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Directed Evolution of Highly Selective Proteases by Using a Novel FACS‐Based Screen that Capitalizes on the p53 Regulator MDM2
Author(s) -
Yoo Tae Hyeon,
Pogson Mark,
Iverson Brent L.,
Georgiou George
Publication year - 2012
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201100718
Subject(s) - proteases , protease , regulator , peptide , selectivity , chemistry , fluorophore , biochemistry , directed evolution , microbiology and biotechnology , computational biology , combinatorial chemistry , enzyme , biology , fluorescence , gene , mutant , physics , quantum mechanics , catalysis
We describe a novel method for the isolation of selective protease variants displayed on the surface of E. coli. The method relies on the electrostatic capture of an autoinhibited protein on the cell surface, combined with external labeling using a fluorophore‐conjugated binding peptide (PMI‐FL). Using this method, we isolated an OmpT variant that hydrolyzes a target sequence (between Ala‐Arg) with high selectivity.