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A method for directed evolution and functional cloning of enzymes.

H Pedersen·S Hölder·D P Sutherlin·U Schwitter·D S King·P G Schultz
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Abstract

A general scheme is described for the in vitro evolution of protein catalysts in a biologically amplifiable system. Substrate is covalently and site specifically attached by a flexible tether to the pIII coat protein of a filamentous phage that also displays the catalyst. Intramolecular conversion of substrate to product provides a basis for selecting active catalysts from a library of mutants, either by release from or attachment to a solid support. This methodology has been developed with the enzyme staphylococcal nuclease as a model. An analysis of factors influencing the selection efficiency is presented, and it is shown that phage displaying staphylococcal nuclease can be enriched 100-fold in a single step from a library-like ensemble of phage displaying noncatalytic proteins. Additionally, this approach should allow one to functionally clone natural enzymes, based on their ability to catalyze specific reactions (e.g., glycosyl transfer, sequence-specific proteolysis or phosphorylation, polymerization, etc.) rather than their sequence- or structural homology to known enzymes.

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Amino Acid SequenceBacteriophagesBase SequenceCloning, MolecularDNA PrimersDirected Molecular EvolutionEnzymesMolecular Sequence DataJournal ArticleResearch Support, Non-U.S. Gov'tResearch Support, U.S. Gov't, P.H.S.

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H Pedersen, S Hölder, D P Sutherlin, U Schwitter, D S King, P G Schultz. 1998-09-01. A method for directed evolution and functional cloning of enzymes.. https://doi.org/10.1073/pnas.95.18.10523

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