EXPRESSION STREAM

7th Annual

Difficult to Express Proteins April 30 - May 1

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 cooperative and to increase yield and performance.

Day 1 | Day 2

SUNDAY, APRIL 29

4:00 - 6:00 pm Main Conference Registration

MONDAY, APRIL 30

7:00 am Registration and Morning Coffee

SOLVING MEMBRANE PROTEIN PROBLEMS

8:30 Chairperson's Opening Remarks

9:10 Peptide Surfactants for Cell-Free Production of Functional G Protein-Coupled Receptors

Shuguang Zhang, Ph.D., Associate Director, Center for Biomedical Engineering, Massachusetts Institute of Technology

We report using peptide surfactants in commercial E. coli cell-free systems to rapidly produce milligram quantities of soluble G protein-coupled receptors (GPCRs). The GPCRs expressed in the presence of the peptide surfactants were soluble and had α-helical secondary structures, suggesting that they were properly folded. These short and simple peptide surfactants may be able to facilitate the rapid production of GPCRs, or even other membrane proteins, for structure and function studies.

9:40 Expression of the Transmembrane Domain of the Human APP Binding Protein LR11 for �gin Situ�h NMR Structural Analysis

Fang Tian, Ph.D., Assistant Professor, Department of Biochemistry and Molecular Biology, College of Medicine, Pennsylvania State University

Information about protein structure in biological environment is scarce.To date, most membrane structure determinations have been carried out in detergent preparations and synthetic lipid bilayers. Using a new MBP expression vector, we successfully expressed the transmembrane domain of the human APP binding protein LR11 at high yields for a direct structural characterization in native Escherichia coli membranes.

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

11:10 Engineering Membrane Proteins for Better Expression in E. coli

Morten Nørholm, Ph.D., Senior Scientist, Novo Nordisk Center for Biosustainability, Technical University of Denmark

In this study we show that fusion of an N-terminal peptide to a poorly-expressed membrane protein of E. coli or human origin, can significantly improve over-expression levels. Further, we could mimic the effect of the N-terminal peptide by re-engineered the 5' mRNA with favorable synonymous mutations. These simple changes significantly improve over-expression of the native protein and provide a design principle for engineering better expressing membrane proteins.

11:40 Crystallization Chaperone Strategies for Membrane Proteins

Jennifer A. Maynard, Ph.D., Assistant Professor, Chemical Engineering, University of Texas Austin

From G protein-coupled receptors to ion channels, membrane proteins represent over half of known drug targets, yet structure-based drug discovery is hampered by the lack of available three-dimensional models. Here, we present a novel, generic solution: development of a toolbox of engineered antibodies recognizing short peptides to chaperone crystallization of membrane proteins presenting the peptide ligand in a permissive loop.

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

Claes Gustafsson, Ph.D., Chief Operating Officer, DNA2.0, Inc.

Advances in gene design and synthesis have enabled greater insight into the workings of the genetic code. Full control over features such as codon bias and mRNA structure allows 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, yeasts, bacteria, etc. We show predictive relationships between gene sequence features and expression that provide the basis for gene design algorithms that far outperform previous methods.

12:25 Sponsored Presentation (Opportunity Available)

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

NOVEL HOSTS AND PLATFORMS

2:00 Chairperson's Remarks

2:05 Retention of Thrombin Inhibitory Activity by Recombinant Serpins Expressed as Integral Membrane Proteins Tethered to the Surface of Mammalian Cells

William P. Sheffield, Ph.D., Professor, Departments of Pathology and Molecular Medicine, McMaster University

Both TR- and AR-α(1) PI M358R were enriched in the integral membrane fraction of transfected COS-1 or HEK 293 cells, and formed inhibitory complexes with thrombin, although less rapidly than soluble α(1) PI M358R.Two of three thrombin-inhibitory serpins retained functionality when expressed as integral membrane proteins. Our findings could be applied to create and screen hypervariable serpin libraries expressed in mammalian cells, or to confer protease resistance on engineered cells in vivo.

2:35 A Novel Expression and Purification Platform for the Production of Soluble Human Lysozyme in E. coli

John Lamppa, Chemical and Biomolecular Engineering, Thayer School of Engineering, Dartmouth College

Pre-clinical assessment of novel lysozyme variants requires a robust, efficient, and scalable expression system. E. coli is accessible, efficient and a scalable platform, but expression of soluble lysozyme is toxic to these cells. To capitalize on the numerous benefits of this bacterial host, we have developed an anti-toxin co-expression system that yields a 1000-fold increase in soluble lysozyme relative to prior reports.

3:05 Daedalus: A Robust, Turnkey Platform for Rapid Production of Decigram Quantities of Active Recombinant Proteins in Human Cell Lines Using Novel Lentiviral Vectors

Ashok Bandaranayake, Ph.D., Center for Immunity and Immunotherapies, Seattle Children's Hospital Research Institute

We describe a novel system for the rapid production of recombinant mam-malian proteins, including immune receptors, cytokines and antibodies, in a human cell line culture system. The inclusion of minimized ubiquitous chromatin opening elements in the transduction vectors is key for preventing genomic silencing and maintaining the stability of decigram levels of expression.

3:35 Talk Title to be Announced, Pfenex End User

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

4:45 Problem Solving Breakout Discussions

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

Day 1 | Day 2