High-level production and purification of clostripain expressed in a virulence-attenuated strain of Clostridium perfringens.

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.