Research project C3/018 (Research action C3)
Context
Over a period of twenty years, biomolecules have taken up an important position within the spectrum of pharmaceuticals. The advance of these compounds was significantly stimulated because of the development of cell lines and organisms that can serve as expression hosts for heterologous proteins. In this regard, yeast and fungi could play an important role. Compared to animal cell lines, they are more easy to manipulate and much cheaper in use during the protein production process. On top of that and unlike prokaryotic organisms such as E. coli, they can perform post-translational modifications such as the addition of asparagine- or N-linked glycans. The latter modification can play an essential role in the correct folding, bio-activity and pharmaco-kinetic stability of the produced heterologous protein.
Project description
Objectives
N-glycosylation in yeast and fungi differs significantly from that in mammals. Since the effect of the N-glycans on a protein’s bio-activity and pharmacokinetics is also influenced by their specific structure, this project aims to generate plasmids that allow the production of sufficient amounts of interesting heterologous proteins in fungal organisms with a homogenous mammalian-compatible type of N-glycosylation. This objective can be divided into different parts:
- Valorization of existing BCCM/LMBP plasmids for glycan engineering.
- Generation and deposit of new plasmids that allow the modification of the glycosylation synthesis pathway in yeast and fungi via a minimum of steps.
- N-glycan analysis of a large collection of fungal organisms from BCCM/MUCL (partner 2), in order to identify potential industrial yeast strains with interesting N-glycan profiles.
- Introduction of a surface display system in Pichia pastoris for the selection of better secreting protein variants (muteins) or better producing yeast strains. For this, thrombomodulin and endosialin will be used as model proteins.
Methodology and interaction between the different partners
- Different enzymes involved in the N-glycan synthesis of higher eukaryotes need to be introduced into the fungal organism in order to convert their typical high-mannose carbohydrates into more mammalian-like structures such as GalGlcNAcMan5GlcNAc2. To reduce the number of transformation steps and number of introduced expression cassettes, partner 1 will try to generate fusions between some of these enzymes. The functionality of these fusions will in first instance be tested in the yeast Pichia pastoris and the filamentous fungus Trichoderma reesei. Attention will be paid in testing different interesting localization signals as well as linker peptides between the different partners of the fusion protein.
- Using a technique developed in the lab of partner 1, it is now possible to do an N-glycan profiling on a relative small amount of a biological sample. This allows for the analysis of the carbohydrate structures of an extensive number of yeast strains. A collection of different strains, representing all phylogenetic groups within the MUCL fungal library, will be grown after which the N-glycans will be analyzed and evaluated by partner 1 via capillary electrophoresis.
- Experiments have established a correlation between the level of surface expression of a given protein on a yeast cell and the efficiency by which it would be secreted by the same cell. The production of the lectin domains of thrombomodulin and endosialin, two proteins placed at the project’s disposition by partner 3, is rather low for the expression systems tested. Partner 1 will put these proteins on the surface of Pichia pastoris and, after mutagenesis of the display host, screen for a better producing strain via FACS (Fluorescent Assisted Cell Sorting) analysis. In an alternative approach, a mutant library of both lectins can be expressed on the surface of Pichia pastoris. FACS screening can then be used to pick up better secreting variants (muteins) of the heterologous protein. The functionality of the produced proteins will be tested by partner 3 via in vitro and in vivo assays.
Expected results and/or products
- A Pichia and Trichoderma strain which produces homogenous GalGlcNAcMan5GlcNAc2 in a minimum of transformation steps. The heterologous therapeutic proteins will be less immunogenic for men and (after in vitro addition of sialic acid) have a longer half-life in the circulation. The strains will also be valuable tools in the elucidation of the effect of the different glycan structures on the functionality of the underlying protein.
- The N-glycan analysis of a large collection of fungi could contribute to a better understanding of fungal glycosylation and will generate a subcollection of better-characterized organisms. Moreover, it can result in the identification of one or more strains with interesting properties for heterologous protein production.
- The Pichia surface display will be valuable for affinity maturation, modulation of enzymatic activities and the selection of better secreting variants or strains of a specific protein. Data generated with the thrombomodulin and/or endosialin domains, produced within the project, can have a strong impact on the development of new therapies for several inflammatory or malignant diseases.
Partners
Activities:
- Partner 1 (LMBP) contains a collection of 2500 characterized plasmids. The host institute of LMBP is, amongst others, involved in the modification of the N-glycosylation profile of fungus-produced therapeutic glycoproteins, in the investigation of the role of glycans in eukaryotic signaling and the bio-activity of proteins and in the application of glycan analysis as a diagnostic tool for diseases and aging.
- Partner 2 contains a collection of about 28.000 yeast and fungal strains. The host laboratory of partner 2 investigates amongst others the use of those organisms in the food industry, in agriculture and in the protection of the environment.
- Partner 3 uses animal models and functional genomics for the elucidation of clinically relevant medical questions. They are specifically interested in angiogenesis, which plays a central role in numerous cardiovascular and inflammatory diseases.
Contact information:
- Promotor partner 1:
Prof. Dr. Roland Contreras
Unit for Fundamental and Applied Molecular Biology
Department for Molecular Biomedical Research (gastlabo BCCM/LMBP)
Ghent University and VIB
Technologiepark 927
9052 Gent-Zwijnaarde
Tel.: 09/33.13.633
Fax.: 09/33.13.502
E-mail: roland.contreras@dmbr.UGent.be
http://www.dmbr.ugent.be; http://bccm.belspo.be/about/lmbp.php
- Promotor partner 2:
Prof. Dr. Anne-Marie Corbisier
Unité de Microbiologie (MBLA)
Université catholique de Louvain (gastuniversiteit BCCM/MUCL)
Place Croix du Sud 3
1348 Louvain-la-Neuve
Tel.: 010/47.82.61
Fax.; 010/45.15.01
E-mail: corbisier@mbla.ucl.ac.be
http://bccm.belspo.be/about/mucl.php
- Promotor partner 3:
Dr. Edward Conway
Center for Transgene Technology and Gene Therapy
KU Leuven en VIB
Campus Gasthuisberg O&N
Herestraat 49
3000 Leuven
Tel.: 016/34.57.83
Fax.: 016/34.59.90
E-mail: ed.conway@med.kuleuven.ac.be
http://www.kuleuven.be/kuleuven/index.htm
Users committee
- Dr. Thierry Dauvrin, Senior Research Manager, Beldem, Andenne
- Dr. Marc Logghe, Expert Scientist bioinformatics, DevGen NV, Gent-Zwijnaarde
- Prof. Dr. Philippe Büscher, Instituut voor Tropische Geneeskunde, Antwerpen
- Dr. Geert De Jaeger, Department of Plant systems Biology, Universiteit Gent en VIB, Gent-Zwijnaarde
- Dr. Florence Xhonneux, Biologics R&D Services Manager, Eurogentec s.a., Seraing
The members of the users committee are familiar with one or more aspects of the project. New developments within the project can constitute a surplus value for their production processes or within their own research domains: expression of heterologous proteins in modified strains; identification of new expression systems; the use of the Pichia surface expression system for the identification of enzymes with changed properties, for the study of protein-protein interactions,…
