title: Function-based isolation of novel enzymes from a large library.
abstract: Here we describe a high-throughput, quantitative method for the isolation of enzymes with novel substrate specificities from large libraries of protein variants. Protein variants are displayed on the surface of microorganisms and incubated with a synthetic substrate consisting of (1) a fluorescent dye (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 partner while the fluorescent product is retained on the cell surface, allowing isolation of catalytically active clones by fluorescence-activated cell sorting (FACS). Using a synthetic substrate with these characteristics, we enriched Escherichia coli expressing the serine protease OmpT from cells expressing an inactive OmpT variant by over 5,000-fold in a single round. Screening a library of 6 x 10(5) random OmpT variants by FACS using a FRET peptide substrate with a nonpreferred Arg-Val cleavage sequence resulted in the isolation of variant proteases with catalytic activities enhanced by as much as 60-fold. This approach represents a potentially widely applicable method for high-throughput screening of large libraries on the basis of catalytic turnover.
authors.affiliation: Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, TX 78712, USA.
authors.forename: M J
authors.initials: MJ
authors.lastname: Olsen
authors.forename: D
authors.initials: D
authors.lastname: Stephens
authors.forename: D
authors.initials: D
authors.lastname: Griffiths
authors.forename: P
authors.initials: P
authors.lastname: Daugherty
authors.forename: G
authors.initials: G
authors.lastname: Georgiou
authors.forename: B L
authors.initials: BL
authors.lastname: Iverson
mesh_terms: D001896:Boron Compounds / Q000378:metabolism; D002384:Catalysis; D003001:Cloning, Molecular / Q000379:methods*; D004735:Energy Transfer; D004798:Enzymes / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005434:Flow Cytometry; D005453:Fluorescence; D005456:Fluorescent Dyes / Q000378:metabolism; D014644:Genetic Variation / Q000235:genetics; D007700:Kinetics; D009154:Mutation / Q000235:genetics; D019151:Peptide Library; D012235:Rhodamines / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D001896:Boron Compounds; D004798:Enzymes; D005456:Fluorescent Dyes; D019151:Peptide Library; D012235:Rhodamines; C005358:tetramethylrhodamine; D012697:Serine Endopeptidases; C057840:omptin outer membrane protease