Renaturation of reduced ribonuclease A with a microsphere-induced refolding system

Hidenobu Shimizu, Keiji Fujimoto, Haruma Kawaguchi

Research output: Contribution to journalArticle

25 Citations (Scopus)

Abstract

We intended to refold reduced ribonuclease A (RNase A) using polymeric microspheres. Polymeric microspheres were allowed to react with dithiothreitol (DTT) to immobilize the disulfide and thiol moieties on their surface. The fully reduced RNase A was added to the dispersion of the modified microspheres. Protein refolding and renaturation were estimated by the change in the number of disulfide bonds of RNase A and the recovery of the enzymatic activity, respectively. Without microspheres, the activity gradually recovered with the increase in the number of disulfide bonds. However, the formation of disulfide bonds of reduced RNase A was accelerated by adding the modified microspheres, and the rate of renaturation was increased depending on the amount of charged DTT and the reaction time of the immobilization. These results indicate that modified microspheres significantly catalyze the recovery of active RNase A from the reduced form. The protein adsorption data demonstrated that the disulfide moieties of the modified microspheres react with the thiol moieties of the reduced RNase A to form a mixed disulfide. The thiol/disulfide exchange reaction can possibly proceed at the microsphere/protein interface, resulting in the formation of a correct three-dimensional structure.

Original languageEnglish
Pages (from-to)248-253
Number of pages6
JournalBiotechnology Progress
Volume16
Issue number2
DOIs
Publication statusPublished - 2000 Jan 1

ASJC Scopus subject areas

  • Biotechnology

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