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Science 21 May 1993:
Vol. 260. no. 5111, pp. 1113 - 1117
DOI: 10.1126/science.8493554
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Articles

Science, Vol 260, Issue 5111, 1113-1117
Copyright © 1993 by American Association for the Advancement of Science

articles

Substrate phage: selection of protease substrates by monovalent phage display

DJ Matthews and JA Wells

Department of Protein Engineering, Genentech, South San Francisco, CA 94080.

A method is described here for identifying good protease substrates among approximately 10(7) possible sequences. A library of fusion proteins was constructed containing an amino-terminal domain used to bind to an affinity support, followed by a randomized protease substrate sequence and the carboxyl-terminal domain of M13 gene III. Each fusion protein was displayed as a single copy on filamentous phagemid particles (substrate phage). Phage were then bound to an affinity support and treated with the protease of interest. Phage with good protease substrates were released, whereas phage with substrates that resisted proteolysis remained bound. After several rounds of binding, proteolysis, and phagemid propagation, sensitive and resistant substrate sequences were identified for two different proteases, a variant of subtilisin and factor Xa. The technique may also be useful for studying the sequence specificity of a variety of posttranslational modifications.


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