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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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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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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DOI: https://doi.org/10.1038/nmeth.1262
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