蛋白質表達技術

4月28日 (一)

7:00 am - 5:45 pm Registration Open

8:30 am Chairperson's Opening Remarks
Paul Wengender, CEO, Blue Sky Biotech, Inc.

8:40 Recombinant Proteins and Antibodies in Mammalian Cells: From DNA to Process at Large Scale - Today and Tomorrow
Florian M. Wurm, Ph.D., rer. nat., Dipl. Biol., Professor of Biotechnology, Faculty of Life Sciences, Ecole Polytechnique Federale de Lausanne
Mammalian cells in bioreactors have become the leading principle for high yield production for pharmaceutical applications. The talk will summarize the scientific and technological steps that lead to hundreds of milligram and now grams/liter in yield in extended batch processes. It will also evaluate further options for speeding up time lines and reducing costs for manufacturing during early and late phases in pre-clinical and clinical development. This includes novel gene transfer systems (large scale transient gene expression) as well as novel bioreactor technology that does not rely entirely on fixed installations but applies disposable materials, from banking to large scale processing.

NOVEL VECTORS AND HOSTS

9:10 New Vector Technology for Membrane Protein Production 
Larry DeLucas, Ph.D., Center Director, CBSE-UAB, The Center for Biophysical Sciences and Engineering
Novel technologies for improved production for membrane and aqueous proteins and generation of inducible animal models have been recently developed. One of the latest development centers around a unique, advanced lentiviral-based vector capable of regulating gene over-expression and silencing in vitro and in vivo. This technology has demonstrated the capacity to produce milligram quantities of membrane proteins for drug discovery addressing an unmet production scale need for membrane protein drug targets. The latest success will be dis-cussed regarding the production of the Cystic Fibrosis Transmembrane Regulator Protein.

9:40 An E. coli Vector-Based Antigen Expression System for the Construction of Novel Vaccines
Eric Huang, Ph.D., Associate Professor, Medicine, University of California, San Diego
The transition from protein target selection, protein expression/purification to vaccine creation is still moving slowly partly due to lack of an effortless platform that can efficiently express antigens at low cost and a larger scale. Here, we present a platform using an E. coli vector-based antigen expression system for expediting the evaluation of protein immunogenicity and antibody detection. A tetanus toxin C fragment (Tet-c) was used as a representative antigen to establish this platform. A cell wall-anchoring sialidase-like protein (SLP)of Propionibac-terium acnes (P. acnes) and a spore coat associated protein (SCAP) of Bacillus anthracis were utilized to assess the efficacy of this platform. Our results demonstrated that detectable antibodies were elicited in mice six weeks after intranasal administration of UV-irradiated E. coli vector-based vaccines. The antibody production of Tet-c, SLP or SCAP was significantly elevated after boosting. Notably, the platform with main benefits of using E. coli itself as a vaccine carrier provides a critical template for applied proteomics aimed at screening novel vaccine targets. In addition, the immunogenic SLP and SCAP potentially serve as novel antigen candidates for the development of vaccines targeting P. acnes-associated diseases and anthrax attack.

10:10 Grand Opening Coffee Break in the Exhibit Hall

11:10 Dual-Promoter Lentiviral System allows Inducible Expression of Noxious Proteins in Macrophages 
Igor Kramnik, Ph.D., Associate Professor, Immunology and Infectious Diseases, Harvard School of Public Health
Many proteins, which play an important role in innate immunity and are expressed by activated or infected macro-phages, possess cytotoxic, anti-proliferative or pro-apoptotic activities. Therefore, generation of stable macrophage cell lines that constitutively express those proteins for in-depth biochemical and functional analysis is often impossible. We have developed a lentiviral system for inducible gene expression both in macrophage cell lines and in primary macrophages based on a dual-promoter lentiviral vector, in which expression of a “gene-of-interest” is driven by a doxycycline-inducible promoter and the expression of a selectable surface marker is driven by an independent consti-tutive promoter. A transgenic mouse strain as well as macrophage cells lines that expresses reverse tetracycline transactivator (rtTA) under the control of macrophage-specific promoter were generated. Using this system, we per-formed analysis of dynamic protein-protein interactions in macrophages during activation with interferons and infection with virulent Mycobacterium tuberculosis. These tools will be especially useful for the discovery of modifiers of innate immunity in a physiologically-relevant context of macrophage interactions with pathogens.

11:40 Chaperna Technology: Use of RNA as Molecular Chaperone 
Baik Seong, Ph.D., CEO, Biotechnology, Protheon
The post-genome research initiatives on structural proteomics require a robust technical platform for protein ex-pression. So far, expression of functionally active proteins in E. coli remains a formidable task due to misfolding into inclusion bodies. Here, we report a novel chaperone type of protein folding facilitated by interaction with RNA. The technology is by far superior to MBP-fusion and extremely robust for soluble expression of a variety of proteins of human origin. This folding technology could be usefully implemented for high-throughput protein expression for functional and structural genomic research initiatives.

12:10 pm New High-Yield Eukaryotic Protein Expression System Based on the Protozoan Leishmania Tarentolae
Sergei Mureev, Ph.D., Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology
Production of functional eukaryotic proteins in recombinant form is a bottle-neck in various post-genomic appli-cations and in life science in general. At least partially, this is due to the problems associated with the use of en-dogenous RNA polymerase II for high-level transcription of heterologous genes in eukaryotic expression systems. To circumvent these problems we developed an inducible protein expression systems based on a protozoan Leishmania tarentolae. In this system, high level transcription of protein coding genes can be mediated by a re-pressor controlled T7 RNA polymerase. From this perceptive the system represents a eukaryotic analogue of the most successful bacterial expression architecture. We demonstrate that the system can inducibly over-express intracellular proteins with a yield of > 10% to >total cellular protein (ca. 300mg protein per liter of culture). The system is also suitable for production of secreted mammalian-like glycosylated proteins as shown on the example human erythropoietin. Once established the recombinant strains are stable for hundreds of generations, which qualifies them for large scale fermentation. L. tarentolae handling procedures are amenable to automation thus making it a new and attractive host for production of recombinant proteins for various applications.

12:40 Luncheon Workshop (Sponsorship Available) or Lunch on Your Own

1:40 Break

THE STATE-OF-THE-ART

2:00 Chairperson's Remarks 

2:05 Biosynthesis of Folded Cyclotides Inside Living Bacterial Cells 
Julio Camarero, Ph.D., Biomedical Scientist /PI, Biosciences and Biotechnology Division, Lawrence Livermore National Laboratory
The cyclotide MCoTI-II is a powerful trypsin inhibitor recently isolated from the seeds of Momordica cochinchinensis, a plant member of cucurbitaceae family. We report for the first time the in vivo biosynthesis of natively-folded MCoTI-II inside live E. coli cells. Our biomimetic approach involves the intracellular backbone cyclization of a linear cylotide-intein fusion precursor mediated by a modified protein splicing domain. The cyclized peptide then sponta-neously folds into its native conformation. The use of genetically engineered E. coli cells containing mutations in the glutathione and thioredoxin reductase genes considerably improves the production of folded MCoTI-II in vivo. Bio-chemical and structural characterization of the recombinant MCoTI-II confirmed its identity. Biosynthetic access to correctly-folded cyclotides allows the possibility of generating cell-based combinatorial libraries that can be screened inside living cells for their ability to modulate or inhibit cellular processes.

2:35 Solutions Showcase I: Largest Comparative Study on Expression Improvement by Gene Optimization: Results and Applications	Sponsored by
Marcus Graf, Ph.D., Co-Founder Manager Operations, GENEART AG

2:50 Solutions Showcase II: What's on TAP at Blue Sky Biotech?... A Novel Tandem Affinity Purification System!	Sponsored by
Rajiv Gangurde, Ph.D., Senior Research Scientist, Blue Sky Biotech, Inc.

3:05 Refreshment Break in the Exhibit Hall

3:45 Using an Engineered Solubilization Tag to Achieve Enhanced Protein Expression and Purification Based on Covalent Immobilization 
Rachel Friedman-Ohana, Ph.D., Senior Research Scientist, Research and Development, Cellular Proteomics, Promega Corporation
Using rational design and molecular evolution principles, we have developed a novel fusion tag (HaloTag7) to increase yield of soluble recombinant protein and enable efficient purification through rapid covalent immobilization onto solid-phase media. In benchmark studies on a large panel of difficult to express proteins in E. coli, a greater number of theses were solubilized by HaloTag7 compared to His Tag, GST and MBP (3, 2 and 1.5-fold more, respectively). The HaloTag7 rapidly bonds covalently to a chloroalkane ligand, allowing simple quantitation through stoichiometric fluorescent labeling or specific capture onto agarose beads (HaloLink). The protein of interest is released from HaloLink through an optimized cleavage method, providing approximately 80% yield of the expressed soluble protein free of the tag.

4:15 SuggestES: A Tool for Selecting the Best Expression System 
Jaime Prilusky, Ph.D., Head Bioinformatics ISPC, Bioinformatics Unit, Weizmann Institute of Science
SuggestES, a tool for selecting the best Expression System for a given protein, scans a large database with protein sequences with known results for different expression systems. At the time of generating a suggestion, suggestES takes into consideration several parameters: Similarity: how similar is your sequence to the existing data in the database?. The expression systems used on sequences similar to yours are preferred when creating the list of suggestions. Recentness: how recently was a given expression system used?. The older the record of the us-age of a given expression system, the less this system will influence the final result. This will provide visibility to recently appearing system. Frequency: how frequently a given expression system has been used?

4:45 Problem-Solving Break-Out Sessions 

5:45 – 6:45pm Networking Cocktail Reception in the Exhibit Hall