Nohara D, Sugiura H, Sakakibara H, Matsubara M, Kojima S, Miura K, Sakai T
Faculty of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, Nagoya, 467-8603, Japan.
J Biochem. 1999 Feb;125(2):343-7. doi: 10.1093/oxfordjournals.jbchem.a022292.
Refolding of reduced and denatured Streptomyces griseus trypsin (SGT) was investigated. In the standard buffer of 50 mM Tris-HCl, the refolding yield of 1 microg/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, 1 M Tris, 1 M diethanolamine, and 1 M 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.
对还原变性的灰色链霉菌胰蛋白酶（SGT）的复性进行了研究。在50 mM Tris-HCl标准缓冲液中，1 μg/ml SGT的复性产率不超过15%。这种低产率主要被认为是由于复性过程中同时发生的自催化水解和/或聚集所致。基于这一假设，将SGT固定在琼脂糖凝胶上以抑制此类分子间相互作用，并考察了各种复性介质减少错误折叠的能力。结果，选择了1 M Tris、1 M二乙醇胺和1 M三乙醇胺，将它们应用于溶液体系后，复性产率显著提高，达到约45%。当将经工程改造作为SGT临时抑制剂的突变型枯草芽孢杆菌蛋白酶抑制剂（SSI，C71SM73KC101S）添加到溶液体系中以抑制复性过程中的自催化水解时，产率进一步大幅提高至85%。临时抑制剂的应用不仅在提高产率方面可能非常有效，而且在选择蛋白酶复性的介质方面也可能非常有效。
