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Directed enzyme evolution via small and effective neutral drift libraries

Abstract

Small libraries for directed evolution can be obtained by neutral drifts that maintain the protein's original function, yielding highly polymorphic, stable and evolvable variants. We describe methods for preparing such libraries, using serum paraoxonase (PON1). An optimized GFP variant fused to PON1 reported levels of soluble, functional enzyme, enabling selection by flow cytometry and identification of enzyme variants exhibiting improved specific and total activities toward several substrates, including toxic organophosphates.

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Figure 1: Correlation of fluorescence and enzymatic activities in fusions of PON1 variants with three different GFP-variant reporters.
Figure 2: Library screens.

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Acknowledgements

Financial support by US National Institutes of Health (81XWH-07-2-0020) and Defense Threat Reduction Agency (HDTRA 1-07-C-0024) is gratefully acknowledged. We thank O. Khersonsky for substrate synthesis, D. Tal for assistance with protein purification and N. Tokuriki for help in creating the GFP constructs.

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Correspondence to Dan S Tawfik.

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Supplementary Figures 1–2, Supplementary Tables 1–4, Supplementary Note, Supplementary Methods (PDF 466 kb)

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Gupta, R., Tawfik, D. Directed enzyme evolution via small and effective neutral drift libraries. Nat Methods 5, 939–942 (2008). https://doi.org/10.1038/nmeth.1262

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