Oxidative refolding from inclusion bodies

Research output: Contribution to journalArticle

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Abstract

This protocol describes the growth and purification of bacterial inclusion body proteins with an option to selenomethionine label the targeted protein through feedback inhibition of methionine biosynthesis in common (non-auxotrophic) strains of E. coli. The method includes solubilization of inclusion body proteins by chemical denaturation and disulfide reduction, renaturation of the solubilized material through rapid dilution by pulsed injection into refolding buffer containing arginine and a mixture of oxidized and reduced glutathione, recovery of the recombinant protein using a stirred cell concentrator, and removal of the aggregated or misfolded fraction by passage over size-exclusion chromatography. The quality of the resulting protein can be assessed by SDS-PAGE.

Original languageEnglish
Pages (from-to)145-157
Number of pages13
JournalMethods in Molecular Biology
Volume1140
DOIs
StatePublished - Jan 1 2014
Externally publishedYes

Keywords

  • Aggregation
  • Cell lysis
  • Escherichia coli
  • Feedback inhibition
  • Inclusion bodies
  • Induction
  • Protein concentration
  • Protein purification
  • Protein refolding
  • Recombinant protein expression
  • Selenomethionine labeling
  • Size-exclusion chromatography
  • Solubilization

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