Modulation of infectivity in phage display as a tool to determine the substrate specificity of proteases

Chembiochem : a European Journal of Chemical Biology
Javier F Chaparro-RiggersUwe Bornscheuer

Abstract

Proteases play an important role in human and animal diseases. Rapid determination of substrate specificity is possible through the use of substrate phage display; however, current methods possess several drawbacks. They require phage-immobilization and cannot be used for infectivity-destroying or affinity tag-destroying proteases; this can make entire libraries useless. To overcome these limitations, here we introduce infectivity-modulated phage display (IMOP). IMOP uses a protease-resistant and infectivity-reducing tag fused to substrate-displaying polyvalent phages, and the specific cleavage of the substrate increases the infectivity of the phages by releasing the infectivity-reducing tag. The resulting phages were first tested with the infectivity-destroying detergent protease subtilisin; this resulted in a highly specific substrate at a 200-fold enrichment. In a second example, the protease ompT was used and led to an enrichment of the known double-arginine motif. The IMOP system thus substantially improves and simplifies previous systems.

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Citations

Nov 1, 2007·Chemical Communications : Chem Comm·Jacob Kofoed, Jean-Louis Reymond
Apr 8, 2010·Current Protocols in Neuroscience·Andrew R M Bradbury

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