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High level expression and purification of peptide methionine sulfoxide reductase in Escherichia coli.

Authors
  • Rahman, M A
  • Brot, N
  • Weissbach, H
Type
Published Article
Journal
Cellular and molecular biology
Publication Date
Aug 01, 1992
Volume
38
Issue
5
Pages
529–542
Identifiers
PMID: 1468111
Source
Medline
License
Unknown

Abstract

The enzyme peptide methionine sulfoxide reductase catalyzes the conversion of methionine sulfoxide residues in proteins to methionine. The 636 nucleotide coding region of the peptide methionine sulfoxide reductase gene has been amplified from a genomic clone using the polymerase chain reaction and the product was subcloned into plasmid pGEX-2T downstream of the glutathione S-transferase gene under control of the tac promoter. Escherichia coli XL1-Blue cells transformed with this plasmid and induced with isopropylthio-beta-galactoside expressed high levels of the fusion protein. The protein was soluble and was purified to homogeneity by affinity binding to a glutathione-agarose resin followed by cleavage of the fusion protein with thrombin. Both the fusion protein and the purified peptide methionine sulfoxide reductase protein showed high peptide methionine sulfoxide reductase activity.

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