Objective
This project aims to develop a system for the display of complex eukaryotic proteins on the surface of insect baculovirus particles, thus complementing the powerful bacterial "phage display" technology which is already so successful. This latter system was developed to isolate specific ligands from pools of different binding proteins expressed on the viral surface as fusion proteins with a phage coat protein. This approach, however, has severe limitations when the proteins under study are large complex, eukaryotic proteins that cannot be processed correctly in a bacterial expression system.
Surface display has been performed in eukaryotic cells via the use of yeast, retroviral vectors and plant viruses but a system of comparable power to the bacteriophage has yet to be developed.
The insect baculovirus, Autographa californica nuclear polyhedrosis virus (AcMNPV), is utilized for the expression of recombinant proteins. The virus has a single major envelope glycoprotein, gp64. As a prominent surface projection, gp64 has recently been shown to be suitable for the display of foreign proteins on the baculovirus surface. Moreover, AcMNPV can accommodate large DNA insertions and grow to high titre, making it satisfactory for the generation of display libraries representing a variety of large proteins such as surface receptors, viral glycoproteins, ion channels, and enzyme complexes in a stable and functional form. Before this goal can be realized, however, further research needs to be done to understand the basis of fusion protein pick-up on the virus surface and to improve the technology for recombinant formation to enable the construction of complex libraries. This application addressed these needs with the following specific objectives:
- To determine the three dimensional structure of AcMNPV gp64 and to identify suitable sites for the optimization of insertion of foreign coding regions for display purposes.
- To develop new methods for the construction of baculovirus display libraries that represent a high level of complexity.
- To develop methods for the efficient selection of baculovirus bearing a specific surface display ligand from a background of non specific recombinants
The skills of the four partners in this collaboration encompass a broad range of experience with baculovirus expression systems including basic virology, genetics and vector design for foreign protein production. In addition, one of the partners has an established reputation within the field of phage display, its development and applications . Three of the groups are not only working on similar areas, but individually they all have initiated the development of the baculovirus surface display system, although each group has chosen somewhat different practical approaches. By developing the eukaryotic surface display system described in this proposal together, not only would the output be greater in a shorter period of time, but also the variety of different model systems would demonstrate the wide applicability of the system. The project forms an ideal target for funding under the EC Biotechnology programme.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques.
- natural sciencesbiological sciencesmicrobiologyvirology
- natural sciencesbiological sciencesgeneticsDNA
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
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Call for proposal
Data not availableFunding Scheme
CSC - Cost-sharing contractsCoordinator
40014 JYVASKYLA
Finland