Wednesday, 26 September
17:00-18:00 Early Registration
Thursday, 27 September
07:30 Registration, Poster Setup and Morning Coffee
08:30 Welcome by Session Chairperson
08:45 New High-Yield Eukaryotic Protein
Expression System Based on the Protozoan Leishmania Tarentolae
Kirill Alexandrov, Ph.D., Department of Physical Biochemistry, Max-Planck-Institute for Molecular Physiology
Production of functional eukaryotic proteins in recombinant form is a bottle-neck in various post-genomic applications and in life science in general. At least partially this is due to the problems associated with the use of endogenous RNA polymerase II for high-level transcription of heterologous genes in eukaryotic expression.To circumvent these problems we developed an inducible protein expression system based on a protozoan Leishmania tarentolae. In this system high level transcription of protein coding genes can be mediated by a repressor controlled T7 RNA polymerase. From this perspective the system represents a eukaryotic analogue of the most successful bacterial expression architecture. We demonstrate that the system can inducibly over-express intracellular proteins with a yield of > 10% to >total cellular protein (ca. 300mg protein per liter of culture). The system is also suitable for production of secreted mammalian-like glycosylated proteins as shown on the example human erythropoietin. Once established, the recombinant strains are stable for hundreds of generations which qualifies them for large scale fermentation. L. tarentolae handling procedures are amenable to automation thus making it a new and attractive host for production of recombinant proteins for various applications.
09:15 Co-Expression of Endogenous and Heterologous Genes in Hansenula polymorpha
Jens Klabunde, Molecular Biology, Artes-Biotechnology GmbH
The methylotrophic yeast Hansenula polymorpha is known for its strong expression efficiency of heterologous protein whereas total amounts of secreted or intracellulary deposed products exceed the productivity of other microbial hosts as demonstrated by many examples. H. polymorpha strains expressing various commercially relevant proteins, and containing additional copies of different bottleneck genes, displayed significantly enhanced secretion of all proteins tested from already high initial levels. Results from shake flask cultures could be reproduced on high cell density pilot fermentation scale. In addition, the excellent suitability of H. polymorpha for co-expression strategies was successfully applied in a biocatalysis approach to convert D-fructose to D-mannitol. Thus, co-expression approaches may provide a generally applicable tool of enhancing the production of any type of recombinant protein or bio-transformation of renewable starting material to valuable products using the well established H. polymorpha platform.
09:45 Development of Ophiostoma Floccosum and Ophiostoma Piliferum as Hosts for Recombinant Protein Expression
Lisa Robson, Researcher, Biological Sciences, University of Waikato-New Zealand
Ophiostoma floccosum and Ophiostoma Piliferum are dimorphic ascomycete fungi found throughout the world colonizing timber. Both species are important economically as they are known to cause discoloration of wood thus reducing its aesthetic value and subsequently price. Albino variants of the two species are used as biological control agents to prevent sap staining and have been used commercially for the past 15 years to reduce pitch/wood extractives in paper manufacturing. Extracellular xylanases and lipases were characterized from these species and have been demonstrated to have post translational modifications. Due to the capability of these Ophiostoma species to be fermented and their biotechnology applications, they are particularly suitable as hosts capable of excreting extracellular recombinant proteins. Our research is aimed at developing the two species as hosts for protein expression for both biotechnological application and to better understand protein expression in these organisms. Recombinant strains of both species have been produced using various transformation methods. To date, vectors used in these transformations have contained heterologous promoters and termination sequences. To optimize the efficiency of these systems we have sought to identify promoters in both species that may be applied to a vector system. Using expressed tag sequence analysis (EST’s), abundantly transcribed proteins and proteins of interest have been identified. Promoters and various transcriptional elements such as secretion signals have been elucidated from selected proteins using chromosome walking methods. Current efforts are focused on incorporating these host transcriptional elements into select vectors.
10:15 Morning Coffee, Poster and Exhibit Viewing
11:00 Suspension-Cultured Plant Cells as an Efficient Production System for Glyco-Allergens
Veronique Gomord, CR, Biology, CNRS UMR 6037, Université de Rouen, UFR des Sciences
The replacement of crude allergen extracts by selected major allergens currently represents a major goal to improve allergy diagnosis and immunotherapy. Indeed, the development of better defined products devoid of non allergic molecules would facilitate both standardization and enhance batch to batch reproducibility and treatment specificity. In this study we have investigated the potential of plant cells to produce biologically active forms of the two major allergens Der p1 and Der p 2 from the house dust mite Dermatophagoides pteronyssinus. After purification from cell culture media, a detailed characterization of such plant-made allergens has shown similar proteolytic maturation, folding and glycosylation as well as comparable immuno-reactivity to their natural counterparts. Specifically, our study demonstrates the value of plant cells over yeasts or E. coli for production and post-translational maturations of these allergens. Altogether, these results illustrate that suspension-cultured BY2 tobacco cells represent a low cost and environmentally-safe expression system suitable to produce the two major allergens Der p1 and Der p2 from D.pteronyssinus under a form appropriate for diagnostic and therapeutic purposes.
11:30 Production of Pharmaceutical Proteins in Plants and Plant Suspension Cells
Stefan Schillberg, Ph.D., Head of Department Plant Biotechnology, Fraunhofer-Institut für Molekularbiologie und Angewandte Oekologie IME
Plants and plant suspension cells provide an inexpensive and convenient system for the large-scale production of valuable recombinant proteins. The principle has been demonstrated by the success of a diverse repertoire of proteins, with therapeutic molecules showing the most potential for added value, and many others are currently under development. Plants have many advantages over other production systems, particularly in terms of practicality, economy and safety. However, several constraints that hinder the widespread use of plants as bioreactors remain to be addressed. The production of pharmaceutical proteins in plants will only realize its huge potential if the products are provided at consistent high quality levels, allowing the delivery of clinical grade proteins that will gain regulatory approval and which can be used routinely in clinical trials.
12:00 Technology Focus (Sponsorship Available)
|12:15 Luncheon Technology Workshop
13:25 Comments by Session Chairperson
Stefan R. Schmidt, Ph.D., AstraZeneca GPS&S
13:30 Development of a Generic Recombinant Protein Production Platform: A Fast Route to Soluble, Secreted Proteins at High Titre
Ian Hodgson, Ph.D., Head of Molecular Biology, ECS, Avecia Biologics
The production of proteins for therapeutic development, or structure/characterization studies, share a common need - speed, less process development time, coupled with high titre of soluble biologically active material. We have developed a novel generic protein production platform - pAVEwayTM that provides a family of expression systems, with a broad host range, designed for high level recombinant protein expression, including toxic proteins/membrane proteins, with minimal process development to achieve high titre. The design and construction of the systems will be described, together with case studies exemplifying performance, in both microbial and mammalian expression hosts, for the production of a wide range of therapeutically useful proteins (cytokines, growth factors, antibody fragments, vaccines etc) at titres >10g/L.
14:00 Adapting the High Level Type I Secretion Apparatus of Caulobacter Crescentus for Recombinant Protein Export or Surface Display
John Smit, Ph.D., Professor of Microbiology, Microbiology and Immunology, University of British Columbia
Caulobacter Crescentus is a harmless freshwater bacterium that produces a useful two-dimensional crystalline assembly on its outer surface, composed of a single highly expressed protein. Because it uses the flexible Type I secretion mechanism for export of this protein, we have been able to adapt the export system to enable secretion of a wide variety of proteins (using the C-terminal secretion signal) or for display of peptides and proteins on the cell surface, using the entire S-layer gene. Display of peptides of 10-300 amino acids is commonly accomplished. Because of the crystalline nature of the S-layer successful presentation can occurs at densities of 40,000 copies/cell. In one application we have displayed a complete Protein G binding capability (involving 3 IgG binding domains and spacer peptides), enabling use of the bacteria for pull-down immunoassays. Other applications of the S-layer display in development include whole cell vaccines for veterinary or anti-cancer applications, and infection blocking agents. Cloning and expression is based on small, high copy number plasmids that readily shuttle to E. coli. Protein secretion by fusion to the C-terminal secretion signal can also take advantage of a built-in purification mechanism; the aggregate forming properties of the S-layer monomer secretion signal enable rapid purification by simple filtration.
14:30 From Dust Balls to Biocatalysts: A New View of Bacterial Inclusion Bodies
Salvador Ventura, Ph.D., Associate Professor, Institute of Biotechnology and Biomedicine, Department of Biochemistry and Molecular Biology, Universitat Autonoma de Barcelona
The aggregation of polypeptides into insoluble polypeptide chains as inclusion bodies (IBs) during protein production in bacteria is of major concern in biotechnology. It is a widespread belief that IB proteins are biologically inert and therefore useless in bioprocesses, accordingly many biologically relevant proteins have been disregarded for commercialization. Nevertheless, in the last few years an increasing body of evidence comes to contradict this traditional view of IBs. Here we will discuss these new data, which opens an opportunity to tune in vivo protein aggregation and to produce highly active IBs for biotechnological processes.
15:00 Alternatives to CHO: Protein Accumulation Strategies in Eukaryotic Cell Factories
Miriam Bastida, Ph.D., ERA Biotech S.A
ERA Biotech has developed a designed storage organelle technology which enables new classes of protein products and enhances manufacturing of existing ones. This new paradigm in protein production is based on the Zera family of assembler peptides which facilitate the stable in vivo accumulation of fully assembled, folded eukaryotic proteins. Fusions of the Zera domain to heterologous proteins results in the formation of synthetic membrane-bound structures (StorPro organelles) containing high concentrations of the respective target proteins. Zera is derived from a GRAS (generally recognized as safe) source and forms non-random aggregates which retain folding and function in situ of tethered recombinant proteins. StorPro organelles can be induced to form in most eukaryotic cells, including standard mammalian host cell lines, insects and insect cells transfected using baculovirus, filamentous fungi, and plant cells. Encapsulation within StorPro organelles serves to both insulate target proteins from endogenous metabolic and proteolytic activities as well as to insulate the host cell from any adverse biological activities of the target protein. The resulting ca. 1 micron stable structures are highly dense and can be readily separated from lysed cell debris using non-chromatographic means. Zera technology is currently being explored by more than 25 global commercial, academic and public-sector partners.
15:30 Technology Focus (Sponsorships Available)
15:45 Afternoon Refreshment Break, Poster and Exhibit Viewing
16:15 A High-Throughput Protein Production Pipeline Applied to the Characterization of a Pan-Genome Atlas of Transcription Factor DNA-Binding Specificity in Ciona Intestinalis
Renaud Vincentelli, Head, Methods in Structural Genomics, AFMB-UMR CNRS 6098
More than 600 sequence-specific transcription factors (TFs) have been annotated in the genome of the ascidians Ciona Intestinalis. This organism has been selected by a consortium of three groups in Europe and one in Japan as a model for the first characterization of the full repertoire of DNA Binding domain (DB) specificity of all TFs in a metazoan genome. The strategy is to express milligrams of all the TF DNA BDs in E. Coli and characterize in vitro their DNA binding specificity by automated (HTP) SELEX (Systematic Evolution of Ligands by EXponential enrichment) in 96 well formats followed by Gel-shift assays. After extensive and multiple optimizations of expression parameters in E. Coli, a cheap and easy consensus production and purification protocol could be applied on a first set of 330 DNA BD TF at a purification rate of 48-96 proteins/week. Most of the proteins could be purified in milligram amounts. This production protocol, which ,was developed for the transcription factors, has since been successfully applied to hundreds of proteins of multiple origins. The result of the i characterization of the DNA specificity of the TF DNA BDs should provide a global view of the repertoire of DNA-binding specificity in a chordate. Furthermore, evidences suggest that these DNA binding consensus sequences are likely to be conserved within other organisms including vertebrates.
16:45 Networking Reception in Exhibit Hall
18:00 End of Day one