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7:00 am Registration and Morning Coffee
8:30 Chairperson’s Opening Remarks
Norman Garceau, Ph.D., Chief Scientific Officer, Blue Sky Biotech, Inc.
8:40 KEYNOTE PRESENTATION
Breaking the Cell Wall Barrier for Difficult-to-Produce Natural and Supernatural Products
James R. Swartz, Ph.D., Bioengineering, Stanford University
Scaleable, cost-effective technologies now enable cell-free protein production for commercial applications. With this comes the exciting ability to modify and control protein expression and folding for difficult-to-express pharmaceuticals. Examples will illustrate advantages for co-factor and disulfide bond containing proteins as well as membrane proteins. Even more exciting is a new freedom to construct “supernatural” proteins with novel functionalities. Complex fusion proteins; conjugated and precisely modifi ed proteins; and large, precisely assembled protein complexes for vaccines, drug delivery, imaging, and diagnostics will be described.
9:10 Screening Approaches to Solving Expression and Solubility Problems Using Novel Tagging and Detection System
Geoffrey Waldo, Ph.D., Team Leader, Biosciences, Los Alamos National Laboratories
We use a 15 amino acid tag, ‘FPmicroTag’, from a novel fluorescent protein to screen for soluble, stable protein complexes, find compact soluble protein domains and well-behaved multidomain proteins in living cells and in the test tube. The fluorescent detection system is stable and even 9 M urea does not perturb fusion protein behavior. We apply this to protein trafficking, protein interaction detection, high-throughput measurement of soluble protein in living cells, and engineering proteins for stability and solubility.
9:40 Applications and Expansions of Expression Modules That Tag Mammalian Membrane Proteins
Li Lin, Ph.D., Cardiovascular Sciences, National Institute on Aging, NIH
The recombinant expression of mammalian membrane proteins has been a major stumbling block in efforts to dissect their biological functions and determine their structures. It is also often difficult to generate effective antibodies to membrane proteins. To overcome these difficulties, we have designed, and generated, a set of expression modules that facilitate subcloning, expression, and detection of mammalian membrane proteins. The applications of these modules are further expanded to study the configuration of membrane proteins on cell surface, and to study signal transduction.
10:10 Grand Opening Coffee Break in Exhibit Hall
11:10 Evaluating Host Cell Differences for Difficult to Generate Proteins
Jennitte Stevens, Ph.D., Senior Scientist, Protein Science, Amgen
11:40 Meeting the Gene Expression Challenges Posed by Heterologous Polyketide Biosynthesis
Blaine A. Pfeifer, Assistant Professor, Chem. & Biological Eng., Tufts University
The last 15 years have seen a steady increase in efforts to heterologously produce polyketides through engineering-friendly organisms like Escherichia coli. However, many polyketide synthases are large proteins (>300 kDa) with unique structural and higher order assembly characteristics. In addition, the pathways needed for successful heterologous reconstitution may require up to 20 coordinately expressed genes before full biosynthesis would be expected. These challenges remain key technical hurdles to realizing the full potential of heterologously produced polyketide natural products, and this presentation will specifically address routes our group has taken to meet these challenges.
12:10 pm Dual Purpose Aminoacyl-tRNA Synthetases
A. James Link, Ph.D., Professor, Chemical Engineering & Molecular Biology, Princeton University
Much effort in the last decade has been placed on the engineering of aminoacyl-tRNA synthetases (aaRS) for the incorporation of unnatural amino acids into recombinant proteins. In this talk we describe strains of E. coli that harbor a single genomic copy of an engineered methionyl-tRNA synthetase (MetRS). The MetRS can ligate either the unnatural amino acid, azidonorleucine, or its natural substrate, methionine to methionyl-tRNA. Thus, these strains can be considered “dual purpose” organisms, the genetic code of which changes as a function of environment. The use of these strains for protein production and several other studies will be discussed.
12:40 Optimizing Saccharomyces Cerevisiae for the Secretion of Difficult Proteins
Chris Finnis, Ph.D., Manager, Molecular Biology, Novozymes Inc
Yeast strains originally engineered by Novozymes Biopharma UK Ltd for the commercial production of Recombumin® (recombinant human albumin USP-NF*) have been further developed for the secretion of both recombinant transferrin and a range of albumin fusion proteins (albufuse®). Manipulation of chaperone expression in these strains, focusing on the genes PDI1, JEM1 and ORM2, has led to large increases in the expression of previously difficult to express proteins, with transferrin production increased at least 50-fold to above 2g/L. Combined with collections of yeast strains designed to control O-linked glycosylation and proteolysis, high throughput expression studies can be used to rapidly identify characteristics beneficial for production strains.
12:55 Luncheon Presentation I
Human In Vitro Translation Systems for Rapid, High Fidelity Protein Production
Brian Webb, Ph.D., Platform Manager, Proteomics R&D, Thermo Fisher Scientific
Current in vitro expression systems suffer from low yields or inaccurate post-translational modifications. E.coli- and wheat germ-based in vitro systems cannot glycosylate proteins and protocols involving rabbit reticulocyte lysates in combination with canine microsomal membranes produce low amounts of protein and are inefficient at glycosylation. We report here the development of novel in vitro systems derived from human cell lines that yield biologically active glycoproteins with up to 15-fold more protein than rabbit reticulocyte lysates.
1:25 Luncheon Presentation II (Sponsorship Opportunities Available) or Lunch on Your Own
2:00 Chairperson’s Remarks
Philip Laible, Ph.D., Argonne National Laboratory
2:05 Assessment of the Importance of Sample Purity on Membrane-Protein Crystallization in Different Systems
Philip Laible, Ph.D., Principal Investigator, Biosciences, Argonne National Laboratory
Practical guidelines are needed to increase the efficiency of membrane protein crystallization. The difficulty in purifying active membrane protein samples and the high costs associated with producing such samples require extremely pragmatic approaches. We have investigated the effects of commonly encountered impurities on various membrane protein crystallization regimes, and we report that the lipidic-cubic-phase based crystallization methodology is more robust than crystallization in detergent environments in its ability to tolerate contaminations in the forms of protein, lipid, or other general membrane components.
2:35 Expression, Refolding and Purification of a Human IL-17A Variant for Structural Studies
Bingyuan Wu, Ph.D., Research Scientist, Molecular and Protein Biosciences, Centocor, Inc.
In this study, the expression of a human IL-17A variant in E. coli was optimized. The protein was isolated as inclusion bodies and refolded in a buffer containing arginine, glycerol and a redox coupling agent. The refolded rhIL-17A variant was subsequently purified using a combination of cation-exchange, reversed phase and fluoroapatite chromatography. The purified product was active, homogeneous and crystallizable. This presentation describes an example of expression and purification method development to overcome the challenges of a difficult protein.
3:05 Refreshment Break, Poster and Exhibit Viewin
3:45 Production of Recombinant Proteoliposomes for Therapeutic Uses
Jean-Luc Lenormand, Ph.D., HumProTher Laboratory, Université Joseph Fourier
The use of recombinant proteoliposomes containing therapeutic membrane proteins is a recently developed technology that allows biologically active proteins to penetrate across the plasma membrane of eukaryotic cells. One of the bottlenecks in this powerful delivery system lies in the production of functional therapeutic membrane proteins, mainly due to their biophysical characteristics. This presentation describes the methodology for the production of bioactive proteoliposomes containing therapeutic, proapoptotic membrane proteins synthesized with an optimized cell-free expression system. This system can be easily adapted for producing “difficult to express proteins.”
4:15 Engineering N-Glycosylation in the Baculovirus Expression System
Christoph Geisler, Department of Molecular Biology, University of Wyoming
Insect cell hosts used in the baculovirus expression system typically produce glycoproteins with truncated N-glycans, whereas glycoproteins from mammalian cells bear extended N-glycans. This difference is caused by the presence of a deleterious processing enzyme as well as a lack of glycosyltransferases in insect cells relative to mammalian cells. We have engineered insect cells to express glycosyltransferases, thus allowing the production of mammalian-like N-glycans. Recently, we identified genes encoding processing enzymes in commonly used cells lines, which allows us to further improve their glycosylation potential.
4:45 Problem Solving Break-Out Sessions
Table 6: Enhancing Cytoplasmic Expression in Mammalian Cells
Moderator: Dominic Esposito, Ph.D. (Contractor), Principal Scientist, Group Leader, Clone Optimization Group, Protein Expression Laboratory, Advanced Technology Program, SAIC-Frederick, Inc.
Effects of promoters, enhancer, and other elements on transient protein production
Transient transfection vs lentiviral transduction
Ways to monitor protein expression and solubility using fusion tags
High-throughput mammalian protein expression techniques
Table 7: Getting a Handle on Proteins Using the Right Tag
Moderator: Geoffrey Waldo, Ph.D., Team Leader, Biosciences, Los Alamos National Laboratories
Tagging objectives: Purification or detection or both.
Library screening requirements (host, protein, library size, property to be screened for).
Survey of some protein tagging systems (pros and cons, specificity, size, expense, readout format).
Examples of tagging and library screening
Table 8: Mammalian membrane proteins: how to break the bottleneck
Moderator: Li Lin, Ph.D., NIH/NIA/IRP
Choice of expression hosts
Ways to monitor/detect membrane protein in cells and tissues (epitope tagging and generating effective antibodies)
Proteomics studies of membrane protein complexes
Structure-function study of membrane proteins
Membrane protein and signal transduction
Table 9: Novel Analytical Techniques/Tools for the Characterization of Biopharmaceuticals
Moderator: Jennifer F. Nemeth, Ph.D., Principal Research Scientist and Head, Discovery Mass Spectrometry, Centocor Research and Development
New and improved technologies
Software characterization programs
Bioinformatics options for biopharmaceutical characterization
5:45 Networking Cocktail Reception in the Exhibit Hall
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