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Function-based isolation of novel enzymes from a large library

Nature Biotechnology volume 18, pages 1071–1074 (2000)Cite this article

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 × 105 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.

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Figure 1: FRET substrate binding and fluorescence emission upon catalytic turnover or binding to protein displayed on the cell surface.
Figure 2
Figure 3: Flow cytometric discrimination of E. coli on the basis of OmpT activity.

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Acknowledgements

We would like to thank W.F. Mangel, M. Ostermeier, and C.B. Mullins for reading the manuscript and for many useful comments. We also thank the University of Texas Peptide Synthesis Facility, and the University of Texas Mass Spectrometer Facility. This work was supported by grants from the National Science Foundation BES, DARPA(Defense Advanced Research Projects Agency)(MDA972-97-10009), US Department of Energy (DE-96ER62322), Texas Higher Education Coordinating Board (ATP 3658-0423-1999), and the Welch Foundation (G.G.).

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  1. Patrick Daugherty

    Present address: Fred Hutchinson Cancer Research Center, Division of Molecular Medicine, P.O. Box 19024, Seattle, WA, 98109-102

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, The University of Texas at Austin, Austin, 78712, TX

    Mark J. Olsen & Brent L. Iverson

  2. Department of Chemical Engineering, The University of Texas at Austin, Austin, 78712, TX

    Daren Stephens, Patrick Daugherty & George Georgiou

  3. The Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, 78712, TX

    Daren Stephens, Devin Griffiths, George Georgiou & Brent L. Iverson

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  1. Mark J. Olsen
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Correspondence to George Georgiou or Brent L. Iverson.

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Olsen, M., Stephens, D., Griffiths, D. et al. Function-based isolation of novel enzymes from a large library. Nat Biotechnol 18, 1071–1074 (2000). https://doi.org/10.1038/80267

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