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Protein Eng Des Sel. 2009 Oct;22(10):607-13. doi: 10.1093/protein/gzp034. Epub 2009 Jul 21.

Tying up the loose ends: circular permutation decreases the proteolytic susceptibility of recombinant proteins.

Author information

1
Department of Chemical Engineering, University of California, 201 Gilman Hall, Berkeley, CA 94720, USA.

Abstract

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 beta-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.

PMID:
19622546
DOI:
10.1093/protein/gzp034
[Indexed for MEDLINE]

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