High-level expression of his-tagged clostridial collagenase in Clostridium perfringens

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• 作者：Eiji Tamai ; Shigeru Miyata ; Hiroaki Tanaka ; Hirofumi Nariya ; Motoo Suzuki ; Osamu Matsushita ; Naoya Hatano and Akinobu Okabe
• 关键词：Collagenase ; Clostridium histolyticum ; Clostridium perfringens ; Protein purification
• 刊名：Applied Microbiology and Biotechnology
• 出版年：2008
• 出版时间：September, 2008
• 年：2008
• 卷：80
• 期：4
• 页码：627-635
• 全文大小：218.6 KB

文摘

Clostridium histolyticum collagenase is used to isolate cells from various organs and tissues for tissue engineering, and also to treat destructive fibrosis; thus, the demand for high-grade enzyme preparations is increasing. In this study, we constructed a plasmid encoding C. histolyticum type II collagenase (ColH) with a C-terminal hexahistidine tag (ColH-his) to facilitate the purification of the enzyme through immobilized metal affinity chromatography (IMAC). When ColH-his was expressed in a protease-deficient mutant of Clostridium perfringens, it was produced in the culture supernatant more efficiently than the untagged ColH. ColH-his exhibited the same hydrolytic activity as ColH against 4-phenylazobenzyloxy-carbonyl-Pro-Leu-Gly-Pro-d-Arg (Pz peptide), a synthetic collagenase substrate. From 100 ml of the culture supernatant, approximately 1 mg of ColH-his was purified by ammonium sulfate precipitation, IMAC, and high-performance liquid chromatography on a MonoQ column. When IMAC was performed on chelating Sepharose charged with Zn2+ instead of Ni2+, a potential carcinogenic metal, the specific activities against Pz peptide and type I collagen decreased slightly. However, they were comparable to those reported for other recombinant ColHs and a commercial C. histolyticum collagenase preparation, suggesting that this expression system is useful for large-scale preparation of high-grade clostridial collagenases.