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Laboratory-Directed Protein Evolution

Department of Plant and Soil Sciences, and Kentucky Tobacco Research and Development Center, University of Kentucky, Cooper and University Drives, Lexington, Kentucky 40546,¹ Verdia Research Campus, Pioneer International, A Dupont Company, 700A Bay Road, Redwood City, California 94063,² Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637³

Systematic approaches to directed evolution of proteins have been documented since the 1970s. The ability to recruit new protein functions arises from the considerable substrate ambiguity of many proteins. The substrate ambiguity of a protein can be interpreted as the evolutionary potential that allows a protein to acquire new specificities through mutation or to regain function via mutations that differ from the original protein sequence. All organisms have evolutionarily exploited this substrate ambiguity. When exploited in a laboratory under controlled mutagenesis and selection, it enables a protein to "evolve" in desired directions. One of the most effective strategies in directed protein evolution is to gradually accumulate mutations, either sequentially or by recombination, while applying selective pressure. This is typically achieved by the generation of libraries of mutants followed by efficient screening of these libraries for targeted functions and subsequent repetition of the process using improved mutants from the previous screening. Here we review some of the successful strategies in creating protein diversity and the more recent progress in directed protein evolution in a wide range of scientific disciplines and its impacts in chemical, pharmaceutical, and agricultural sciences.

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