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The switch-like regulation of protein activity by molecular signals is abundant in native proteins. The ability to engineer proteins with novel regulation has applications in biosensors, selective protein therapeutics, and basic research. One approach to building proteins with novel switch properties is creating combinatorial libraries of gene fusions between genes encoding proteins that have the prerequisite input and output functions of the desired switch. These libraries are then subjected to selections and/or screens to identify those rare gene fusions that encode functional switches. Combinatorial libraries in which an insert gene is inserted randomly into an acceptor gene have been useful for creating switches, particularly when combined with circular permutation of the insert gene. Methods for creating random domain insertion libraries are described. Three methods for creating a diverse set of insertion sites in the acceptor gene are presented and compared: DNase I digestion, S1 nuclease digestion, and multiplex inverse PCR. A PCR-based method for creating a library of circular permutations of the insert gene is also presented. Copyright © 2013 Elsevier Inc. All rights reserved.


Manu Kanwar, R Clay Wright, Amol Date, Jennifer Tullman, Marc Ostermeier. Protein switch engineering by domain insertion. Methods in enzymology. 2013;523:369-88

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PMID: 23422439

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