Conference Registration

Optimizing Expression

1:45 Chairperson’s Opening Remarks

1:50 Performance by Design: Engineering Functionality into Biopharmaceutical Products

Susan Dana Jones, Ph.D., Vice President and Senior Consultant, BioProcess Technology Consultants, Inc.

Most cell line development technologies focus on obtaining high titers quickly with little to no attention paid to post-translational modifications and potential product heterogeneity. A more effective approach would be to use cells engineered to produce the desired post-translational modifications as well as achieve high titers. This “Performance by Design” truly reflects the goals of Quality by Design by enabling a rational approach to the production of product with desired quality attributes.

2:20 Engineering Type III Secretion in Salmonella

Daniel Widmaier, Researcher, Voigt Lab, Chemistry and Chemical Biology Graduate Program, University of California, San Francisco

The Type III Secretion System (T3SS) of Salmonella Typhimurium is a unique secretion system in eubacteria capable of exporting proteins through both membranes to the extracellular environment. Genetic circuits are presented to control the timing and magnitude of expression and secretion of heterologous protein through the T3SS. Specifically, the spider silk family of fibroin proteins are used to probe export ability and the physiochemical constraints of secreted proteins.

2:50 Theory and Application of Solubility-Enhancement Tags in Biomolecular NMR

Pei Zhou, Ph.D., Associate Professor of Biochemistry, Duke University Medical Center

The application of solubility-enhancement tags (SETs) has been highly effective in overcoming solubility and sample stability issues and has enabled structural studies of important biological systems previously deemed unapproachable by solution NMR techniques. Here, we reflect the motivation behind SETs and give a theoretical analysis of the solubility enhancement effect. Additionally, we briefly survey recent development and successful applications of the SET strategy in biomolecular NMR.

Sponsored by
3:20 Pichia Pastoris Expression System: Taking it to the Limit
Roland Weis, Head of R&D, VTU Technology GmbH
VTU Technology employs its proprietary AOX1-promoter library for the high-level expression of a variety of proteins in Pichia pastoris. Reliable microscale cultivation and screening enables a weekly throughput above 20.000 clones. VTU-AOX1 promoters with increased expression profile on methanol as compared to WT-AOX1 promoter give rise to ultra-high yields of intact secreted target proteins, as e.g. 18g/L for HSA and up to 9g/L of HSA-fusion proteins. Novel 2nd generation VTU-AOX1-promoter variants enable methanol-free, glycerol-driven expression of many target proteins to often g/L levels, outperforming GAP-driven expression by far.

3:35 Refreshment Break, Exhibit and Poster Viewing

4:30 The Use of SUMO-Tag to Improve Protein Expression and Factors Affecting Its Removal

Bingyuan Wu, Ph.D., Research Scientist, Biologics Research, Centocor R&D

The use of SUMO-tag has the potential of increasing protein solubility and heterologous expression. In addition, the tag can be removed specifically by SUMO-protease, which recognizes the three dimensional structure of the SUMO protein rather than a linear sequence of a few amino acids as other proteases do. However, the efficiency of the tag removal could be affected by the structure of the target protein and the digestion condition. Here the optimization of the digestion for one SUMO-tagged fusion protein will be presented.

5:00 Efficient Tag Combinations for Tandem Affinity Purification

Yifeng Li, Ph.D., Technical Director, Protein Production Core Facility University of Texas Health Science Center, San Antonio

TAP (tandem affinity purification) allows rapid and clean isolation of tagged protein along with its interacting partners from cell lysates. The key of TAP is the use of a dual affinity tag, which is fused to the protein of interest. As the technique has been widely exploited, a number of TAP tags based on various affinity handles have been developed. This presentation will give an overview of some practically used tag combinations with a highlight on those that result in higher protein recovery.

5:30 A Rational Design of 5’ UTR for the Optimization of Protein Expression in Prokaryotes

Sunjae Lee, Researcher, Bio and Brain Engineering, KAIST

To control the protein expression in prokaryotes, there has been considerable attention in the transcriptional element; however, the translational element, which is located at 5’ UTR, could hamper the optimization of protein expression. Furthermore, secondary structure of mRNA makes it difficult to identify consensus sequence for optimal expression, and therefore methodology for rational design of 5’ UTR becomes indispensable.

6:00 Close of Day

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Links to Companion Meetings

Targeting Genes, Engineering Vectors, Designing Constructs and Optimizing Clones 

Choosing, Designing, and Optimizing Hosts and Platforms 

Membrane Proteins