The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
High Performance in Refolding of Streptomyces griseus Trypsin by the Aid of a Mutant of Streptomyces Subtilisin Inhibitor Designed as Trypsin Inhibitor
Daisuke NoharaHiroyuki SugiuraHiromu SakakibaraMamoru MatsubaraShuichi KojimaKin-ichiro MiuraTomoya Sakai
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1999 Volume 125 Issue 2 Pages 343-347

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Abstract

Refolding of reduced and denatured Streptomyces griseus trypsin (SGT) was investigated. In the standard buffer of 50mM Tris-HCl, the refolding yield of 1 μg/ml of SGT did not exceed 15%. This low yield was assumed to be due 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 refolding media were examined for their ability to minimize misfolding. As a result, 1M Tris, 1M diethanolamine, and 1M triethanolamine were chosen, and their application to the solution system increased the refolding yield considerably, to ca. 45%. A further dramatic increase in yield, to 85%, was observed when a mutant Streptomyces subtilisin inhibitor (SSI, C71SM73KC101S), engineered as a temporary inhibitor of SGT, was added to the solution system to suppress autoproteolysis during refolding. The application of a temporary inhibitor may be greatly effective in not only improvement of yield but also selection of media for the refolding of protease.

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© The Japanese Biochemical Society
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