Achievement of renaturation of subtilisin BPN' by a novel procedure using organic salts and a digestible mutant of Streptomyces subtilisin inhibitor.
The pro-sequences of proteases have been considered to be required for the refolding of denatured proteases. However, here we report achievement of almost complete restoration of enzymatic activity of subtilisin BPN' in the absence of its pro-sequence. The presence of 2 M potassium acetate in the folding medium enhanced the refolding efficiency of guanidine hydrochloride (GdnHCl)-denatured subtilisin BPN' by up to 28%, and other organic salts were also found to be useful, suggesting that general contribution of the bulky hydrophobic moieties of the salts to the formation of a favorable environment required for folding. This finding will provide new insights into the folding mechanisms not only of proteases but also of various other proteins. Almost complete restoration of enzymatic activity of denatured subtilisin in the organic salt solution was accomplished by further addition of mutated Streptomyces subtilisin inhibitor (SSI), which had been converted to a digestible temporary inhibitor by removal of the disulfide bridge near the reactive site.
Matsubara
M
M
Department of Chemical Reaction Engineering, Faculty of Pharmaceutical Sciences, Nagoya City University, Japan.
Kurimoto
E
E
Kojima
S
S
Miura
K
K
Sakai
T
T
D000085:Acetates / Q000494:pharmacology; D019342:Acetic Acid; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000494:pharmacology; D008969:Molecular Sequence Data; D009154:Mutation; D009842:Oligopeptides / Q000737:chemistry; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation; D013302:Streptomyces / Q000235:genetics; D013379:Substrate Specificity; D013381:Subtilisins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000187:drug effects
D000085:Acetates; D001426:Bacterial Proteins; D009842:Oligopeptides; C018150:subtilisin inhibitor protein, Streptomyces; C043307:succinyl-alanyl-alanyl-prolyl-phenylalanine-4-nitroanilide; D013381:Subtilisins; D019342:Acetic Acid