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

Eiji Tamai, Shigeru Miyata, Hiroaki Tanaka, Hirofumi Nariya, Motoo Suzuki, Osamu Matsushita, Naoya Hatano, Akinobu Okabe

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

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 Zn 2+ 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.

Original languageEnglish
Pages (from-to)627-635
Number of pages9
JournalApplied Microbiology and Biotechnology
Volume80
Issue number4
DOIs
Publication statusPublished - Sep 2008
Externally publishedYes

Keywords

  • Clostridium histolyticum
  • Clostridium perfringens
  • Collagenase
  • Protein purification

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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