論文 - 詳細
| RRC ID | 15993 |
|---|---|
| 著者 | Tanaka H, Nariya H, Suzuki M, Houchi H, Tamai E, Miyata S, Okabe A. |
| タイトル | High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens. |
| ジャーナル | Protein Expr Purif |
| Abstract |
Clostripain (CLO) produced by Clostridium histolyticum is an arginine-specific endopeptidase with the potential for applicability to diverse medical and industrial uses. In this study, we developed an expression system allowing high-level production and efficient purification of recombinant CLO (rCLO). Our expression system comprises pCLO, an rCLO expressing vector, and Clostridium perfringens 13Δ6, an in-frame deletion strain as to six genes encoding major virulence factors and secretory proteins. rCLO was purified from the culture supernatant of C. perfringens 13Δ6/pCLO by ammonium sulfate precipitation, hydroxyapatite chromatography, and affinity chromatography on benzamidine-Sepharose. From 200 ml of culture supernatant 4.5 mg of purified rCLO was obtained. N-Terminal amino acid sequencing and molecular mass determination of the purified rCLO and commercially available CLO revealed that the two enzymes have identical subunits, a 38.1-kDa heavy chain and a 15.0-kDa light chain, indicating that rCLO is processed in the same manner as CLO. Analysis of the enzymatic activities toward N-benzoyl-L-arginine p-nitroanilide and acyl-L-lysine p-nitroanilide showed that rCLO and CLO exhibit strict specificity for arginine at the P1 position, and that the specific activity of the former is approximately 2-fold higher than that of the latter. These results indicate that the new method involving a virulence-attenuated C. perfringens strain is useful for preparing large amounts of high-grade rCLO. |
| 巻・号 | 76(1) |
| ページ | 83-9 |
| 公開日 | 2011-3-1 |
| DOI | 10.1016/j.pep.2010.10.002 |
| PII | S1046-5928(10)00279-2 |
| PMID | 20940055 |
| MeSH | Amino Acid Sequence Chromatography, Affinity Clostridium perfringens / genetics Clostridium perfringens / metabolism* Clostridium perfringens / pathogenicity Cysteine Endopeptidases / biosynthesis* Cysteine Endopeptidases / isolation & purification Recombinant Proteins / biosynthesis* Recombinant Proteins / isolation & purification Virulence |
| IF | 1.513 |
| 引用数 | 6 |
| WOS 分野 | BIOTECHNOLOGY & APPLIED MICROBIOLOGY BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY |
| オルトメトリクス指標 |
オルトメトリクス指標項目
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| 最多言及媒体 | Patent(IFI CLAIMS) |
| 各媒体での言及数の合計 | 4 |
| 過去6か月間でのオルトメトリクス指標の変動値 | 0.0 |
| リソース情報 | |
| 一般微生物 | JCM 1403 |