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

Timothy A. Whitehead, Lisa M. Bergeron, Douglas S. Clark

Research output: Contribution to journalArticle

  • 19 Citations

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 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.

Original languageEnglish (US)
Pages (from-to)607-613
Number of pages7
JournalProtein Engineering, Design and Selection
Volume22
Issue number10
DOIs
StatePublished - Oct 2009
Externally publishedYes

Profile

Recombinant Proteins
Proteins
Recombinant proteins
Proteolysis
Omsk Hemorrhagic Fever
Escherichia coli
Frangula
Thermosomes
Group II Chaperonins
Methanocaldococcus
Protein Engineering
Periplasm
Genetic Engineering
beta-Lactamases
Fermentation
Genetic engineering
Transmission electron microscopy
Dental Pulp Calcification

Keywords

  • βlactamase
  • Chaperone
  • Circular permutation
  • Protein engineering
  • Proteolysis

ASJC Scopus subject areas

  • Biochemistry
  • Biotechnology
  • Bioengineering
  • Molecular Biology

Cite this

Tying up the loose ends : Circular permutation decreases the proteolytic susceptibility of recombinant proteins. / Whitehead, Timothy A.; Bergeron, Lisa M.; Clark, Douglas S.

In: Protein Engineering, Design and Selection, Vol. 22, No. 10, 10.2009, p. 607-613.

Research output: Contribution to journalArticle

Whitehead, Timothy A.; Bergeron, Lisa M.; Clark, Douglas S. / Tying up the loose ends : Circular permutation decreases the proteolytic susceptibility of recombinant proteins.

In: Protein Engineering, Design and Selection, Vol. 22, No. 10, 10.2009, p. 607-613.

Research output: Contribution to journalArticle

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