Genetic reporter system for the detection and characterization of site specific proteases
Abstract
Site specific proteases hold great promise for several industrial and medical applications. The growing demand has prompted the development of high throughput assays that are capable of efficiently reporting on proteolytic activity. A bacterial genetic reporter system is described for the detection and characterization of site-specific proteases. The system relies on the successful transcription of a genetic reporter in the presence of active protease which leads to the survival of the host under selective pressure. An engineered single chain repressor, composed of two DNA binding domains, contains a variable hinge region where substrates can be incorporated as proteolytic bait. Intact SCR interferes with transcription and leads to host cell death. Otherwise, degradation of the SCR by a co-expressed protease will not interfere with the transcription of the reporter genes required for survival on selective media. The sensitivity of this system to report on catalytic efficiency towards different substrates was assessed using tobacco etch virus protease as a model. The genetic reporter system was characterized and selection conditions were optimized via reporter gene based assays, co-expression and biochemical assays. The applicability of this system as a genetic selection assay was tested to establish its ability to identify improved proteolytic activity from a pool of variants.
Degree
Ph.D.
Advisors
Savinov, Purdue University.
Subject Area
Biochemistry
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