title: Extracellular production of human cystatin S and cystatin SA by Bacillus subtilis.
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 (DeltaaprE 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 DeltaaprE 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 DeltaaprE 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.
authors.affiliation: Biological Science Laboratories of Kao Corporation, 2606 Akabane, Ichikai, Tochigi 321-3497, Japan.
authors.forename: Shunichi
authors.initials: S
authors.lastname: Akiba
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Hayashi
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Hakamada
authors.forename: Keiji
authors.initials: K
authors.lastname: Endo
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Ara
authors.forename: Shuji
authors.initials: S
authors.lastname: Kawai
authors.forename: Eiichi
authors.initials: E
authors.lastname: Saitoh
mesh_terms: D001412:Bacillus subtilis / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D019149:Bioreactors / Q000382:microbiology; D015891:Cystatins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D017353:Gene Deletion; D015870:Gene Expression; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics; D010957:Plasmids / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D055333:Salivary Cystatins
chemical_list: C088074:AprE protein, Bacteria; D001426:Bacterial Proteins; C525172:CST2 protein, human; C525173:CST4 protein, human; D015891:Cystatins; D026901:Membrane Transport Proteins; D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D055333:Salivary Cystatins