Effective renaturation of denatured and reduced immunoglobulin G in vitro without assistance of chaperone.
IgG is an oligomeric protein that consists of two heavy and two light chains. To form the oligomer, a highly concentrated protein would be required on renaturation. On the other hand, refolding of proteins at high concentration often led to aggregation. Therefore, denatured and reduced oligomeric protein scarcely refolded to the native structure. As was expected, the folding yield of the denatured and reduced IgG was below 5% under the condition employed in rapid dilution. The low folding yield was elucidated to be due to assembly or aggregation. Using a renaturation method previously developed to depress aggregation effectively by means of slow dialysis, the refolding yield of the denatured and reduced IgG at above 1 mg/ml was above 70%. Most of the refolded IgG was identical with the intact material based on analyses by affinity chromatography and SDS-PAGE.
Maeda
Y
Y
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.
Ueda
T
T
Imoto
T
T
D002846:Chromatography, Affinity; D003956:Dialysis; D004220:Disulfides; D004591:Electrophoresis, Polyacrylamide Gel; D007074:Immunoglobulin G / Q000737:chemistry*; D008623:Mercaptoethanol; D018832:Molecular Chaperones / Q000737:chemistry; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000737:chemistry
D004220:Disulfides; D007074:Immunoglobulin G; D018832:Molecular Chaperones; D008623:Mercaptoethanol; D014508:Urea