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PEDS Advance Access originally published online on September 15, 2009
Protein Engineering Design and Selection 2009 22(11):691-698; doi:10.1093/protein/gzp052
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

An in vitro selection strategy for conferring protease resistance to ligand binding peptides

Bill Eldridge1,3, R. Neil Cooley1, Richard Odegrip2, Duncan P. McGregor3, Kevin J. FitzGerald1 and Christopher G. Ullman1,4

1 Isogenica Ltd., The Mansion, Chesterford Research Park, Little Chesterford, CB10 1XL, UK 2Department of Genetics, Microbiology and Toxicology, Stockholm University, Svante Arrheniusväg 16E, Floor 5, Room E549, 106 91 Stockholm, Sweden 3Cyclogenix, Rowett Institute, Greenburn Road, Aberdeen AB21 9SB, UK

4 To whom correspondence should be addressed. E-mail: chris.ullman{at}isogenica.com

One drawback to the use of peptides as therapeutics has been their susceptibility to proteolysis. Here, we have used an in vitro display technology, CIS display, to enhance the proteolytic resistance of ligand-binding peptides by selection of protecting motifs from a large peptide library. The premise to this selection was that certain linear peptides within a library could form structures capable of preventing the access of proteases to defined cleavage sites without affecting ligand binding. A diverse 12-mer peptide library was inserted between a FLAG epitope motif and a thrombin cleavage site and this construct was fused to the bacterial initiator protein RepA for CIS display selection. After five rounds of selection, protection motifs were isolated that were capable of preventing proteolytic cleavage of the adjacent thrombin site. Some of the selected peptides were also resistant to more promiscuous proteases, such as chymotrypsin and trypsin, which were not used in the selection. The observed resistance to thrombin, trypsin and chymotrypsin translated into increased resistance to plasma proteases in vitro and to an increase in circulating half-lives in rats. This method can be applied to enhancing the in vivo stability of therapeutic peptides.

Keywords: CIS display/in vitro selection/peptide/protease resistance/stability

Received January 6, 2009; revised August 10, 2009; accepted August 11, 2009.


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