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Development of high-throughput in vitro expression and capture methodologies for the identification and characterisation of proteases

Reference	BBS/B/01375
Principal Investigator / Supervisor	Professor Christopher Scott
Co-Investigators / Co-Supervisors	Professor Brian Walker
Institution	Queen's University of Belfast
Department	Sch of Pharmacy
Funding type	Research
Value (£)	181,422
Status	Completed
Type	Research Grant
Start date	01/07/2004
End date	30/06/2007
Duration	36 months

Abstract

We propose to develop inhibitor-based approaches for the identification of protease species, employing in vitro transcription/translation methodologies. In essence, the approach will exploit the use of synthetic, substrate-derived affinity labels, for the covalent capture of recombinant cysteine proteases, produced from cDNA libraries in cell-free protein expression lysates. Firstly, this will involve the global expression of protein in vitro from a cDNA library, and subsequent interrogation with biotinylated affinity labels to irreversibly bind cysteine protease species. Disclosure/profiling of the expressed protease species will be achieved using electrophoretic and streptavidin-based western blotting methods developed by our group. Secondly, we propose to achieve the rapid and complete characterisation of these captured cysteine proteases at the gene level, using a novel application of ribosome display.

Summary

unavailable

Committee	Closed Committee - Engineering & Biological Systems (EBS)
Research Topics	X – not assigned to a current Research Topic
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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