タンパク質発現に関する会議 - 3日目

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アジェンダ (英語PDF)

Wednesday, January 9

7:00am-5:30pm Registration Open 

7:30 Breakfast Workshop:  A Revolution in Mammalian Gene Expression: Reaching Extremely High Protein & Antibody Expression (60-120pg/cell/day) in Three Weeks in Fast-Growing CHO Cells Matthew Hui, Ph.D., M.D., Founder & Chief Scientific Officer, AmProtein Corporation

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AmProtein Corporation has just discovered a DNA structure-based common mechanism for possibly all eukaryotic gene expression. Based on this discovery, AmProtein has rapidly generated more than 10 cell lines, which produced more than 10 different proteins and antibodies at levels of 60-120pg/cell/day. All of these were achieved in three weeks in fast-growing CHO-S cells after stable gene transfections. These results have strongly suggested that this discovery has revolutionized the protein production industry and made mammalian protein production rapid, affordable and dominant over other transgenic production methods. Currently, AmProtein is working on the more detailed mechanism and is applying this discovery to plant, yeast and insect gene expression.

8:15 Chairperson’s Remarks
Norm Garceau, Ph.D., President & CSO, Blue Sky Biotech Inc.

8:20 High-Throughput, Small-Scale Protein Expression Optimization in Baculoviral Expression System
Jim King, Ph.D., Principal Scientist, Biologics & Biomolecular Sciences, Boehringer Ingelheim Pharmaceuticals
We have optimized and automated a high-throughput, small-scale baculoviral expression process to quickly and effectively assess protein expression. After one step affinity purification, the resulting protein is analyzed using a Caliper LabChip90, and is suitable for follow-up characterization to prioritize large-scale expression. We have applied this technique to many projects to scan domain boundary variants and point mutants.

8:50 High-Throughput Protein Expression in Insect Cells
Linda King, Ph.D., Professor, Oxford Expression Technologies Ltd.
This paper will present recent advances in high throughput, robotic technologies for the production of proteins in insect cells. The new advances include modifications to the baculovirus expression vector (flashBAC) to delete non-essential genes that results in enhanced yield and quality of many proteins. These advances are complemented by a new, automated, virus titration assay.

9:20 The Proteomic Code: A Molecular Recognition Code for Proteins
Jan C. Biro, M.D., Ph.D., Homulus Foundation
The Proteomic Code is a set of rules by which information in genetic material is transferred into the physico-chemical properties of amino acids. It determines how individual amino acids interact with each other during folding and in specific protein-protein interactions. The Proteomic Code is part of the redundant Genetic Code. A novel cloning method for the design and production of specific, high-affinity-reacting proteins (SHARP) is presented. This method is based on the concept of proteomic codes and is suitable for large-scale, industrial production of specifically interacting peptides.

9:50 Coffee Break in the Exhibit Hall

10:45 Expression of mRNA Destabilizing Protein Tristetraprolin and its Homologues in Bacterial and Mammalian Cells
Heping Cao, Ph.D., Research Biologist, Human Nutrition Research Center, USDA-ARS
Tristetraprolin (TTP) family proteins are mRNA binding and destabilizing factors with high specificity towards pro-inflammatory cytokine mRNAs coding for tumor necrosis factor, granulocyte-macrophage colony-stimulating factor, and others. They have the potential to be therapeutic targets for the prevention and treatment of inflammation-related diseases. TTP family proteins have proven to be difficult to express in various expression systems. Previous studies showed that the great majority of recombinant TTP family proteins were degraded and precipitated following induction in the cells. We have successfully expressed active TTP and its homologue ZFP36L1 proteins in E. coli and human cells. The over-expressed proteins are functionally active with the capacity of binding to tumor necrosis factor mRNA AU-rich sequence in a zinc- and concentration-dependent manner.

11:15 New Tools for Mammalian Protein Expression
Dominic Esposito, Ph.D., Senior Scientist, Protein Expression Laboratory, SAIC-Frederick, Inc.
Mammalian protein expression is typically done using a standard set of vectors with a CMV promoter. We have developed a set of new vectors that easily allows testing of a variety of mammalian promoters and expression elements, and have demonstrated that expression of proteins can be dramatically enhanced through the use of these different elements. In addition to effects on transient expression, these tools can be used to create novel lentiviral vectors which have been successfully used in vivo. We will discuss the technology, our current results on promoter activity, and possible future uses of these new vectors.

11:45 The Uptake and Trafficking of Transiently Transfected DNA in CHO Cells
Athena Wong Ph.D., Associate Scientist, Early Stage Cell Culture, Genentech, Inc.
This presentation will discuss the endocytosis and trafficking of two cationic liposome transfection reagents in CHO cells; DMRIE-C and Lipofectamine LTX. We found that DMRIE-C-transfected DNA is internalized via caveo-lae, while LTX-transfected DNA is internalized by clathrin-mediated endocytosis, with both pathways converging at the late endosome/lysosome. Since transfections using the LTX produce higher titers this suggests alternative nuclear delivery mechanisms among DMRIE-C and LTX with the latter being more efficient. This work was per-formed with the goal of enhancing our understanding of the trafficking pathways for transiently transfected DNA in an effort to identify the step(s) that affect transfection efficiency.

12:15pm Close of Morning Session

12:30 Luncheon Workshop (Sponsorships Available) or Lunch on Your Own

1:45 Chairperson’s Remarks
Ajith Kamath, Ph.D., Jubilant BioSys

1:50 FASTR and EESYR Technologies for the Expression of Antibodies in CHO Cells
Gang Chen, Ph.D., Director, Cell Technologies, Regeneron Pharmaceuticals, Inc.
We have developed FASTR and EESYR technologies for the construction and isolation of recombinant CHO cells for antibody production. FASTR (Flow cytometry-based autologous secretion trap) is a novel screening method that enables efficient isolation of production CHO cell lines for large-scale manufacturing. In EESYR (Enhanced expression and stability region), the proteins of interest are expressed from a defined genomic locus. EESYR en-ables high-throughput construction of antibody-producing stable CHO lines.

2:20 Use of the Baculovirus Expression System for the Production of rAAV Vectors
Joseph Rininger, Ph.D., Protein Sciences Corp.
The Baculovirus Expression Vector System (BEVS) is now emerging as a key manufacturing platform capable of safely producing complex products such as recombinant adeno-associated virus (rAAV) vectors with significant advantages over mammalian production platforms. One of the key advantages is the more simplistic means of large scale production for target indications that will require high quantities of rAAV due to the need for targeted systemic delivery of rAAV particles to the appropriate organ system. This presentation will review baculovirus-based protein production, outline the strategy and data for baculovirus-based
rAAV production and development of a cGMP production process for such an indication, Duchenne’s Muscular Dystrophy (DMD). 

2:50 Antibody Engineering and Expression Optimization for Research Reagents and Production Cell Lines
Jill M. Carton, Ph.D., Senior Scientist, Department of Molecular Discovery Technologies, Centocor Inc.
Three essential features of recombinant therapeutic proteins are their biological activity, biochemical and bio-physical properties and the level of production. In order to impact level of production, we developed a codon engineering process that has a positive influence on antibody expression levels. The engineering strategy has been incorporated into our expression process for research and therapeutic reagents. This presentation describes our antibody expression process and current methods of expression optimization.

3:20 Technology Spotlight Optimizing Protein Expression by  Rationale Gene Design and de novo Construction

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Speaker to Be Announced, GENEART AG Efficiency and yields in protein expression are often critically influenced by the encoding DNA sequence itself. Natural genes obtained from clone selection, or by amplifying genes from natural sources or cDNA libraries are often difficult to express outside their original context, since the frequency with which different codons are used varies significantly between organisms and/or different tissues. This has been solved in the past by changing the expression system or by co-expressing limiting tRNAs. However, changing the expression system is often not fea-sible for high-throughput analysis systems; and cis-acting elements such as splice sites or ribosomal entry sites may demolish expression even when tRNAs are available. The very same holds true for simple codon optimization approaches using an artificial gene based on a back translated amino acid sequence, or a sequence which codon usage was adapted to the codon frequency of the expression host. Here, we compare different gene optimization strategies and show that high performance gene optimization is needed to modulate many expression properties in parallel, including correct weighting of certain parameters over others. In head-to-head expression studies, the performances of differently optimized genes are compared to natural gene expression resulting sometimes in up to 100-fold increase in expression yields. Combined with a high-throughput, high-capacity and fast-turnaround synthesis platform, rationally designed and de novo constructed artificial genes thus provide a quick and reliable source for genetic research material at modest costs, and lift research in life sciences up to a new level of productivity.

3:35 Refreshment Break in the Exhibit Hall

4:45 Poster Awards in the Exhibit Hall

5:00 A Platform for Recombinant Expression of Human Proteases Targeted to the Secretory Pathway
Daniele Carettoni, Ph.D., Head, Biochemistry, Axxam SpA
Proteases concurrently represent one of the most relevant therapeutic enzyme classes for drug discovery, and one of the most challenging protein class for recombinant expression. In order to generate an array of HTS-compatible functional assays for screening purposes, a streamlined research program has been undertaken for the heterologous expression and assay configuration of human proteases targeted to the secretory pathway, including both secreted and transmembrane enzymes. A subset of 124 proteases has been selected on the basis of their expression profile and pathophysiological relevance, and sequentially subcloned using a restriction-free protocol, recombinantly expressed in insect cells, purified by affinity chromatography, and configured as fluorescence-based enzymatic assays. Miniaturization, parallelization and automation-amenable approaches have been developed and applied at different steps along the process, to increase reproducibility, consistency and through-put. At present, 94 proteases have successfully entered the expression phase, and the enzymatic activity of 13 of them has been fully configured as HTS-compatible assay, contributing with new insights on their biochemistry and functionality.

5:30 Optimization of Recombinant Protein Secretion in HEK-293 Cells by Protein Engineering
Shaoqiu Zhuo, Ph.D., Protein Science, diaDexus Inc.
HEK-293 cell line has been widely used to produce recombinant proteins. However, many human proteins are cell-type dependent in expression and post-translational processing. We have developed protein engineering methods for the expression and secretion of recombinant proteins in HEK-293 cells. Examples will be shown in our presentation. One of the case studies is to enhance the expression and secretion of the mature macrophage inhibitory cytokine-1 (MIC-1). We have identified the rate-limiting step for the folding of the protein and are able to significantly increase the secretion of the mature dimmer of the protein. The transient expression process can be easily adapted into 510K GMP.

6:00 Close of the Protein Expression Conference