title: High-level expression of his-tagged clostridial collagenase in Clostridium perfringens.
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 Ni(2+), 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.
authors.affiliation: Department of Infectious Diseases, College of Pharmaceutical Sciences, Matsuyama University, 4-2 Bunkyo-cho, Matsuyama, Ehime, 790-8578, Japan.
authors.forename: Eiji
authors.initials: E
authors.lastname: Tamai
authors.forename: Shigeru
authors.initials: S
authors.lastname: Miyata
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nariya
authors.forename: Motoo
authors.initials: M
authors.lastname: Suzuki
authors.forename: Osamu
authors.initials: O
authors.lastname: Matsushita
authors.forename: Naoya
authors.initials: N
authors.lastname: Hatano
authors.forename: Akinobu
authors.initials: A
authors.lastname: Okabe
mesh_terms: D001483:Base Sequence; D047009:Hathewaya histolytica / Q000201:enzymology*; D003016:Clostridium perfringens / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D020784:Matrix Metalloproteinase 8 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
chemical_list: C471213:His-His-His-His-His-His; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D006639:Histidine; D020784:Matrix Metalloproteinase 8