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WEDNESDAY, JANUARY 14
7:00am – 5:30pm Registration
7:30 Breakfast Workshop (Sponsorship Available)
PROTEIN EXPRESSION WITH A DOWNSTREAM FOCUS
蛋白质表现与下游之聚焦
8:15 Chairperson’s Remarks
Dennis M. Kraichely, Ph.D., Centocor R & D, Inc.
8:20 Codon Harmonization Leads to Enhanced Heterologous Protein Expression
由密码子之调和化促进异种蛋白质表现
Evelina Angov, Ph.D., Chief, Laboratory Molecular Parasitology USMMVP-WRAIR
For Plasmodium falciparum, codon harmonization has yielded dramatic improvements in plasmid stability, overall expression levels and soluble protein yield (indicator of correct folding) compared to expression by the non-recoded native genes in E. coli. Since adjustments to relative codon usage improve the reliability of functional protein expression, this approach may represent a paradigm shift for heterologous protein expression, with important consequences for both structural biology and biotechnology.
8:50 Transgenic Plant Systems for the Production of Structural Proteins
构造蛋白质生成用植物系转换
Julio A. Báez, Ph.D., Senior Research Fellow, FibroGen USA
Plant-based transgenic systems could offer a cost effective system to deliver thousands of metric tons per year by producing recombinant structural proteins as by-products of biorefining. Data describing the expression of recombinant collagen-related proteins in transgenic corn and barley seeds will be discussed indicating the feasibility of using these plant-based transgenic production systems for making animal-component free structural proteins with consistent composition and structure.
9:20 Automated Small-Scale Transfections to Scale Up Lead Molecule
因小规模转换自动化产生lead分子规模扩大
Matthew Husovsky, Senior Associate Scientist, Protein Sciences, Centocor, A Johnson & Johnson Company
We have a rapid system that can go from screening clones to characterizing lead molecules in less than 4 months. By automating 96-well DNA production, along with multi-well transfections, we have the potential to screen thousands of variants per week. We are working with a robust transient transfection system that allows us to express and purify up to 40 lead candidates in the 5mg scale in one-week. We are using large-scale transient CHO transfections to characterize 2-8 lead molecules, and generating 50mg of each protein prior to NME declaration.
9:50 Coffee Break in the Exhibit Hall
10:45 Post-Transformational Vector Amplification in the Yeast Pichia pastoris
酵母形质转换后带菌量增加
James M. Cregg, Ph.D., Keck Graduate Institute of Applied Life Sciences
11:15 Development of a Protease Production Platform for Structure-Based Drug Design
基于构造的药剂设计用蛋白酶生成平台开发
Edward Fox, Ph.D., Vertex Pharmaceuticals
11:45 Panel Discussion: Looking Downstream Early to Prevent Problems Later
小组讨论：早期注意下游以避免后续问题
12:15 Close of Morning Session
12:30 Luncheon Workshop Sponsored by 
Difficult to Express Proteins: Crown Bioscience Strategy
蛋白质表现之困难度:最高水准的生物科学策略
Faming Zhang, CTO Crown Biosciences, Adjunct Professor University of Indiana
CASE STUDIES
案例学习
1:45 Chairperson’s Remarks
Dennis M. Kraichely, Ph.D., Centocor R & D, Inc.
1:50 Expression and Purification of the Functional
Domain of DKK2 in E. coli
大肠菌的DKK2机能领域之发现与精制
Jie Zheng, Ph.D., Associate Member, Structural Biology, St. Jude Children's Research Hospital
Dickkopf (Dkk) proteins inhibit the canonical Wnt signaling pathway. In this study, we developed a protocol to express the C-terminal domain in E. coli and to purify it through chromatography. We demonstrated that this protocol allows us to effectively generate s highly purified and fully activated protein for structural biology studies. Therefore, this protocol represents an important advancement that will enable more efficient research into the structural and functional properties of DKK proteins.
2:20 Large-Scale Biopharmaceutical Production without the Constraints of Living Cells
无生体细胞限制之大规模生物医药品制造
James Swartz, D.Sc., Professor, Chemical Engineering and Bioengineering, Stanford University; Sutro Biopharma
Sutro Biopharma's cell-free production technology previously offered low cost and robust protein folding, even for proteins that are difficult to produce using conventional in vivo technologies. Now, for the first time, we announce the ability to scale-up this powerful and economical system for commercial biopharmaceutical production. We will show how conventional bioprocessing equipment can be used to produce pharmaceutical quality GMCSF at the 100-liter scale, a development that many biotechnologists felt was impossible.
2:50 Technology Watch
REEMAC: An Platform for Recombinant Protein Production in Cultured Mammalian Cells
REEMAC：培养哺乳类细胞之重组蛋白质生成平台
Qiang Li, Ph.D., Chief Scientific Officer, ATGCell, ATGCell Inc.
We developed an exogenous expression platform REEMAC to quickly and efficiently produce recombinant proteins from cultured mammalian cells by creating a set of new expression vectors and utilization of our proprietary ExpressBoost reagents. In this platform, we examined a variety of expression elements and optimized the entire expression cassette structure. We also formulated a set of chemical cocktails for further expression enhancement. We have applied REEMAC to express over 100 target proteins, including cytokines, growth factors, hormones, cell surface antigen receptors, proteases and monoclonal antibodies in CHO and HEK cells.
3:05 Consistent Development of Stable-High Expressing Mammalian Cell Lines in Four Months Using GPEx®
4个月以来利用GPEx®的哺乳类细胞株之安定且高发现性：稳定确实之进展
Greg Bleck, Ph.D., Senior Director Cell Line Engineering, Gala Biotech, A Catalent Pharma Solutions Company
The GPEx® method of cell line engineering generates high-expressing, genetically stable cells for all mammalian cell types. Antibiotic selection is not needed as part of the procedure, so multiple gene constructs can be added individually, at different gene ratios without any requirement for antibiotic resistance markers. Specific productivities of GPEx® antibody producing cell lines range from 30 -70 picograms/cell/day without upstream process development, while cell lines producing non-antibody proteins as expected have a much broader range of productivities, but if the protein has no secretion problems and does not inhibit cell growth, specific productivities similar to antibodies are obtained.
3:20 Technology Spotlight Sponsored by 
Optimization of Gene Design for Heterologous Expression
用于异种发现之遗传因子设计最佳化
Mark Welch, Ph.D., Senior Scientist, DNA 2.0
Optimization of heterologous expression systems is critical for efficient production of high value protein products and can be essential for generating sufficient material for structure based drug design and functional studies. While much work has been done in expression vector and host strain development, surprisingly little has until now been accomplished in the area of gene sequence optimization. With support from NSF and multiple collaborations across the biotechnology industry, DNA2.0 has actively studied the relationship of gene design to heterologous expression yield. Using its gene synthesis capacity, DNA2.0 has created large systematically varied gene sets for multiple gene types and analyzed expression in several commonly utilized host systems. Striking differences are observed for the dependence of expression on synonymous codon usage in different hosts. In E. coli, protein expression levels varied over two orders of magnitude for each of two gene sets tested. Our data show that codon bias is a strong determinant of expression level in E. coli; however, the preferred codon bias is distinctly different from that of genes naturally highly expressed in the bacterium and no correlation is seen between expression and the codon adaptiveness index. With collaborators we have also synthesized and tested systematically varied gene sets for multiple target proteins in multiple expression hosts. The correlation between codon bias and protein expression in multiple systems is compared.
3:35 Refreshment Break in the Exhibit Hall
4:15 Poster Awards in the Exhibit Hall
EMERGING TECHNOLOGY
新技术
4:30 An Efficient Screening Method of Libraries for Expression of Soluble and Crystallizable Proteins
and Domains in E. coli
大肠菌之可溶性、结晶化蛋白质及领域发现库有效的筛选手法
Pär Nordlund, Ph.D., Evitaproteoma AB
Using a library of different protein constructs, the SPOTLIGHT system is able to detect constructs with soluble expression at the colony level. This is performed by applying a separation step through a filter at lysis, followed by immunological detection. The method constitutes an efficient HTP tool for screening for soluble protein expression which requires no automation. The method also reflects the situation at scale-up since detection is performed on lysate and no fusion reporter is used. In addition, it potentially increases the probability of obtaining well-diffracting crystals through its capacity to identify many different constructs in one single experiment.
5:00 Production of (Therapeutic) Non-Enzyme Proteins Using a Versatile PAOX1-Promoter Library of Pichia pastoris
利用万能的酵母促进(治疗)非酵素蛋白质生成
Thomas Purkarthofer, PhD, Head R&D, Biotechnology, VTU Technology
Employing our proprietary promoter library of the strongest promoter known to date, the Alcohol Oxidase 1 promoter (AOX1) from Pichia pastoris, controllable expression for several proteins at once is feasible. Co-expression of e.g. chaperones for correct protein folding along with particular target proteins eventually requires different expression characteristics for the helper and target protein, respectively, which can be achieved employing our PAOX1-library. Apart from several examples of high-level expression of industrial enzymes, we will focus on case studies of successful production of highly relevant (therapeutic) non-enzyme proteins.
5:30 Close of Conference
6:00 PepTalk Dinner “BuzZ in the Pipeline”
8:00 Close of Day
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