Chemical methods for producing disulfide bonds in peptides and proteins to study folding regulation

Masaki Okumura, Shigeru Shimamoto, Yuji Hidaka

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)


Disulfide bonds play a critical role in the folding of secretory and membrane proteins. Oxidative folding reactions of disulfide bond-containing proteins typically require several hours or days, and numerous misbridged disulfide isomers are often observed as intermediates. The rate-determining step in refolding is thought to be the disulfide-exchange reaction from nonnative to native disulfide bonds in folding intermediates, which often precipitate during the refolding process because of their hydrophobic properties. To overcome this, chemical additives or a disulfide catalyst, protein disulfide isomerase (PDI), are generally used in refolding experiments to regulate disulfide-coupled peptide and protein folding. This unit describes such methods in the context of the thermodynamic and kinetic control of peptide and protein folding, including (1) regulation of disulfide-coupled peptides and protein folding assisted by chemical additives, (2) reductive unfolding of disulfide-containing peptides and proteins, and (3) regulation of disulfide-coupled peptide and protein folding using PDI.

Original languageEnglish
Article number28.7
JournalCurrent Protocols in Protein Science
Issue numberSUPPL.76
Publication statusPublished - 2014


  • Additive
  • Disulfide
  • Folding
  • Glutathione
  • Protein disulfide isomerase


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