TY - JOUR
T1 - Nonaggregating refolding of ribonuclease A using reverse micellar dialysis
AU - Ono, Tsutomu
AU - Nagatomo, Mai
AU - Nagao, Tomoaki
AU - Ijima, Hiroyuki
AU - Kawakami, Koei
PY - 2005/2/5
Y1 - 2005/2/5
N2 - 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.
AB - 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.
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U2 - 10.1002/bit.20329
DO - 10.1002/bit.20329
M3 - Article
C2 - 15625675
AN - SCOPUS:14244267169
VL - 89
SP - 290
EP - 295
JO - Biotechnology and Bioengineering
JF - Biotechnology and Bioengineering
SN - 0006-3592
IS - 3
ER -