title: Nonaggregating refolding of ribonuclease A using reverse micellar dialysis.
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 beta-cyclodextrin washing. These findings should facilitate the production of a continuous protein refolding membrane reactor.
authors.affiliation: Department of Chemical Engineering, Kyushu University, 6-10-1 Hakozaki, Fukuoka 812-8581, Japan. tono@chem-eng.kyushu-u.ac.jp
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Ono
authors.forename: Mai
authors.initials: M
authors.lastname: Nagatomo
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Nagao
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Ijima
authors.forename: Koei
authors.initials: K
authors.lastname: Kawakami
mesh_terms: D003956:Dialysis / Q000295:instrumentation / Q000379:methods*; D008823:Micelles; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000302:isolation & purification*; D014462:Ultrafiltration
chemical_list: D008823:Micelles; D012259:Ribonuclease, Pancreatic