Function-based isolation of novel enzymes from a large library

来自 EBSCO

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作者：

MJ Olsen，D Stephens，D Griffiths，PS Daugherty，BL Iverson

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摘要：

Here we describe a high-throughput, quantitative method for the isolation of enzymes with novel substrate specificities from large libraries of variants. 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 , allowing isolation of catalytically active clones by fluorescence-activated sorting (). Using a synthetic substrate with these characteristics, we enriched expressing the serine protease from expressing an inactive variant by over 5,000-fold in a single round. Screening a library of 6 x 10(5) random variants by using a FRET substrate with a nonpreferred Argcleavage sequence resulted in the isolation of variant proteases with 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.

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关键词：

flow cytometry directed enzyme evolution fluorescence

DOI：

10.1038/80267

被引量：

348

年份：

2000