Function-based isolation of novel enzymes from a large library

Here we describe a high-throughput, quantitative method for the isolation o f enzymes with novel substrate specificities from large libraries of protei n variants. Protein variants are displayed on the surface of microorganisms and incubated with a synthetic substrate consisting of (1) a fluorescent d ye (2) a positively charged moiety (3) the target scissile bond, and (4) a fluorescence resonance energy transfer (FRET) quenching partner. Enzymatic cleavage of the scissile bond results in release of the FRET quenching part ner while the fluorescent product is retained on the cell surface, allowing isolation of catalytically active clones by fluorescence-activated cell so rting (FACS). Using a synthetic substrate with these characteristics, we en riched Escherichia coli expressing the serine protease OmpT from cells expr essing an inactive OmpT variant by over 5,000-fold in a single round. Scree ning a library of 6 x 10(5) random OmpT variants by FAGS using a FRET pepti de substrate with a nonpreferred Arg-Val cleavage sequence resulted in the isolation of variant proteases with catalytic activities enhanced by as muc h as 60-fold. This approach represents a potentially widely applicable meth od for high-throughput screening of large libraries on the basis of catalyt ic turnover.