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Day 1 | Day 2 MONDAY, 5 OCTOBER 16:00-18:30 Conference Registration 18:30 BIOTECHNICA Opening and EUROPEAN BIOTECHNICA AWARD Ceremony plus Reception
TUESDAY, 6 OCTOBER
08:30-19:00 Conference Registration
Progress in Protein Expression 10:05-10:35 Cell-Free Expression of Membrane Proteins Frank Bernhard, Ph.D., Lab Leader, Institute of Biophysical Chemistry, Goethe University-Frankfurt Cell-free expression eliminates most central problems associated with the conventional cellular production of membrane proteins and it allows completely new expression approaches by the direct synthesis of membrane proteins into defined artificial environments like detergent micelles or liposomes. We demonstrate the cell-free production of diverse groups of membrane proteins involved in transport, efflux, signaling, metabolism or biosynthesis in mg amounts by automated throughput optimization strategies. The quality of selected membrane proteins including eukaryotic solute carriers, G-protein coupled receptors and transporters has been evaluated by a number of complementary techniques. 10:35-11:00 Coffee Break Sponsored by 11:00-11:30 Secreted Expression of Self-Assembling Proteins in Pichia pastoris Catarina Ferreira da Silva, M.Sc., Bioprocess Engineering, Wageningen University and Research Centre Custom-made self-assembling proteins, resembling proteins like collagen or silk, may have a broader medical and pharmaceutical application if they could be produced in an animal-free way. The optimization of Pichia pastoris as a host organism for the production of these proteins will provide a new system for the synthesis of innovative protein materials with well-defined conformations and properties. 11:30-12:00 Lemo21(DE3): A Generic E. coli-based Protein Overexpression Platform Guaranteeing Maximum Yields Jan-Willem de Gier, Ph.D., Associate Professor, Department of Biochemistry & Biophysics, Stockholm University/Xbrane Bioscience AB A simple generic method for optimizing protein overexpression in Escherichia coli is still lacking. Therefore, we have engineered the Lemo21(DE3) strain. Lemo21(DE3) is tunable for protein overexpression and conveniently allows optimizing overexpression of any given soluble and membrane protein by using only a single strain rather than a multitude of different strains. Sponsored by Hans Buegl, Ph.D., Head, Marketing and Sales, GENEART AG We report the largest gene expression study on synthetic optimized genes in mammalian cells to Date. Fifty human genes from the NCBI Entrez database representing different protein classes such as protein kinases, cytokines, membrane proteins and transcription factors, were optimized for increased mRNA half-life and protein expression in human cells. Expressed protein levels in HEK 293T cells were quantified and compared. The results clearly indicate a significant improvement of expression yield with optimized constructs compared to respective wildtype versions. Therefore, gene synthesis is not only a versatile manner to obtain individualized genes but also allows for autologous expression increase in most cases. 12:15-12:30 Sponsored Presentation (Opportunity Available) 12:30-13:45 Lunch for purchase in the Exhibit Hall and Exhibit Viewing
Protein Expression for CMC and 14:00-14:05 Chairperson’s Remarks Trevor Wilkinson, Ph.D., Head, Protein Sciences, MedImmune Ltd 14:05-14:35 EnBase: Novel High Cell Density Culture-Based Screening Platform for Recombinant Protein Production and Bioprocess Development Peter Neubauer, Ph.D., Professor, Department of Bioprocess Technology, Institute of Biotechnology, Technische Universität Berlin EnBase is a unique microbial cultivation platform for high cell density growth in micro-well plate and shake flask formats. It is based on the principle of the glucose-limited fed-batch technology but applies an enzyme controlled internal delivery system for the controlled supply of glucose which allows easy scaling to any cultivation volume, including microwell cultures. Here we demonstrate that EnBase works well for the production of a large number of recombinant proteins in various shake flask and micro-well plate formats. Interestingly, aside from 10-20x higher cell densities compared to shaken cultures, and an equal growth in parallel cultures, in a number of examples the amount of the soluble active form of the target product was significantly increased per cell unit. Scalability of the Enbase technology has been shown in 100 liter cultivations. 14:35-15:05 Helmholtz Protein Sample Production Facility for Joop van den Heuvel, Ph.D., Group Leader, Structural Biology - Protein Sample Production Facility, Helmholtz Centre for Infection Research The Helmholtz PSPF is a unique infrastructure for the production of pure proteins in adequate amounts for biochemical and 3-dimensional structural studies by X-ray crystallography and NMR spectroscopy. The PSPF is a German-wide open access support facility for structural biologists and will participate in the European infrastructure initiative INSTRUCT (EU Frameworkprogram 7). A broad package of advanced techniques are now available that allow protein expression in a range of cultivation systems. 15:05-15:35 Sponsored Presentation (Opportunity Available) 15:35-16:00 Refreshment Break Sponsored by
Alexei Yeliseev, Ph.D., Staff Scientist, LMBB, NIH/ NIAAA The peripheral cannabinoid receptor, CB2, a heptahelical G protein-coupled membrane receptor, has become one of the most sought after pharmaceutical targets. Structural studies on CB2 by NMR spectroscopy and other biophysical techniques will contribute greatly into the development of novel specific ligands targeting this receptor. In order to study CB2 at functional conditions, reconstitution of the purified receptor into liposomes is required. The ability of CB2-proteoliposomes to activate G-proteins in response to agonist binding was studied as a function of lipid composition and detergent concentration. The fermentation protocol was adapted to expression in minimal media supplemented with stable isotope-labeled tryptophan, and yielded 2 mg of 15N-tryptophan-labeled CB2 from 1L of culture. An efficient incorporation of the isotope was confirmed by mass spectrometry. We further adapted fermentation procedures for uniform labeling of CB2 with 13C and 15N that produced over 3 mg/L of labeled material. Functionally active 15N/13C labeled CB2 was reconstituted into liposomes, and is being currently analyzed by solid state NMR. 16:30-17:00 Library-Based Construct Screening for Difficult-to-Express Proteins: Influenza Polymerase as a Case Study Darren Hart, Ph.D., Team Leader, High-Throughput Protein Technologies, European Molecular Biology Laboratory The ESPRIT construct screening technology has been developed at EMBL to identify soluble constructs of “difficult-to-express” protein targets that resist the classical approach of bioinformatics and PCR cloning. In each experiment, 30,000 individual constructs are assayed in parallel for yield and solubility using a highly automated colony array format. Results will be presented on the influenza polymerase that has, prior to this study, proved intractable due to the absence of homologues required for multiple sequence alignments. Previously unsuspected domains were expressed and characterized structurally by X-ray crystallography and NMR, providing insights into the mechanisms of virus replication. 17:00-17:30 Tackling Difficult-to-Express Proteins for Structural Studies: A Case Study of Using Library Methods Chris Meier, Ph.D., Prinicipal Scientist, Crystallography, UCB Celltech Availability of multi-milligram quantities of soluble and monodispersed protein is an absolute requirement for crystallographic studies. Traditionally, Bioinformatics methods have been used to identify suitable expression constructs. Recently, a number of alternative techniques have been developed which use a different approach: instead of bioinformatically designing expression constructs, large genetic libraries are screened for gene fragments which give high-level soluble expression. This talk discusses the study of a difficult-to-express protein. In collaboration with Domainex, a library screening experiment identified a construct with excellent expression and crystallization properties. Analysis of the crystal structures explains some of these properties.
17:30-17:45 Move to Breakout Discussion Groups
17:45-19:00 Interactive Breakout Discussion Groups Mammalian Expression Systems: Options for Producing Moderator: Trevor Wilkinson, Ph.D., Head, Protein Sciences, MedImmune Ltd Mammalian expression systems - current options
GPCRS: Overcoming Cell Free Expression Challenges Moderator: Frank Bernhard, Ph.D., Lab Leader, Institute of Biophysical Chemistry, Goethe University Frankfurt Expression of GPCRs has traditionally been difficult, no matter the host chosen.
Saving Time and Costs for Antibody Expression in Mammalian Cells Moderator: Gerald Casperson, Ph.D., BioTherapeutics Center of Emphasis, Pfizer Discovery Research Discussion will include:
E. coli Expression Systems: Making the Right Choices to Enhance Expression Moderator: Ian Hodgson, Ph.D., Head, Molecular Biology, Research and Development, Avecia Biologics As the workhorse host for protein expression, E. coli has been used both at the bench level and at the industrial level. However, there are still many choices to be made during the process to enhance the eventual outcomes. We will discuss:
19:00-21:00 CHI Reception Sponsored by |
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