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Optimisation of membrane protein expression in E.coli and yeast: quantitation of the host response

Reference	BB/D524832/1
Principal Investigator / Supervisor	Professor Peter Henderson
Co-Investigators / Co-Supervisors	Professor Roslyn Bill
Institution	University of Leeds
Department	Inst of Molecular & Cellular Biology
Funding type	Research
Value (£)	167,234
Status	Completed
Type	Research Grant
Start date	01/12/2005
End date	31/08/2006
Duration	9 months

Abstract

Membrane proteins in general, and those from eukaryotes in particular, have always been the `high-hanging fruit¿ of structural biology, with most groups traditionally working on a single protein or protein family that they have found to be highly-abundant in native cellular membranes and amenable to, usually functional, and occasionally structural, analysis in their hands. With the advent of structural genomics and the realisation that membrane proteins comprise approximately 30 per cent of genomic information, however, the need to develop tools to study the structural biology of membrane proteins as a class has come into sharp focus, and is a major goal of current BBSRC initiatives. Since it is clear that the vast majority of membrane proteins are not available in sufficient quantities from their natural sources, the lack of suitable systems to overproduce recombinant membrane proteins is a substantial bottleneck in the pipeline from gene to structure, which must be relieved. The development of generic production systems that can be applied to a wide range of membrane proteins, rather than those that have previously been found to be effective on a case-by-case basis, is thus an area of research of the utmost importance. Since Grisshammer and Tate¿s seminal 1995 review (Grisshammer and Tate, 1995) is still regarded as the primary reference for membrane protein production, it is clear that the field as a whole has not advanced as rapidly as might have been hoped. Although more recent articles have covered the use of bacteria (Ward et al., 2000; Kunji et al. 2003), yeast (Bill, 2001), insect cells (Bosman et al., 2003), mammalian cells (Lundstrom, 2003) and cell-free systems (De Kayzer et al., 2002) as production hosts for membrane proteins (Sarramegna et al., 2003), no generic strategies for milligram-scale membrane protein production have been published. The widely-applicable solution for soluble proteins, the pET-based system of vectors from Novagen in an E. coli host, has not been applicable to membrane proteins (Wang et al., 2003), and no alternative membrane protein-specific panacea has been discovered. This means that membrane protein production is recognised by biologists as the primary bottleneck in contemporary structural genomics programs. Our proposal is to elucidate the reasons for successes and failure in recombinant membrane protein production in the prokaryote, Escherichia coli, and the eukaryotes, Saccharomyces cerevisiae and Pichia pastoris, at the level of the host cell. Using a matrix-based design of experiments, we will systematically quantify parameters of growth and metabolic activities in parallel high-performance bioreactors under tightly-defined growth regimes. The outcome will be a substantial acceleration of information about structures and molecular mechanisms of membrane proteins, which will greatly strengthen the UK¿s leading position in the structural genomics of membrane proteins.

Summary

unavailable

Committee	Closed Committee - Biomolecular Sciences (BMS)
Research Topics	X – not assigned to a current Research Topic
Research Priority	X – Research Priority information not available
Research Initiative	Research Equipment Initiative 2005 (RE5) [2005]
Funding Scheme	X – not Funded via a specific Funding Scheme

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