NDLI: A transposase strategy for creating libraries of circularly permuted proteins

Content Provider	PubMed Central
Author	Mehta, Manan M. Liu, Shirley Silberg, Jonathan J.
Copyright Year	2012
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.
Related Links	http://dx.doi.org/10.1093/nar/gks060
Starting Page	71
File Format	PDF
ISSN	13624962
e-ISSN	13624962
Journal	Nucleic Acids Research
Issue Number	9
Volume Number	40
Language	English
Publisher	Oxford University Press
Publisher Date	2012-05-01
Access Restriction	Open
Rights Holder	Oxford University Press
Subject Keyword	Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Genetics

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