High performance in refolding of Streptomyces griseus trypsin by the aid of a mutant of Streptomyces subtilisin inhibitor designed as trypsin inhibitor

Refolding of reduced and denatured Streptomyces griseus trypsin (SGT) was i nvestigated. In the standard buffer of 50 mM Tris-HCl, the refolding yield of 1 mu g/ml of SGT did not exceed 15%, This low yield was assumed to be du e mainly to autoproteolysis and/or aggregation occurring concurrently with refolding. On the basis of this assumption, SGT was immobilized on agarose gel in order to suppress such intermolecular interactions, and various refo lding media were examined for their ability to minimize misfolding. As a re sult, 1 M Tris, 1 M diethanolamine, and 1 M triethanolamine were chosen, an d their application to the solution system increased the refolding yield co nsiderably, to ca. 45%. A further dramatic increase in yield, to 85%, was o bserved when a mutant Streptomyces subtilisin inhibitor (SSI, C71SM73KC101S ), engineered as a temporary inhibitor of SGT, was added to the solution sy stem to suppress autoproteolysis during refolding, The application of a tem porary inhibitor may be greatly effective in not only improvement of yield but also selection of media for the refolding of protease.