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Nonaggregating refolding of ribonuclease A using reverse micellar dialysis
Author(s) -
Ono Tsutomu,
Nagatomo Mai,
Nagao Tomoaki,
Ijima Hiroyuki,
Kawakami Koei
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20329
Subject(s) - chemistry , rnase p , urea , ribonuclease , chromatography , membrane , reagent , ultrafiltration (renal) , glutathione , biochemistry , organic chemistry , enzyme , rna , gene
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. © 2004 Wiley Periodicals, Inc.