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

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 origin...

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Bibliographic Details
Published in:Protein engineering, design and selection design and selection, 2009-10, Vol.22 (10), p.607-613
Main Authors: Whitehead, Timothy A., Bergeron, Lisa M., Clark, Douglas S.
Format: Article
Language:English
Subjects:
beta-Lactamases - chemistry
beta-Lactamases - genetics
beta-Lactamases - metabolism
chaperone
circular permutation
Escherichia coli
Escherichia coli - genetics
Kinetics
Methanocaldococcus jannaschii
Models, Molecular
Penicillin Amidase - chemistry
Penicillin Amidase - genetics
Penicillin Amidase - metabolism
protein engineering
Protein Engineering - methods
Protein Stability
proteolysis
Recombinant Proteins - chemistry
Recombinant Proteins - genetics
Recombinant Proteins - metabolism
Thermosomes - chemistry
Thermosomes - genetics
Thermosomes - metabolism
β lactamase
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Summary: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.
ISSN:1741-0126
1741-0134
DOI:10.1093/protein/gzp034