2013 Archived Content

Expression Stream

9th Annual
Difficult to Express Proteins

Harnessing Innovation to Improve Expression and Function

April 29-30, 2013

Membrane proteins, ion channels, toxic or insoluble proteins, low abundance protein complexes, vaccines and other “finicky” proteins often are the best choices for therapeutics, yet their successful expression is a minefield of failures and challenges. This conference will present innovative and imaginative methods to make those difficult to express proteins more “expressible” and functional.

Day 1 | Day 2 | Download Brochure 



7:00am Conference Registration and Morning Coffee

Kinases and GPCRs 

8:30 Chairperson’s Opening Remarks

Stephen Bottomley, Ph.D., Research Fellow, Biochemistry and Molecular Biology, Monash University


Strategies for the Generation of Difficult to Express Recombinant Proteins

Ian HuntIan Hunt, Ph.D., Head, Protein Sciences, Center for Proteomic Chemistry, NIBR

Topics covered will include a review of the pros and cons of E. coli and baculovirus expression systems, the use of co-expression strategies to enhance expression levels and the utility of multi-parallel screening and characterization tools for the identification of well-behaved recombinant tool proteins. Case studies will be presented.

9:10 De novo Synthesis and Characterization of Functional  Kinases and GPCRs for Structural Biology

Matthew ColemanMatthew Coleman, Ph.D., Professor, Radiation Oncology, UC Davis

Our process is easily adapted to HT technologies and may prove an ideal approach for feeding the membrane structural biology pipeline. This represents a unique solution to solubility and purification problems for characterizing both function and structure for multiple difficult to express and characterize membrane proteins.

9:40 A Robust and Rapid Method of Producing Soluble, Stable, and Functional G-Protein Coupled Receptors

Karolina CorinKarolina Corin, Ph.D., Researcher, Center for Biomedical Engineering, MIT

Commercial cell-free systems can reduce or eliminate large entry barriers associated with membrane protein production.  However, selection of appropriate detergent is critical. Using Brij-35, 13 GPCRs were expressed in cell-free systems. Analyses showed that purified receptors were soluble, properly folded, and bound their ligands. This simple, robust method can facilitate the study of membrane proteins.

10:10 Grand Opening Coffee Break in Exhibit Hall with Poster Viewing

Refolding and Disulfide Bonds 

11:10 REFOLD: Providing an Insightful Way to Refold Your Protein

Stephen BottomleyStephen Bottomley, Ph.D., Research Fellow, Biochemistry and Molecular Biology, Monash University

The REFOLD database consists of over 2000 entries detailing numerous ways to refold your protein. This fully searchable database provides an immense amount of information about how to refold your protein based upon your protein's characteristics. Using REFOLD it is possible to generate a starting protocol for refolding your protein and to trouble shoot your current protocols.

11:40 Bioengineering of Coagulation Factor VIII for Efficient Expression through Elimination of a Dispensable Disulfide Loop

Randal KaufmanRandal J. Kaufman, Ph.D., Director, Degenerative Disease, Neuroscience, Aging, and Stem Cell Research Center, Burnam Medical Research  Institute

Heterologous expression of factor VIII (FVIII) is about two to three orders of magnitude lower than similarly sized proteins. Bioengineering strategies aimed at different structural and biochemical attributes of FVIII have been successful in enhancing its expression levels. Combined targeted bioengineering strategies facilitate more efficient production of recombinant FVIII and lower costs as well.

12:10 pm Maximizing Recombinant Protein Expression through Systematic Gene Design

Mark Welch, Ph.D., Director, Research & Development, DNA2.0, Inc.Advances in gene design and synthesis allow full control over features such as codon bias and mRNA structure, enabling systematic study of how gene sequence impacts expression of encoded proteins. We present studies on how gene design variables affect heterologous protein expression for a wide range of protein targets and host organisms, including mammalian, insect cells, yeasts, bacteria, etc.

12:40 Luncheon Presentations (Sponsorship Opportunities Available) or Lunch on Your Own 

Rescuing Proteins and Peptides and Improving Stability 

2:00 Chairperson’s Remarks

Alan Dombkowski, Ph.D., Assistant Professor, Division of Clinical Pharmacology and Toxicology, Pediatrics, Wayne State University School of Medicine

2:05 Improving Protein Stability and Function through Disulfide Engineering: A Computational Approach

Alan DombkowskiAlan Dombkowski, Ph.D., Wayne State University School of Medicine

Protein stability and function can be enhanced in a wide range of biomedical applications through disulfide bond engineering. Computational methods have proven to be effective for designing strategically placed disulfides. We will discuss structural considerations for successful use of this technology, recent advances in our computational method for rational disulfide design, and applications.


Human Cytomegalovirus Full-Length gB Produced in BEVS

Marco Patrone, Instituto de Biologia Experimental e Tecnológica (iBET)

Membrane Protein Expression and Modification in Eukaryotic Cell-Free Systems by Cotranslational Incorporation of Non-Canonical Building Blocks

Robert Benjamin Quast, Fraunhofer Institute of Biomedical Engineering (IBMT)

3:05 A Novel Method for the Large-Scale Production of PG-CNP37, a C-Type Natriuretic Peptide Analogue

Shinong LongShinong Long, Ph.D., Senior Scientist, BioMarin Pharmaceutical, Inc.

We have developed a novel, simple and cost effective method to produce a CNP analogue, PG-CNP37, at a large scale from E. coli. A PG-CNP37 fusion protein was over-expressed as inclusion bodies in E. coli, which were purified then cleaved by formic acid to release the PG-CNP37 peptide. 0.5 grams of 95% pure, soluble and active PG-CNP37 peptide was produced from 1 liter of culture.

3:35 Structural Biology Perspectives and Strategies for Rescuing Insoluble Protein Expression

Stephen Nakazawa HewittiStephen Nakazawa Hewitt, Ph.D., Head, Bioreactor Core Facility,  University of Washington

This study offers large-scale evidence that MBP can improve the soluble expression of previously non-expressing proteins from a variety of eukaryotic and prokaryotic organisms. While the behavior of the cleaved proteins was disappointing, further refinements in MBP tagging may permit the more widespread use of MBP-fusion proteins in crystallographic studies.

4:05 Refreshment Break in the Exhibit Hall with Poster Viewing

4:45 Problem Solving Breakout Discussions

Concurrent problem solving breakout discussions, open to all attendees, speakers, sponsors, and exhibitors, provide a forum for discussing key issues and meeting potential collaborators. Plan to take part and explore these topics in-depth. Please pick a topic of your choice, find your table and join in.

Current obstacles, approaches, and potential solutions to efficient expression of disulfide-dependent proteins

Moderator: Alan Dombkowski, Ph.D., Wayne State University School of Medicine

   •  High-yield molecular machinery
   •  Cell-free systems: a viable approach?
   •  Optimizing the secretion system

Is it worth the effort to refold your protein?

Moderator: Stephen Bottomley PhD, Senior Research Fellow, Monash University

   •  Is there a universal refolding protocol?
   •  What factors determine when you should give up trying to refold your protein?
   •  How to tell if your protein is refolded
   •  What are the best strategies for refolding your protein?

Solving solubility and purification problems in kinases

Moderator: Matthew Coleman, Ph.D., Senior Scientist, LLNL

   •  Are there better methods for production of kinases?
   •  What additive reagents and cofactors are required for solubilization and stability?
   •  Do we need to tailor purification to meet downstream applications?

5:45-6:45 Welcoming Reception in the Exhibit Hall with Poster Viewing

Day 1 | Day 2 | Download Brochure 

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