Difficult to Express Proteins
EXPRESSION STREAM May 9-10
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SUNDAY, MAY 8
4:00 - 6:00 pm Main Conference Registration
MONDAY, MAY 9
7:00 am Registration and Morning Coffee
8:30 Chairperson’s Opening Remarks
» Opening KEYNOTE Presentation
8:40 Novel Tools for the Overexpression of Transport Proteins
 Raimund Dutzler, Ph.D., Professor, Biochemistry, University of Zurich
The recent success in the structure determination of membrane proteins is tied to the possibility to characterize large numbers of homologues of a particular protein family in different expression systems to identify candidates with superior biochemical properties. I will discuss a novel tool that allows the rapid generation of expression constructs for all common pro- and eukaryotic expression systems and discuss its application to different ion transport families.
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9:10 The Importance of Characterization in Successful Protein Production
Jeff Culp, Ph.D., Associate Research Fellow, Primary Pharmacology Group, Pfizer Research and Development
It is much easier to express a protein than it is to produce a fully functional protein for use in Drug Discovery. Specific examples will be described of the importance of proper protein charaterization to eliminate potential mistakes. Guidlines will be suggested to aid the protein production specialist. Examples will include proteins intended for use in target screens, NMR, protein crystallization and biophysical characterization.
9:40 Refolding, Purification, and Characterization of the Ectodomain Complex of the CGRP Receptor
Norzehan Abdul-Menan, Ph.D., Research Fellow I, Gene Expression, Vertex Pharmaceuticals, Inc.
The calcitonin gene-related peptide receptor is a heterodimer of two membrane proteins: calcitonin receptor-like receptor (CLR) and receptor activity-modifying protein 1 (RAMP1). CLR is a B G-protein-coupled receptor, possessing a large N-terminal extracellular domain (ECD) for ligand recognition and binding. Heterodimerization of CLR with RAMP1 provides specificity for CGRP peptide. The expression, purification, and refolding of the heterodimer of the ectodomain from inclusion bodies will be presented. The refolded complex forms a stable, monodisperse complex and is competent to bind ligands.
10:10 Grand Opening Coffee Break in the Exhibit Hall with Poster Viewing
11:10 Integrated Upstream and Downstream Optimization as the Key for the Development of Complex Biologics
Michaela Wendeler, Ph.D., Scientist I, Biopharmaceutical Development, MedImmune
For complex biotherapeutics with extensive posttranslational modifications, changes in upstream process conditions can significantly impact product quality and alter requirements for downstream processing. This case study illustrates the challenges of understanding and controlling the impact of process conditions on protein attributes and demonstrates the course of integrated upstream and downstream development that led to the successful implementation of a robust manufacturing process.
11:40 Poster Spotlight Presentation: Der P7 Allergan Expression and Characterization
Lori Edwards, Biologist, Protein Expression Core Facility, Laboratory of Structural Biology, National Institute of Environmental Health Sciences (NIEHS)
Sponsored by
12:10 pm Advancing Synthetic Gene Design for Optimal Protein ExpressionMark Welch, Ph.D., Director, Gene Design, DNA2.0Advances in gene design and synthesis have enabled greater insight into the workings of the genetic code. This includes how changes in gene sequences can impact the expression of encoded proteins through mechanisms including codon bias, mRNA stability, and translation initiation. Natural gene sequences have been shaped in response to many different evolutionary pressures, but are rarely optimal for "biotechnological fitness". Here we present how gene design variables such as codon choice and mRNA structure predictably affect the yield of heterologous protein expression, often by up to orders of magnitude increase in expression levels. Host systems validated include mammalian cell lines (CHO/HEK293), yeast (S.cerevisiae/P.pastoris/K.lactis),
E.coli and more.
12:25 Sponsored Presentation (Opportunity Available)
12:40 Luncheon Presentation I
Sponsored by
High-Yield in vitro Protein Expression System for Functional Protein Synthesis using Immortalized Human Cell Lines
Penny Jensen, Ph.D., Research Scientist, Proteomics Research and Development, Thermo Fisher Scientific
Culturing of mammalian cells for the purpose of protein expression is a time consuming and expensive process. An effective alternative is cell-free expression (i.e., in vitro translation) using extracts prepared from mammalian cells. Several immortalized human cell lines, including HeLa, HuH7, and HEK293 have been used to prepare translationally competent extracts. These results along with information regarding our optimized in vitro expression system based on HeLa extracts for producing several hundred micrograms of recombinant protein per ml of reaction, expression of multiple proteins in a single reaction, and high-throughput compatibility of our system will be discussed.
1:10 Luncheon Presentation II (Sponsorship Opportunity Available) or Lunch on Your Own
1:40 Break
2:00 Chairperson’s Remarks
2:05 Expression and Purification of Membrane Protein Diacylglycerol Acyltransferase
Heping Cao, Ph.D., Principal Research Scientist, Southern Regional Research Center, US Department of Agriculture
DGAT knockout mice are resistant to diet-induced obesity and lack milk secretion. DGAT genes have been isolated from many organisms, but progress in characterization of the enzymes has been slow because DGATs are membrane-associated and difficult to express and purify. We developed a procedure for full-length DGAT expression in E. coli and yeast. This study represents the first description of a procedure for producing full-length recombinant DGAT protein from any species using an E. coli expression system.
2:35 Recent Progress in Production of Human Membrane Protein Targets and Use in Drug Discovery
Niek Dekker, Ph.D., Principal Scientist, Discovery Enabling Capabilities & Sciences, AstraZeneca R&D Molndal
Results will be presented on the expression of human ion channels in various eukaryotic expression systems. Total protein expression levels have been analyzed using Western blotting and radio-ligand binding. Target localization has been analyzed using confocal microscopy, and functional properties have been studied using electrophysiology. The combined approaches provided good insight in quality of produced targets in the various expression systems. The successful mg-scale production of a human ligand-gated ion channel will be presented including biophysical verification of ligand-binding properties using circular dichroism and isothermal titration calorimetry. Progress on crystallization of this target and ongoing engineering efforts will be presented. In addition, examples of production of other membrane proteins including GPCRs will be discussed.
3:05 Refreshment Break in Exhibit Hall with Poster Viewing
3:45 A Sensitive Fluorescent Method for Rapidly Identifying and Characterizing Lead Membrane Protein Constructs
Christopher Koth, Ph.D., Scientist, Structural Biology, Genentech, Inc.
A number of features of membrane proteins render them challenging targets for the structural biologist, among which the most important is the difficulty in obtaining sufficient quantities of properly folded and homogeneous protein. To address this, we have developed a simple, high-throughput procedure to rapidly characterize and optimize membrane protein solubility, homogeneity and aggregation state in various buffers/detergents. This method has aided in the purification of several membrane protein targets including GPCRs and ion channels.
4:15 From Clones to Crystals on a Shoestring Budget
Jian Payandeh, Ph.D., Pharmacology, University of Washington
The success of a membrane protein structural biology project may warrant a “try everything” approach, but this is seldom feasible. I will describe practical aspects to achieving high-level expression and sample homogeneity in a standard laboratory setting. Key considerations in devising a streamlined and cost-effective screen will be highlighted, and examples from our current structural biology efforts will be detailed.
4:45 Problem Solving Breakout Sessions
Concurrent Problem Solving Breakouts are interactive sessions hosted by a moderator to discuss a topic in depth. They are open to all attendees, sponsors, exhibitors, and speakers and provide a forum for discussing key issues and meeting potential partners. Please pick a topic of your choice and join in.
TABLE 8: Membrane Protein Overexpression
Moderator: Raimund Dutzler, Ph.D., Professor of Biochemistry, University of Zurich
• Why are membrane proteins so difficult to express?
• Do eukaryotic membrane proteins require eukaryotic expression hosts?
• How can we improve the stability of purified membrane proteins?
TABLE 9: Is This the Right Time to Invest In and Start the Use of Purified Membrane Proteins in Drug Discovery Projects to Complement Cell-Based Approaches?
Moderator: Niek Dekker, Ph.D., Principal Scientist, Discovery Enabling Capabilities & Sciences, AstraZeneca R&D Molndal • Expression and purification of membrane proteins
• Use of purified membrane proteins in drug discovery process
TABLE 10: Characterize Your Protein Now or Pay for It Later
Moderator: Jeffrey Culp, Ph.D., Associate Research Fellow, Primary Pharmacology Group, Pfizer Research and Development
• What potential and undesirable modifications could occur on your protein?
• How do I detect heterogeneity in my protein?
• How do I eliminate or remove undesired heterogeneity?
TABLE 11: Membrane Protein Purification
Moderator: Heping Cao, Ph.D., Principal Research Scientist, Southern Research Center, USDA
• Why most membrane proteins are expressed as insoluble proteins?
• How to select detergents for membrane protein solubilization?
• What steps to purify membrane proteins from detergent-solubilized solutions?
TABLE 12: Finding the Right Tools for Recombinant Protein Production
Moderator: Ling Yuan, Ph.D., Researcher, Plant and Soil Sciences, University of Kentucky
• What is the ideal tool for the production of my protein?
• What to do when my protein is toxic to the host cells?
• How to modify proteins for desired functions?
TABLE 13: Topic to be Announced
Moderator: Takanori Kigawa, Ph.D., Team Leader, Protein Preparation Screening Team, RIKEN
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5:45 - 6:45 Reception in the Exhibit Hall with Poster Viewing
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