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Although most scientists would argue that protein expression science is mature, challenges still remain. New expression methods and technologies continue to provide improved protein yield, stability, and structure. Cambridge Healthtech Institute’s 13th Annual “Characterization, Purification & Screening” (formerly “Protein Expression”) conference will explore the successful methods and technologies currently being used, or in development, to provide the quantity and quality of proteins necessary for upstream research and drug development. The 2010 Meeting continues the strong tradition of addressing research and development throughout the protein expression pipeline from initial steps, to efficient expression system design. Case studies will shed light on the manufacture of modified gene cells as well as cell clone characterization and purification of proteins from high producers. In its 13th year, Protein Expression, the backbone for PepTalk, provides the foundation for other therapeutic production pipelines.
Recommended Pre-Conference Short Course*
Sunday, January 10
(SC4) Specialized Protein Expression Systems - An Overview
* Separate registration required
Tuesday, January 12
Arrive early and join BuzZ Session Roundtable Discussions, Poster Awards and Short Courses!
2:45 Networking Refreshment Break in the Exhibit Hall
3:15 Poster Awards in the Exhibit Hall
4:15 Close of Optimizing Biologics Formulation Development
4:15 Registration for Short Courses
4:30 – 7:30 Concurrent Short Courses* (SC 5 – SC 8)
Recommended Short Course
(SC7) From Protein and Peptide Characterization to Quantitation: Advancing Mass Spec Beyond Protein Discovery
Wednesday, January 13
7:00 am Registration for Characterization, Purification
7:30 Breakfast Presentation or Morning Coffee
8:15 Chairperson’s Opening Remarks
8:20 KEYNOTE PRESENTATION
Ambient Electrospray-Assisted Laser Desorption Ionization (ELDI) Mass Spectrometry for Biomolecule Characterization
Joseph A. Loo, Ph.D., Professor, Department of Chemistry and Biochemistry, University of California, Los Angeles
ELDI is a soft, ambient ionization method for mass spectrometry and combines features of both electrospray ionization (ESI) and UV/IR matrix-assisted laser desorption ionization (MALDI) to generate multiply charged molecules from dried or wet samples deposited on a sample plate. ELDI is amenable for rapid MS-based top-down and bottom-up characterization of large proteins. ELDI allows on-line chemical reactions to occur during the analyte-droplet mixing process and provides a potentially convenient platform for a variety of applications, including tissue imaging and ligand screening.
9:00 Synthesis of Functional GPCRs Supported in a Nanolipoprotein Disc
Matthew Coleman, Ph.D., Associate Professor, Radiation Oncology, University of California, Davis
We have developed a new biotechnology application leading to formation of nanolipoprotein particles (NLPs) capable of solubilizing GPCR membrane proteins during cell-free synthesis. This talk will demonstrate our cell-free approach for screening and characterizing different classes of GPCR proteins using NLPs. Furthermore, we demonstrate the utility of fluorescent correlation spectroscopy, which can provide functional information on both qualitative and quantitative assessment of membrane proteins never before obtained using other biophysical techniques. This represents a unique solution to solubility, purification, and characterization associated with membrane proteins.
9:30 Achieving the Crystal Structure of Cholesteryl Ester Transfer Protein
Jeff Culp, Associate Research Fellow, Primary Pharmacology Group, Pfizer Global Research & Development
Plasma Cholesteryl ester transfer protein (CETP) has 476 amino acids and a 74 kDa molecular mass, of which 28% by mass is attributed to glycosylation at four sites. The high content of glycosylation, hydrophobic residues (44%) and free cysteines (5) presented significant challenges for structural studies. A robust expression system was created and a high yield purification protocol was developed. Thorough characterization of the expressed CETP facilitated the ability to design new constructs to control glycosylation and free cysteines. The first structure of CETP was solved at 3.5A using crystals of a CETP triple mutant in complex with an antibody Fab fragment. Another round of design and production led to a fully active quadruple mutant yielding a structure at 2.2A resolution without Fab. The structure reveals an elongated “boomerang” shape that forms a continuous tunnel occupied by cholesteryl esters and phosphatidylcholine and provides insights into the mechanism of action of CETP.
10:00 Networking Coffee Break in the Exhibit Hall
10:45 Using GFP Screens to Identify Well-Expressed Membrane Proteins
Daniel L. Minor, Jr., Ph.D., Associate Professor, California Institute for Quantitative Biosciences, University of California, San Francisco
Identification of well-expressed, properly behaved samples remains a bottleneck for membrane protein crystallography. To overcome this limitation, we have been developing a GFP-fusion expression system as a tool for facile construct evaluation. We implemented this assay to screen over 300 membrane proteins from eighteen bacterial and archaeal extremophilic microorganisms. The results demonstrate that the GFP assay accurately predicts over-expression potential and that coupling the assay with expression optimization using a panel of non-GFP constructs yields multi-milligram levels of membrane proteins.
11:15 The Use of Blue Native PAGE in the Evaluation of Membrane Protein Aggregation States for Crystallization
Jichun Ma, Ph.D., Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health
Blue native PAGE (BN-PAGE) was used as a tool to evaluate the protein samples for crystallization. There was a significant correlation between the monodispersity measured by BN–PAGE and the propensity for crystallization of a number of soluble and membrane protein complexes. Moreover, the oligomeric states of proteins as measured by BN–PAGE were matching those obtained from their crystalline forms. This method was proven to be useful when applied to a membrane protein with unknown structure for the screens of various constructs and detergents.
11:45 Purification and Characterization of Membrane Proteins using Tetra Detection
Larry Miercke, Ph.D., Specialist, Biochemistry, University of California, San Francisco
Size Exclusion Chromatography and a Tetra Detector Array (absorbance, refractometer, viscometer and light scattering detectors) are being successfully utilized to monitor and optimize detergent concentration while assaying the Protein Detergent Complex homogeneity during purification and concentration for crystallization. In doing so, the oligomeric state, size, shape and the detergent:protein ratio of the PDC are measured. A variety of different membrane proteins and detergent systems will be presented along with lessons learned in TDA data acquisition and analysis and where improvements can be made.
12:15 pm Close of Morning Session
12:30 Luncheon Presentation
or Lunch on Your Own
1:00 Luncheon Presentation or Lunch on Your Own
2:00 Chairperson’s Remarks
2:05 Expression and Purification of Human G-Protein Coupled Receptors
Mulugeta Mamo, Ph.D., Department of Structural Biology, Abbott Laboratories
Often in industry, we are asked to prepare target membrane proteins within a short time frame. Without many precedents of over-expression of such membrane proteins found in the literature, we have developed different approaches to prepare large amounts in a timely fashion. We present an example of a human type II G-Protein coupled receptor (glucagon-like peptide type 1 receptor) to demonstrate the approach to screen for over-expression in insect cells and the purification in a variety of solution conditions. The selection for expression and purification conditions is based on the protein yield, specific activity, physical heterogeneity, and thermal stability.
2:35 A Reversible Immunocapture Method for Comprehensive SPR Analysis of Antibody-Antigen Interactions
John Kulman, Ph.D., Assistant Member, Puget Sound Blood Center
The applicability of SPR for the evaluation of protein-protein interactions is often limited by the time and expense involved in generating high-quality ligands. To circumvent these limitations, we have developed a method for the reversible on-chip purification of ligands from the medium of transiently transfected mammalian cells. We demonstrate the utility of this approach for determining kinetic and thermodynamic parameters governing antibody-antigen interactions with unsurpassed precision and for a mere fraction of the cost and effort of conventional approaches. Techniques for significant throughput enhancement will be presented, including single-cycle kinetics and interaction array SPR imaging.
3:05 Complementing SPR with SEC in Rational Protein Design
Benjamin J. McFarland, Ph.D., Associate Professor, Department of Chemistry and Biochemistry, Seattle Pacific University
Using RosettaDesign, we rationally designed MICA proteins and assessed affinity for the immunoreceptor NKG2D using surface plasmon resonance (SPR) and size-exclusion chromatography (SEC). MICA mutants exhibited enhanced multiphasic binding kinetics: different on-rates but similar off-rates. SEC “persistence time” discriminated enhanced-affinity/fast-on from low-affinity/slow-on complexes, and was tuned with urea. Unbound MICA retention times correlated with expected protein dynamics, so that SEC analysis provided useful assessment of complex stability. Currently we are combining several RosettaDesigned surface mutations that individually enhance affinity by >1 kcal/mol.
3:35 Elucidating the Mechanism of Action of Therapeutic Antibodies using Surface Plasmon Resonance
Marina Roell, Ph.D., Associate Director of the Molecular Interactions & Biophysics Group, Xoma
Characterization of both target protein and antibody mechanism of action (MOA) can facilitate many phases of therapeutic antibody discovery and development, impacting the strategy used for early stage discovery, in vivo proof-of-concept studies, toxicological assessment, clinical trial design and commercial development. Antibody MOA analysis often starts with the development of methods to monitor target activation such as receptor ligand binding and assembly of an activated signaling complex.
3:50 Networking Refreshment Break
4:30 Amphiphiles and Ligands for Structural Investigation of Membrane Proteins
Qinghai Zhang, Ph.D., Assistant Professor, Molecular Biology, Scripps Institute
High resolution structural determination of integral membrane proteins is challenging. The protein itself, the environment, and bound ligands (in some cases) are the major determinants for the stability of a membrane protein. While careful optimization of protein constructs and screening homologue proteins are common practices, chemistry efforts to address specific ligand requirement and other environmental factors, detergents and lipids in particular, lag in membrane protein structural projects. We will present our study on new chemical tool development for this challenging field.
5:00 GPCRs in Non-Native Environments
Peter Nollert, Ph.D., Director, Emerald BioSystems
We have assessed the properties of G-protein coupled receptors (GPCRs) in non-membrane environments and their utility for characterization, purification and crystallization screening. The stability of receptors that are not located in their native membrane can be enhanced dramatically by carefully adjusting the physical chemical properties of the artificial membrane mimetic. We show how a carefully custom tailored micro-environment aids in the stabilization of GPCRs.
5:30 Reception in the Exhibit Hall
6:30 Close of Day
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