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
|
リソース情報 |
一般微生物 |
JCM 1403 |