PEDS Advance Access originally published online on July 21, 2009
Protein Engineering Design and Selection 2009 22(10):607-613; doi:10.1093/protein/gzp034
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Tying up the loose ends: circular permutation decreases the proteolytic susceptibility of recombinant proteins
Department of Chemical Engineering, University of California, 201 Gilman Hall, Berkeley, CA 94720, USA
1 To whom correspondence should be addressed. E-mail: clark{at}berkeley.edu
Recombinant proteins often suffer from poor expression because of proteolysis. Existing genetic engineering or fermentation strategies work for only a subset of cases where higher recombinant protein expression is needed. In this paper, we describe the use of circular permutation, wherein the original termini of a protein are concatenated and new termini are generated elsewhere with the sequence, as a general protein engineering strategy to produce full-length, active recombinant protein. We show that a circularly permuted variant of the thermosome (Group II chaperonin) from Methanocaldococcus jannaschii exhibited reduced proteolysis and increased expression in three different strains of Escherichia coli. Circular permutation of a different protein, TEM-1 β-lactamase, by a similar method increased the expression lifetime of the protein in the periplasm of E. coli. Both circularly permuted proteins maintained activity near their wild-type counterparts and design criteria for selecting the sites for circular permutation are discussed. It is expected that this method will find broad utility for enhanced expression of recombinant proteins when proteolysis is a factor.
Keywords: β lactamase/chaperone/circular permutation/protein engineering/proteolysis
Received March 4, 2009; revised June 3, 2009; accepted June 8, 2009.