RRC ID 15993
Author Tanaka H, Nariya H, Suzuki M, Houchi H, Tamai E, Miyata S, Okabe A.
Title High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens.
Journal 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.
Volume 76(1)
Pages 83-9
Published 2011-3
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 1.338
Times Cited 5
WOS Category BIOTECHNOLOGY & APPLIED MICROBIOLOGY BIOCHEMICAL RESEARCH METHODS BIOCHEMISTRY & MOLECULAR BIOLOGY
Resource
General Microbes JCM 1403