Nonaggregating refolding of ribonuclease A using reverse micellar dialysis

Tsutomu Ono, Mai Nagatomo, Tomoaki Nagao, Hiroyuki Ijima, Koei Kawakami

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

A hydrophilic ultrafiltration membrane, regenerated cellulose, facilitates the size-selectable permeability of hydrophilic solutes in reverse micellar solution. By using an ultrafiltration membrane with a molecular weight cutoff of 3,500, we demonstrate a nonaggregating protein refolding technique based on the dialysis of reverse micellar solution. This realizes concurrent removal of denaturants, urea and 2-mercaptoethanol, and the supply of redox reagents, reduced and oxidized glutathione (GSH, GSSG), to promote renaturation of proteins. Two mg/ml ribonuclease A (RNase A) was refolded completely without any dilution and aggregation for 60 h. The refolding behavior of RNase A is strongly influenced by the ratio of GSH and GSSG. Moreover, we recovered 90% of the refolded RNase A from AOT reverse micellar solution with acetone precipitation and β-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor.

Original languageEnglish
Pages (from-to)290-295
Number of pages6
JournalBiotechnology and Bioengineering
Volume89
Issue number3
DOIs
Publication statusPublished - Feb 5 2005
Externally publishedYes

Keywords

  • AOT
  • Membrane reactor
  • Oxidative refolding
  • Reverse micelles
  • Ultrafiltration

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology

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