A transposase strategy for creating libraries of circularly permuted proteins

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ID: 297762
2012
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Abstract
A simple approach for creating libraries of circularly permuted proteins is described that is called PERMutation Using Transposase Engineering (PERMUTE). In PERMUTE, the transposase MuA is used to randomly insert a minitransposon that can function as a protein expression vector into a plasmid that contains the open reading frame (ORF) being permuted. A library of vectors that express different permuted variants of the ORF-encoded protein is created by: (i) using bacteria to select for target vectors that acquire an integrated minitransposon; (ii) excising the ensemble of ORFs that contain an integrated minitransposon from the selected vectors; and (iii) circularizing the ensemble of ORFs containing integrated minitransposons using intramolecular ligation. Construction of a Thermotoga neapolitana adenylate kinase (AK) library using PERMUTE revealed that this approach produces vectors that express circularly permuted proteins with distinct sequence diversity from existing methods. In addition, selection of this library for variants that complement the growth of Escherichia coli with a temperature-sensitive AK identified functional proteins with novel architectures, suggesting that PERMUTE will be useful for the directed evolution of proteins with new functions.
Reference Key
openalex_W2116398954 Use this key to autocite in the manuscript while using SciMatic Manuscript Manager or Thesis Manager
Authors Manan Mehta, Shirley Liu, Jonathan J. Silberg
Journal Nucleic Acids Research
Year 2012
DOI
10.1093/nar/gks060
URL
https://doi.org/10.1093/nar/gks060
Keywords Keywords not found

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