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Co-Located with
 BIOTECHNICA 2009, Europe's No.1 Event in Biotechnology and Life Sciences

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

 

 

OPENING KEYNOTE SESSION

09:00-09:05 Chairperson’s Remarks

Jan-Willem de Gier, Ph.D., Associate Professor, Department of Biochemistry & Biophysics, Stockholm University/Xbrane Bioscience AB

Trevor Wilkinson, Ph.D.09:05-09:35 Protein Tools for Antibody Discovery:
Taming Complex and Difficult Proteins

Trevor Wilkinson, Ph.D., Head, Protein Sciences, MedImmune Ltd

The expression and purification of target proteins to support the development of therapeutic antibodies remains a significant challenge. An additional challenge is the requirement to support diverse activities (e.g. biochemical screening, phage and ribosome display) and to produce species variants of the human target proteins to facilitate pharmacology and toxicology studies. To support these activities we have developed a number of platforms to allow expression and purification of challenging proteins. In this presentation I will outline the capabilities developed at MedImmune and provide some case study examples of protein expression and purification

Joerg Standfuss, Ph.D09:35-10:05 Thermostable Mutants for the Crystallographic Study of GPCR Activation

Joerg Standfuss, Ph.D., Senior Post-Doctoral Researcher, MRC-Laboratory of Molecular Biology

G protein-coupled receptors (GPCRs) allow the transmission of chemical signals across the cellular membrane. GPCRs are encoded by ~800 human genes and include ~30% of all known drug targets. Due to their low stability and low natural abundance structural investigations of GPCRs have long been limited to wild-type rhodopsin. To lift this limitation we use wave bag bioreactors for large-scale production of mutant receptors in insect and mammalian cells. Alanine scanning and structural design yielded thermostable mutants that enabled us to crystallize active and inactive receptor conformations and to solve the first structure of a recombinantly produced GPCR.

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. Pharmaceutically important targets such as G-protein coupled receptors or Alzheimer’s disease related proteins can be produced in high quality in less than 24 hours and we present new strategies for their specific labeling and their functional as well as structural evaluation in particular by NMR spectroscopy.

10:35-11:00 Coffee Break Sponsored by Sloning Biotechnology 

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
GeneArt small logo
12:00-12:30 Comparative Study on Autologous Expression Improvement in Human Cells by Gene
Optimization: Results and Applications

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.

12:30-13:45 Lunch for purchase in the Exhibit Hall and Exhibit Viewing

 

Protein Expression for CMC and
Challenging Proteins

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 Large Scale Production of Protein Samples for
Structural Analysis

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  Biosilta




16:00-16:30 Preparation of Stable Isotope-Labeled Cannabinoid Receptor CB2 for NMR Structural Studies

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
Difficult-to-Express Proteins

Moderator: Trevor Wilkinson, Ph.D., Head, Protein Sciences, MedImmune Ltd

Mammalian expression systems - current options

  • Optimising protein expression - current best practice and options
  • Solutions to low expression of target proteins

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.
Expression in cell-free systems can also be difficult. This roundtable will discuss:

  • specific problems in expression of GPCRs
  • challenges and benefits of cell free systems
  • shifting the bottleneck in GPCR production from high level
  • synthesis to appropriate quality control
  • quality improvement of cell-free produced GPCRs

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:

  • Expression challenges in mammalian cells
  • Time-saving strategies
  • Cost-saving strategies
  • Big pharma perspective on savings plans

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:

  • Evaluating and choosing the correct expression route
  • Shortening process development time
  • Increasing yields

19:00-21:00 CHI Reception Sponsored by  Biosilta   DNA 2.0   GeneartNEW

21:00 Close of Day


For more information, please contact:
Elizabeth Lamb, Senior Conference Director
Cambridge Healthtech Institute
E-mail: elamb@healthtech.com 

For exhibit and sponsorship information, please contact:
Carol Dinerstein
Cambridge Healthtech Institute
Email: dinerstein@healthtech.com 
Phone: 781-972-5471