A chemical genetics approach to improved IMP expression in E. coli

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DESCRIPTION (provided by applicant): High-level production of correctly folded integral membrane proteins (IMP's) remains a major obstacle to their biochemical and biophysical characterization. Expression of prokaryotic IMP'S in E. coli is a "hit-or-miss" proposition, with some expressing welt in native form but most expressing poorly or not at all. Unfortunately, the reasons for success or failure in specific cases are not understood, either in terms of the dependency on the sequence and structural features of the target IMP or in terms of the physiological systems controlling the efficiency of the biogenesis process. The situation for eukaryotic IMP's is even worse, with successful expression in E. coli being equally mysterious but substantially less frequent and the leading alternative expression systems being logistically onerous and generally very costly. In this context, improved methods for IMP expression in E. coli would have great practical value. We propose to use "chemical genetics" to explore a pharmacological approach to improving IMP production in E. coli. A limited set of forensic physiological experiments will be performed on IMP-expressing cells to provide preliminary data on the mechanism of action of active compounds.