High Throughput Substrate Phage Display for Protease Profiling

Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 539)

Summary

The interplay between a protease and its substrates is controlled at many different levels, including coexpression, colocalization, binding driven by ancillary contacts, and the presence of natural inhibitors. Here we focus on the most basic parameter that guides substrate recognition by a protease, the recognition specificity at the catalytic cleft. An understanding of this substrate specificity can be used to predict the putative substrates of a protease, to design protease activated imaging agents, and to initiate the design of active site inhibitors. Our group has characterized protease specificities of several matrix metalloproteinases using substrate phage display. Recently, we have adapted this method to a semiautomated platform that includes several high-throughput steps. The semiautomated platform allows one to obtain an order of magnitude more data, thus permitting precise comparisons among related proteases to define their functional distinctions.

Key words

Substrate phage display Substrate Protease Specificity Proteolysis Filamentous phage M13 coat protein 3gene protein 

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Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Center on Proteolytic PathwaysBurnham Institute for Medical ResearchLa Jolla

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