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8:25 Chairperson’s Opening Remarks
Jean-Luc Lenormand, Ph.D., HumProTher Laboratory, Université Joseph Fourier
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8:30 HaloTag® Based Purification of Functional Proteins from Mammalian Cells
Rachel Friedman Ohana Ph.D., Senior Research Scientist, Research and Development: Cellular Proteomics, Promega Corporation
Obtaining high yields of functional mammalian proteins containing relevant post-translational modifications remains a critical challenge; we addressed this with a new method for purification of intracellular mammalian proteins from their native location. This approach is based on a protein fusion tag, HaloTag, which provides efficient protein purification through covalent immobilization coupled with proteolytic tag removal. We have used this method to purify intracellular proteins including 5 kinases from different kinase families and obtained milligram levels of pure proteins from 1 L cultures. All proteins were tested for activity and shown to be functional.
9:00 Employing Rhodobacter sphaeroides to Functionally Express and Purify Human G Protein-Coupled Receptors
Ankita Roy, Ph.D., NIEHS, NIH
Functional production of most recombinant GPCRs is one of the main bottlenecks to obtaining structural information. We used a novel bacterial expression system based on the photosynthetic bacterium Rhodobacter sphaeroides for this purpose. The advantage of employing R. sphaeroides as a host lies in the fact that it provides much more membrane surface per cell compared to other typical expression hosts. We tested this system for the expression of some A GPCRs. The system can be extended for other “difficult to express and crystallize” membrane proteins in general.
9:30 Comparison of High-Throughput Techniques for the Expression of Protein Complexes
Gyorgy Babnigg, Ph.D., Scientist, Biological Sciences, Argonne National Laboratory
While Structural Genomics pipelines have extensive experience with the expression of single proteins in high-throughput fashion, the generation of protein complexes is labor intensive using current techniques. Most of the existing techniques are not compatible with HTP operations or are not economical at that scale. We developed recently a technique compatible with high-throughput operation for the expression of protein complexes and the results of this comparative study are presented.
10:00 Coffee Break, Poster and Exhibit Viewing
10:45 A Robust, Automated, High-Throughput Quantitative HPLC-Based Platform for Glycan Analysis with Computer-Assisted Data Interpretation
Pauline Rudd, B.Sc., LRIC, MA (OXON), Ph.D., NIBRT Professor , Glycobiology, University College Dublin, Medical Sciences, NIBRT
Features include (i) sample immobilization (96-well plates), glycan release, and fluorescent labeling; (ii) quantitative HPLC analysis, including monosaccharide sequence and linkage information for charged and neutral glycans; (iii) automatic structural assignment from HPLC profiles via web-based software that accesses our experimental database (GlycoBase) and (iv) software (autoGU) that progressively analyzes data from exoglycosidase digestions giving a refined list of final structures (v) detection at <0.5% of total glycan pool (vi) sialic acid speciation (vi) compatible with MS and CE technologies.
11:15 Co-Expression of AMPK Subunits in Insect Cells Yields a Stable Heterotrimer
Lata Ramanathan, Ph.D., Senior Scientist, Protein Sciences, Schering-Plough Research Institute
11:45 Elimination of Bacterial Toxicity of the Gene Encoding the Tfg1 Subunit of S. cerevisiae TFIIF
Alfred S. Ponticelli, Ph.D., Associate Professor, Biochemistry, School of Medicine and Biomedical Sciences, State University of New York, Buffalo
Biochemical studies of the yeast S. cerevisiae RNA polymerase II basal transcription machinery have been hampered by difficulties in the production of recombinant transcription factor IIF (TFIIF), specifically due to the extreme toxicity of the gene encoding the Tfg1 subunit in E. coli. I will report on the elimination of TFG1-associated toxicity in E. coli through the identification and mutational inactivation of both a functional E. coli promoter and an internal translation initiation site within the N-terminal coding region.
12:15 pm Expression of a Vaccine Antigen Candidate Protein in the Pseudomonas Fluorescens-Based Pfenex Expression Technology
Bruce Carpick, Ph.D., Principal Scientist, Research Biochemistry, Sanofi Pasteur
Expression of soluble and active protein is critical to the success of any protein based vaccine or therapeutic product development program. This is quite challenging given the complexity of the proteins being expressed in recombinant systems today. This case study will describe the expression of a particularly challenging antigen using Pfenex Expression Technology. The presentation will cover the small scale parallel screening of hundreds of unique expression strains and subsequent in vitro a nd in vivo pre-clinical testing of the antigen produced.
12:45 pm Luncheon Presentation (Sponsorship Opportunities Available) or Lunch on Your Own
2:00 Chairperson’s Remarks
Dominic Esposito, Ph.D., SAIC Frederick, Inc.
2:05 Expression Plasmids and Production of EGFP in Stably Transfected Acanthamoeba
Erik A. Bateman, Ph.D., Research Associate Professor, Microbiology & Molecular Genetics, University of Vermont
Stable transfection of Acanthamoeba castellanii for promoter analysis and protein expression has been characterized. Expression plasmids containing the TBP, TPBF or GAPDH gene promoters from Acanthamoeba were constructed. The promoters for Acanthamoeba TPBF and GAPDH genes were used to drive expression of enhanced green fluorescent protein (EGFP) in stably transfected Acanthamoeba. Purification from lysates of 22-ml cultures yielded approximately 1.1 milligrams of EGFP, a value that extrapolates to 50 milligrams per liter of cell culture. Results suggest that Acanthamoeba is a useful system for production of recombinant proteins.
2:35 The Science of Solubility: Structure/Function Studies of Chimeric NusA Fusion Tags Provides Insight into Protein Solubility Enhancement
Dominic Esposito, Ph.D., Group Leader, Clone Optimization Group, Protein Expression Laboratory, Advanced Technology Program, SAIC-Frederick, Inc.
Enhancement of solubility of heterologous proteins in E. coli is often necessary if these proteins are to be produced at significant levels. We investigated the ability of homologs of the E. coli NusA protein to act as solubility enhancers, and in the process have discovered that very subtle differences in NusA protein sequence can have remarkable effects on the ability to solublize partner proteins. These results help to better understand the characteristics of the NusA protein which lead to its function as a solubility tag, and may lead to the production of more efficient solubility tags.
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3:05 Functional Assembly of Membrane-Associated Protein Complexes on a Derivatized Lipid Template for HTS – a SmartScreen™ Technology Review
Scott Gridley, Ph.D., Head of Product Development, Blue Sky Biotech
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3:20 Advancing Synthetic Gene Design
Mark Welch, Ph.D., Director, Gene Design, DNA2.0, Inc.
Gene synthesis offers immense flexibility in the tailoring of genes for practical uses. Capturing the value of this flexibility, however, is greatly limited by lack of understanding of the interactions between gene sequence features and host expression systems. DNA2.0 has developed a novel approach to interrogate the gene design preferences of expression hosts to maximize production from synthetic genes. Applications of this approach for a number of target proteins in several different host organisms will be discussed.
3:35 Refreshment Break, Poster and Exhibit Viewing
4:15 A Versatile Viral System for Expression and Depletion of Proteins in Mammalian Cells
Eric Campeau, Ph.D., Program in Gene Function and Expression, University of Massachusetts Medical School
The ability to express or deplete proteins in living cells using viral vectors has become essential to the study of biological processes. We recently published a collection of 59 vectors that comprise an integrated system for constitutive or inducible expression of cDNAs, shRNAs or miRNAs, and use a wide variety of drug selection markers. This system can be easily expanded to accommodate new designs and technologies to rapidly screen expression conditions or protein levels that would ensure maximal expression and biological activity.
4:45 Development of Algal Chloroplasts as a Novel Bioreactor for the Production of Pharmaceutical Proteins
Shengwu Ma, Ph.D., Plantigen Inc.; University of Western Ontario
Eukaryotic microalgae have recently received significant attention as a promising alternative to plant, bacteria or mammalian cell bioreactors for the production of recombinant pharmaceutical proteins, because of their simple growth requirements, ease of manipulation and high growth rate. The unicellular green alga Chlamydomonas reinhardtii is particularly attractive as a bioreactor since high levels of foreign protein accumulation have been achieved in its chloroplast. Apart from being easily transformed with foreign DNA, stable transgenic strains and high production volumes in full containment can be obtained with C. reinhardtii within a relatively short time. Furthermore, C. reinhardtii is a green alga which is generally recognized as safe (GRAS) for use as a food ingredient and therefore has the potential as a carrier for direct oral delivery of therapeutic proteins. In this presentation, I will review the progress made in the use of C. reinhardtii chloroplasts for the production of high-value therapeutic proteins, with particular reference to the expression of diabetes-associated autoantigen human glutamic acid decarboxylase (hGAD65) using the algal chloroplast organelle.
5:15 End of Conference
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