蛋白质发现会议 - Day 1

概要 | 短期研讨会 | Day 1 | Day 2 | Day 3

议程(PDF : English)

Monday, January 7

7:30am-6:00pm Registration Open

7:30 Morning Coffee

8:45 Chairperson乫s Opening Remarks
Ajith Kamath, Ph.D., Jubilant BioSys

KEYNOTE PRESENTION

9:00 Antibody Mimetics Derived from Non-HumanMicroprotein Scaffolds: Optimization for Efficacy, Safety and Manufacturability Willem 乬Pim乭 Stemmer, Ph.D., CEO and Founder, Amunix Inc. Amunix has created a platform for the design of antibody mimetics. Since different targets have different requirements, format flexibility is critical. We use binding modules, spacer modules and cross linker modules to create a variety of product formats that optimally fit each target乫s needs. The binding modules are microprotein domains of 20-35AA, which are linked together with spacer modules, unstructured sequences which provide valency and, if long, half life. Unstructured sequences have an increased hydrodynamic radius and show an appar-ent molecular weight that is about 10-fold their actual molecular weight, mimicking the way PEGylation achieves a long serum secretion half life. Our proteins are typically 5-30kD and are expressed at high levels in soluble form in the E. coli cytoplasm. The high disulfide density of microproteins makes our proteins exceptional stable, enabling the use of differential heat-denaturation/precipitation as a one-step purification process. Because of the low sequence complexity of both modules, we can generally reserve specific amino acids, such as Lysines, for exhaustive and efficient chemical conjugation to identical or other proteins, creating a multimer with increased valency, half life and/or effector functions. To minimize the possibility of an immune response which cross reacts with the native human protein, we use non-human microproteins. Using predictive software as well as screening assays, we engineer these domains to minimize their immunogenicity. The resulting freedom to completely change the product乫s amino acid sequence allows us to optimize products for all the desired properties, such as stability, expression, formulation, etc. Finally, the product format must offer optimal patentability, resulting in strong product claims, and optimal freedom of operate, being outside of the antibody-target claims and not covered by existing product format or methods patents. Several product formats aimed at diverse disease targets will be described, combining many desired features.

KEYNOTE PRESENTION

9:50 Engineering Yeast to Produce Protains for Biochemistry and X-ray Crystallography Elizabeth J. Grayhack, Ph.D., Research Associate Professor of Biochemistry and Biophysics, University of Rochester We are developing methods to enhance expression and purification of eukaryotic proteins in the yeast Saccharomyces cerevisiae for biochemical and structural analysis. Yeast is potentially an ideal host from which to obtain high levels of purified proteins based on our observation that over a third of a genomic collection of 5,854 ORF-expressing yeast strains produce ≥ 2 mg of fusion protein per liter and purification effected by affinity tags results in highly purified proteins. To expand the utility of yeast as a source of proteins for x-ray crystallography, we developed a method to obtain protein suitable for anomalous dispersion phasing by resolving the longstanding difficulty with extensive replacement of methionine by selenomethionine in yeast. We developed a general method to incorporate selenome-thionine into proteins expressed in yeast based on genetic manipulation of the appropriate metabolic pathways. Our additional efforts to develop yeast as an expression system include the examination of expression of foreign proteins that are themselves insoluble when expressed in E. coli, the development of vectors for co-expression of protein complexes and an investigation of how manipulation of codon usage improves protein expression.

10:20 Coffee Break

10:45 Vector, Auto-Induction, and Automated Purification Screening of Protein Expression
Brian Fox, Ph.D., Professor, Biochemistry, University of Wisconsin
I will discuss recent developments from the NIH Protein Structure Initiative-funded Center for Eukaryotic Structural Genomic on an expression vector platform that supports bacterial, cell-free, and insect cell expression, provides reliable auto-induction of bacterial expression, and automated small scale protein purification. The system is a bench-top cost effective method to characterization of proteins for structural or functional work.

11:15 The Importance of Post-Transcriptional Steps in the Production of Recombinant Antibody from CHO Cells
James Rance, Ph.D., Senior Scientist, Cell Culture Process Development, Lonza Biologics, plc
High levels of antibody expression result from a combination of many factors, ranging from the choice of expression system to the media used for cell culture fermentation. Lonza Biologics乫 Glutamine Synthetase (GS) Gene Expression System™ has been frequently used to generate cell lines capable of high-level antibody production in fermentation processes. It is possible that recombinant antibody productivity can be increased further by modifying expression vector components that affect transcription and/or downstream steps such as translation, assembly or secretion. For example, one way in which transcription was studied was by replacing the existing promoter in the expression vector with a potentially stronger one. The effects of altering translation efficiency (via gene optimization) and the signal sequence were also looked at. In this talk, the effect of these modifications and their importance in creating highly productive recombinant antibody-producing cell lines will be discussed.

11:45 Cell-free Production of Post-Translationally Modified Virus-like Particles and Disulfide Bonded Proteins
James Swartz, Ph.D., Department of Chemical Engineering, Stanford University
Previously announced advances enable the economical and scalable cell-free production of proteins by activating E.coli-based combined transcription and translation reactions as well as central metabolism and oxidative phosphorylation to supply this energy intensive process. We now show that we can efficiently produce fully assembled virus-like particles, can site-specifically incorporate uniquely reactive, non-natural amino acids, and can attach a variety of ligands. Similarly, we show that a variety of bioactive disulfide bonded proteins can be produced and post-translationally modified. This technology now opens an exciting potential for the production of customized vaccines and unique pharmaceuticals.

12:15pm Close of Morning Session

12:30 Luncheon Technology Workshop Speaker and Talk Title To Be Announced

Sponsored by

2:30 Chairperson乫s Remarks
Walid Qoronfleh, Ph.D., Q3 Consulting LLC.

2:35 Getting the Most out of Inclusion Bodies - A Beginner乫s Guide to Refolding
Stephen Bottomley, Ph.D., Senior Research Fellow, Biochemistry, Monash University
The refolding of recombinant proteins from inclusion bodies represents a bottleneck in the purification of proteins. We have developed the database REFOLD (http://refold.med.monash.edu.au), which is a freely available, open repository for protocols describing the refolding and purification of recombinant proteins. Data in REFOLD is readily accessible using a simple search function, and the database also enables analyses which identify and highlight particular trends between suitable refolding and purification conditions and specific protein properties. This information in turn serves to facilitate the rational design and development of new refolding protocols for novel proteins. In our latest release, due in October, we will have the protocols for over 2000 proteins listed in REFOLD and a number of new predictive functions will be incorporated.

3:05 Variability in the Immunodetection of His-tagged Recombinant Proteins
Arthur Sytkowski, Ph.D., Associate Professor of Medicine, Medicine/Hematology-Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School
Labeling of recombinant proteins with polypeptide fusion partners, or affinity tagging, is a useful method to facilitate protein detection and purification. We report strikingly variable immunodetection of two His-tagged recombinant human erythropoietins (Epo), wild type Epo (Epowt) and Epo containing an R103A mutation (EpoR103A), both of which were engineered to contain a C-terminal six residue His-tag. While the successful in-corporation of the His-tag into our constructs was confirmed by Epo binding to Ni2+-NTA resin and by LC/MS/MS amino acid sequencing, the levels of immunodetection of His-tagged protein varied markedly depending on the particular anti His-tag antibody used. Such variability in His-tag immunorecognition can lead to critical adverse effects on several analytical methods widely used in protein expression technology.

3:35 Large-scale Transfection of 293 and CHO Cells
Yves Durocher, Ph.D., Project Leader, Animal Cell Technology Group, Biotechnology Research Institute, National Research Council Canada
Large-scale transfection of 293 and more recently CHO cells is a powerful method for the very fast production of mg to gram quantities of recombinant proteins. The cationic polymer polyethylenimine (PEI) is very popular for this application due to its efficacy and cost-effectiveness. Even though significant improvements in r-protein titers have been achieved over the last few years, the mechanisms responsible for an efficient and productive transfection are still poorly understood. We have explored the fate of PEI-DNA complexes from binding to the cell surface down to their final intracellular destination. A better understanding of PEI-mediated transfection mechanisms and bottlenecks will certainly allow further improvements in this technology.

4:05 Refreshment Break

4:30 Remedial Strategies in Structural Proteomics: Expression, Purification and Crystallization of the Vav1/Rac1 Complex
Alexei Brooun, Ph.D., Principal Scientist, Structural and Computational Biology, Pfizer, Inc.
The signal transduction pathway involving the Vav1 guanine nucleotide exchange factor (GEF) and the Rac1 GTPase plays several key roles in the immune response mediated by the T cell receptor. Vav1 is also a unique member of the GEF family in that it contains a cysteine-rich segment (CRD) that is critical for Rac1 binding and maximal guanine nucleotide exchange activity, and thus may provide a unique protein-protein interface com-pared to other GEF/GTPase pairs. Here we have applied a number of remedial structural proteomics strategies, such as construct and expression optimization, surface mutagenesis, limited proteolysis, and protein formulation to successfully express, purify, and crystallize the Vav1-DH-PH-CRD/Rac1 complex in an active conformation. We have also systematically characterized various Vav1 domains in a GEF assay, and Rac1 in vitro binding experiments. In the context of Vav1-DH-PH-CRD, the zinc finger motif of the CRD is required for the expression of stable Vav1, as well as for activity in both a GEF assay and in vitro formation of a Vav1/Rac1 complex suitable for biophysical and structural characterization. Our data also indicate that the isolated CRD maintains a low level of specific binding to Rac1, appears to be folded based on 1D-NMR analysis and coordinates two zinc ions based on ICP-MS analysis. The protein reagents generated here are essential tools for the determination of a three dimensional Vav1/Rac1 complex crystal structure and possibly for the identification of inhibitors of the Vav1/Rac1 protein-protein inter-action with potential to inhibit lymphocyte activation

5:00 Microfluidic Devices for Protein Refolding
Brian J. Kirby, Ph.D., College of Engineering, Cornell University

5:30 Welcoming Reception in the Exhibit Hall

7:00 Close of Day