蛋白质发现会议 - Day 2

概要 | 短期研讨会 | Day 1 | Day 2 | Day 3

议程(PDF : English)

Tuesday, January 8

7:30am-6:00pm Registration Open

7:30 Breakfast Workshop  Systematic Analysis of Variables for Designing Highly Expressed Genes

Sponsored by

Claes Gustafsson, Ph.D., Vice President, DNA2.0 Systematically varied sets of synthetic genes were used to identify and explore gene design variables that affect protein expression. The identified intragenic sequence design variables were experimentally shown to control protein expression levels up to 30% of cellmass in independent systems. Previously established variables such as Codon Adaptation Index showed poor correlation to increased protein expression.

8:15 Chairperson乫s Remarks
Ajith Kamath, Ph.D., Jubliant Methods

8:20 Rapid and Efficient Production of Proteins from Cell Lines Using Lentiviral Vectors
Boro Dropulic, Ph.D., Founder & CEO, Lentigen Corporation 

8:50 Expression of Recombinant Proteins and Antibodies in Chloroplasts of Microalgae
Peter B. Heifetz, Ph.D., Chief Scientific Officer, Rincon Pharmaceuticals, Inc.

9:20 Rapid Identification of Effective Host Strains for Biopharmaceutical Production Using the Pseudomonas-based Pfnex Expression TechnologyTM 
Charles Squires, Ph.D., Senior Director, Biopharmaceutical Services, Dowpharma, The Dow Chemical Company
The Pseudomonas-based Pf nex Expression TechnologyTM has proven to be a robust and cost effective platform for the production of numerous classes of therapeutic proteins, including various types of antibody derivatives and 乬scaffold乭 proteins. High cell densities in the fermentor along with high specific protein yields result in high target protein titers. Very high cell densities are also achievable even in small scale growth (96-well plates) now used for expression screening. An extensive tool box of unique host strains has also been developed. These strains have phenotypes selected to impact the amount of target protein produced and its solubility and activity. The high-throughput methods allow dozens of different expression strategies and host cell combinations to be rapidly tested in parallel. Also, efficient periplasmic secretion of proteins has been developed. This capability allows the formation of disulfide bonds, critical to the production of active antibody derivatives, and also the development of simplified downstream processes.

9:50 Coffee Break in the Exhibit Hall

10:45 Elastin-Like Polypeptide Tags for Protein Purification
Ashutosh Chilkoti, Ph.D., Professor of Biomedical Engineering, Mechanical Engineering and Materials Science and Chemistry Associate Director, Center for Biologically Inspired Materials and Materials Systems, Duke University

11:15 Measurements of Reliability and Completeness in Image Analysis of Two-Dimensional Electrophoresis Gels
Ola Forsstrom-Olsson, Ph.D., Ludesi

11:45 Technology Focus

12:15pm Close of Morning Session

12:30 Luncheon Workshop Challenging Protein Expression

Sponsored by

Scott Gridley, Ph.D., Director of Protein Sciences & S. Edward Lee, Ph.D., Blue Sky Biotech Despite more than two decades of investigation, heterologous expression of recombinant proteins continues to present significant challenges in many instances. This discussion will focus on bundled gene to protein process technologies which reduce the time needed to produce proteins for research and development applications. Highlighted technologies include:  Chain Blue Lightning乭 modular progression gene synthesis and nucleic acid sequence refinements (codon optimization)  Bacterial expression of fusion proteins tags which improve solubility and amenability to purification (including our RedSee乭 tagging system)  Optimizing baculovirus expression and scale-up via IKM乭/BIIC乭/TIPS乭  Cost-effective mammalian expression via PEI-based transfection  Novel alternative expression systems

2:00 Chairperson乫s Remarks
Walid Qoronfleh, Ph.D., Q3 Consulting LLC

2:05 A Novel Cumate-Regulated Heterologous Protein Expression System in E. coli
Carlos Miguez, Ph.D., Project Leader, Microbial and Enzymatic Technology, Biotechnology Research Institute, National Research Council of Canada
We have developed a novel tightly regulated gene expression system designed to work in all Escherichia coli production strains by applying the regulatory elements of the Pseudomonas putida F1 cym and cmt operons to control target gene expression at the transcriptional level by using p-cumate as an inducer. This novel expression system includes a specific expression vector, pNEW, that contains a partial T5 phage promoter combined with the P1 synthetic operator and the repressor gene (cymR) driven weakly by a kanamycin promoter (PKm) designed to express the repressor gene constitutively in the host strain. The induction of transcription relies on the addition of the exogenous inducer called cumate which is non-toxic, inexpensive and easy to use. Both high induction of transcription and extremely low basal expression allowed extremely high induction levels, with a degree of control that is far superior to other currently available E. coli expression systems. The results indicated that the present pNEW expression system could be a highly efficient system for the production of recombinant proteins in any type of E. coli strains tested, and expression yields of our novel cumate-regulated system surpassed the comparable but less efficient PT7 IPTG-inducible E. coli expression system.

2:35 Tunable Protein Expression using AOX1-Promoter Libraries for Pichia pastoris
Roland Weis, Ph.D., Head of Biotechnology, VTU Technology
Pichia pastoris emerged as one of the most efficient heterologous hosts for industrial protein expression. While drawbacks of tedious cultivation, restricted selection markers and hyper- as well as non-human glycosylation are already overcome, still few reasonable promoters are available, impeding its speed of development for ever more proteins to be expressed. Employing our proprietary promoter library of the strongest promoter known to date, the Alcohol Oxidase 1 promoter (AOX1) from Pichia pastoris, tunable expression for several proteins at once is feasible. The occurrence of e.g. chaperones for correct protein folding should precede the actual expression of a target protein in order to ensure a defined ratio of helper to acceptor protein. In systems biology, as well, trans-ferred pathways from one organism to another need to follow certain balance rules of protein supply down the route from precursor A to final product Z in order to maximize the efficacy of the pathway.

3:05 TAGZyme for the Removal of N-terminal Affinity Tags from His-tag Proteins
Mikkel Duhrkop, CEO, Unizyme Laboratories AS

3:35 Technology Spotlight Synthetic Biology in Protein Engineering Speaker to Be Announced, Codon Devices, Inc.

Sponsored by

Both in vitro-evolution and directed-mutagenesis approaches to protein engineering require the construction of a large number of variant proteins. Typically, libraries of variants used in evolution experiments are generated by introducing random mutations into protein genes, whereas the much smaller sets of mutants used in directed-mutagenesis approaches are defined based on structural or sequence-based information. The Codon Devices DNA synthesis and assembly technology allows us to combine the large size of in vitro-evolution libraries with the ability to define the sequence of each clone in the library. In addition, our proprietary error-filtering technology results in libraries with error rates significantly below the error rate of oligonucleotide synthesis. Both contribute to an increased probability of success for a library of a given size and, as a consequence, result in faster and more cost-effective therapeutic protein discovery.

3:50 Refreshment Break in the Exhibit Hall

4:30 BuzZ Sessions - Small Group Moderated Discussions Have a topic idea? Improving Protein Expression Throughput Norm Garceau, Ph.D., President & CSO, Blue Sky Biotech Inc. 乬BIIC/TIPS乭 Baculovirus Discussion Mr. William Hermans, Manager, Cell Culture, Blue Sky Biotech Inc.