蛋白质表达技术

4月29日 (二)

8:00 am - 5:15 pm Registration Open

SOLNING PROTEIN EXPRESSION PROBLEMS THROUGH PROTEIN ENGINEERING

8:25 am Chairperson's Remarks

8:30 Soluble Nanolipoprotein Particles (NLPs) Containing Functional Integral Membrane Proteins Form in situ During Cell-Free Protein Synthesis 
Paul Hoeprich, Ph.D., Staff Scientist, CMELS, Livermore National Laboratory
Techniques and methods for isolation, purification, and analysis developed with soluble proteins are not generally transferable or applicable to membrane-associated proteins and protein complexes. We are addressing these problems by focusing on emerging methods for cell-free protein synthesis (IVT) in conjunction with functional re-constitution of membrane proteins into nanolipoprotein particles (NLPs). We and others have shown that membrane protein (MP) containing NLPs can be made by combining apolipoprotein, phospholipid and detergent solubilized MP. We will present two methods for the preparation of functional MP-NLPs - the first involves direct addition of purified apolipoprotein to an IVT reaction mixture producing MP-NLPs and the second involves simultaneous co-expression of both MP and apolipoprotein with concomitant in situ NLP formation. Samples prepared by both methods have been characterized using a suite of advanced, analytical methods including functional assessment.

9:00 Development of a High-Throughput ENaC Ionworks Quattro Assay, using BacMan Technology 
Paul J. Groot-Kormelink, Ph.D., CF/COPD Unit, Novartis Institutes for BioMedical Research
Recently, automated high-throughput electrophysiology systems such as Ionworks Quattro and Q-Patch, have greatly enhanced the feasibility of screening ion channel drug targets. However, these novel systems place new demands on tool generation as every cell needs to express the ion channel and at very high levels. This is difficult to achieve with standard stable cell line generation as ion channels are traditionally difficult to express proteins, of-ten being multimeric and toxic to the cell when over-expressed. We have successfully applied a transient expression system, BacMam technology, to develop an Ionworks Quattro assay for a multimeric epithelial sodium Channel (ENAC) that will be presented.

9:30 Hybrid IL17RA/IL17RC-Fc Fusion Protein as a Soluble Inhibitor of IL17A and IL17F 
Mark Rixon, Ph.D., Principal Scientist, Protein Biochemistry, ZymoGenetics, Inc.
IL17RC has been identified as a receptor for both IL17F and IL17A. Overall secretion of an Fc-fusion form of IL17RC was low while a significant fraction of the protein remained cell-pellet associated. To generate a scalable production process, a series of engineered, variant forms of IL17RC, including directed deletions, amino acid substitutions, and hybrid molecules consisting of portions of IL17RC and IL17RA, were generated based in part on bioinformatic evaluation of the gene structure. A hybrid receptor Fc-fusion protein was identified that demonstrated high productivity in CHO cells. Additionally, the hybrid receptor Fc-fusion protein showed greater affinity for both IL17A and IL17F compared to either IL17RC-Fc or IL17RA-Fc.

10:00 Coffee Break in the Exhibit Hall

APPLICATIONS AVD ISSUES

10:45 P-gel: A DNA Gel That is Able to Produce Proteins without any Living Cell
Dan Luo, Ph.D., Associate Professor, Department of Biological and Environmental Engineering, Cornell University
Our group recently reported an enzyme-catalyzed DNA hydrogel whose scaffolds were composed entirely of branched DNA. Inspired by and based on the success of our DNA hydrogels, we constructed a similar hydrogel using the same X-shaped DNA as crosslinkers but with actual genes as monomers. This gel, termed "P-gel", was able to produce proteins in high yield and efficiency without any living cells. So far we have tested a total of 15 different P-gels and have produced 15 different proteins with very high efficiency and yield.  The mechanisms of P-gel have also been explored. P-gel is envisioned to be an enabling
platform technology for future protein production.

11:15 Orfeus Expression System: A Biorisk-Free Option 
Björn Örvar, Ph.D., CEO, Genetics, ORF Genetics
ORF Genetics has pioneered a unique technology for manufacturing hard-to-produce, recombinant proteins, effectively bypassing both animal cells and bacteria as a production host. Using its proficient, barley grain-based Orfeus™ system the company is the only one able to provide biorisk-free growth factors and cytokines for medical research, cell culture and drug development. The host organism, barley, with its specialized endosperm storage tissue provides many unique features including proficient protein machinery, with eukaryotic folding, and a distinct route for long-term protein protection and storage. A biochemically inert environment, low protease activity and secondary metabolite content, and a simple protein profile, aid in downstream processing. The biorisk-free features include endotoxin-free products, and low pyrogenic and pro-inflammatory activity, as well as lack of human or animal infectious agents according to regulatory authorities. ORF Genetics has now close to 100 human growth factors and cytokines in its pipeline.

11:45 From Neanderthal to Nanobiotech: From Plant Potions to Pharming—Should We Make the Switch to Plant Expression?
Loïc FAYE, Ph.D., Biology, CNRS
Plants were the main source for human drugs until the beginning of the 19th century when plant-derived pharmaceuticals were partly supplanted by drugs produced by the industrial methods of chemical synthesis. During the last decades of the 20th century, genetic engineering has offered an alternative to chemical synthesis, using bacteria, yeasts and animal cells as factories for the production of therapeutic proteins. After a temporary de-crease in interest, plants are rapidly moving back into human pharmacopoeia, with the recent development of plant-based recombinant protein production systems offering a safe and extremely cost-effective alternative to microbial and mammalian cell cultures. In this presentation, we will illustrate that current improvements of plant expression systems for biopharmaceutical production are making them suitable as alternative factories for the production of either simple or highly complex therapeutic proteins.

12:15 pm Luncheon Workshop (Sponsorship Available) or Lunch on Your Own

1:15 Break

CASE STUDIES

2:00 Chairperson's Remarks

2:05 Engineering ScFv with a Lipophilic Tail for Ectoplasmic Insertion into Targeting Liposomes 
Anton-Scott Goustin, Ph.D., Research Scientist, Internal Medicine, Wayne State University
We have experience using stable insect cell transfectants to produce a single-chain variable fragment (ScFv) antibody directed to the same antigen (CD20) as the lymphoma drug Rituxan. We have fused the ScFv ORF to the signal in human placental alkaline phosphatase (PLAP) to create a signal for the insect cells to biosynthetically add a glycosylphosphatidylinositol (GPI) anchor.

2:35 In Vitro Selection and Characterization of DARPins and Fab Fragments for the Co-Crystallization of Mem-brane Proteins
Thomas Huber, MSc., Laboratory of Prof. A. Plueckthun, Department of Biochemistry, University of Zurich
The determination of 3D structures of membrane proteins is still extremely difficult. Co-crystallization with binding proteins may be an important aid to obtain crystals suitable for high resolution structures. The in vitro selection of binding proteins specific for integral membrane proteins and an efficient way to screen their potency for co-crystallization will be presented.

3:05 Solutions Showcase I Novel Small-Molecule Additives to Refold Proteins A Simultaneous and Systematic Evaluation	Sponsored by
Peter A. Leland, Ph.D., R&D Group Leader, Protein Purification and Biochemistry, Novagen Brand, EMD Chemi-cals, Inc.

3:20 Solutions Showcase II (Sponsorship Available)

3:35 Refreshment Break in the Exhibit Hall

4:15 Strategies for the Expression of Difficult Protein Targets at the Midwest Center for Structural Genomics
Gyorgy Babnigg, Ph.D., Scientist Bioinformatics/Biochemist, Scientific Administrator, Midwest Center for Structural Genomics, Argonne National Laboratory
The primary objective of our center is to rapidly determine the structures of large numbers of strategically selected proteins in order to elucidate protein fold space. When a full-length protein does not provide a soluble product for crystallization, truncated versions of the sequence are defined by bioinformatics analyses. We have developed several other strategies in order to enhance protein expression and solubility by providing fusion partners or co-expression interactive proteins. The outcomes of these approaches will be presented.

4:45 Title to Be Determined
Paul Ramage, Ph.D., Novartis Institutes for BioMedical Research 

5:15 pm End of Difficult to Express Proteins Conference