부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.pep.2006.04.005 Byline: Shunichi Akiba (a), Yasuhiro Hayashi (a), Yoshihiro Hakamada (a), Keiji Endo (a), Katsutoshi Ara (a), Shuji Kawai (a), Eiichi Saitoh (b) Keywords: Bacillus subtilis; Endoglucanase; Human salivary cystatin Abstract: We herein describe the development of a Bacillus subtilis system that can be used to produce large quantities of recombinant (r-) human salivary cystatins, a cysteine protease inhibitor of family 2 in the cystatin superfamily. The B. subtilis that lacked the alkaline protease E gene ([DELTA]aprE type mutant strain) was prepared by homologous recombination. The cDNA fragments coding for mature cystatins (S and SA) were ligated in frame to the DNA segment for the signal peptide of endoglucanase in the pHSP-US plasmid vector that was then use to transform the [DELTA]aprE type mutant strain of B. subtilis. The transformants carrying the expression vectors were cultivated in 5-L jar fermenters for 3 days at 30[degrees]C. Both r-cystatin S and r-cystatin SA were successfully expressed and secreted into the culture broth, and were purified using a fast performance liquid chromatography system. The first use of [DELTA]aprE type mutant strain of B. subtilis made it possible to obtain a high yield of secreted protein, which makes this system an improvement over expression in Escherichia coli. We conclude that this system has high utility for expression of commercial quantities of secreted proteins. Author Affiliation: (a) Biological Science Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Tochigi 321-3497, Japan (b) Department of Applied Chemistry and Biotechnology, Niigata Institute of Technology, 1719 Fujihashi, Kashiwazaki, Niigata 945-1145, Japan Article History: Received 16 February 2006; Revised 14 April 2006