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Improving baculovirus expression of multiprotein complexes in insect cells

Reference	BB/C504735/1
Principal Investigator / Supervisor	Professor Polly Roy
Co-Investigators / Co-Supervisors
Institution	London Sch of Hygiene and Trop Medicine
Department	Infectious and Tropical Diseases
Funding type	Research
Value (£)	295,769
Status	Completed
Type	Research Grant
Start date	01/06/2005
End date	31/05/2008
Duration	36 months

Abstract

The baculovirus expression system has well established potential for the production of large amounts of correctly folded eukaryotic proteins for enzymatic and structural studies. However, it is becoming increasingly clear that many, if not most, proteins are active within cells as complexes made up of the products of several distinct genes. Given the acceleration of the pace of discovery of protein structures, there is a real need to establish systems for the rapid and reliable production and purification of protein complexes. This is particularly true of larger complexes that require the simultaneous expression, and eukaryotic folding and processing, of may proteins. We have played a leading role in the development of the baculovirus expression system for the production of multi-protein complexes, and have designed transfer vectors capable of expressing 2, 3 4 and 5 proteins from the same baculovirus genome. Our main goal in these studies was to produce a system capable of synthesising large amounts of viral protein complexes containing non-equimolar amounts of virus encoded proteins for structural and enzymatic studies. One of the outcomes of our previous experience with these systems is that single baculoviruses expressing multiple genes are much more efficient at forming the desired protein complexes than co-infection of insect cells with multiple baculoviruses expressing single genes. In this proposal we will use existing and new technologies for the production of recombinant baculoviruses expressing multiple proteins for the formation of biologically relevant mammalian protein complexes. In particular, we will investigate the optimal arrangement of expression cassettes and promoters within the baculovirus genome and quantify relative expression levels from these promoters when multiple recombinant proteins are synthesised. In addition, we will investigate the use of new bacterial chromosome engineering technologies for the rapid and efficient production of recombinant baculoviruses expressing multiple proteins simultaneously. In order to demonstrate the general utility of baculovirus multiprotein expression technology to the production of useful amounts of mammalian protein complexes, we will use the chaperonin CCT as a model system. CCT is an important functional enzyme complex made up of 8 polypeptides expressed from separate genes but is only found at low levels in most mammalian tissues. Thus it is an ideal example to demonstrate the potential of the baculovirus system for the production of recombinant mammalian protein complexes.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	Industrial Biotechnology, Microbiology, Technology and Methods Development
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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