title: Construction and characterization of a clostripain-like protease-deficient mutant of Clostridium perfringens as a strain for clostridial gene expression.
abstract: The inherent difficulty of expressing clostridial AT-rich genes in a heterologous host has limited their biotechnological application. We previously reported a plasmid for high-level expression of clostridial genes in Clostridium perfringens (Takamizawa et al., Protein Expr Purif 36:70-75, 2004). In this study, we examined the extracellular proteases of C. perfringens strain 13. Zymographic analysis and caseinase assaying of a culture supernatant showed that it contained a protease activated by dithiothreitol and Ca(2+), suggesting that clostripain-like protease (Clp) is the most likely candidate for the major extracellular protease. Disruption of the clp gene by homologous recombination markedly decreased the level of caseinase activity in the culture supernatant. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Clp(-) mutant but not the wild type strain increased the levels of many polypeptides in the culture supernatant after the late exponential growth phase. Such polypeptides included both cytoplasmic and secretory proteins, suggesting proteins secreted or released into the medium were degraded by Clp. To assess the effects of Clp on the productivity and stability of recombinant proteins, 74-kDa NanI sialidase was expressed in the two strains. The mutant strain produced a higher level of NanI activity than the wild type strain. Furthermore, under the conditions where Clp was activated, NanI was degraded easily in the latter culture but not in the former one. These results indicate that the Clp(-) mutant could serve as a useful strain for efficiently expressing and preparing protease-free clostridial proteins.
authors.affiliation: Department of Microbiology, Faculty of Medicine, Kagawa University, 1750-1 Miki-cho, Kita-gun, Kagawa, 761-0793, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Tanaka
authors.forename: Eiji
authors.initials: E
authors.lastname: Tamai
authors.forename: Shigeru
authors.initials: S
authors.lastname: Miyata
authors.forename: Yuki
authors.initials: Y
authors.lastname: Taniguchi
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Nariya
authors.forename: Naoya
authors.initials: N
authors.lastname: Hatano
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Houchi
authors.forename: Akinobu
authors.initials: A
authors.lastname: Okabe
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D049071:Endopeptidase Clp / Q000235:genetics* / Q000378:metabolism; D017353:Gene Deletion; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional; D009439:Neuraminidase / Q000096:biosynthesis / Q000235:genetics; D020543:Proteome / Q000032:analysis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D020543:Proteome; D011994:Recombinant Proteins; D009439:Neuraminidase; D049071:Endopeptidase Clp; D008666:Metalloendopeptidases; C030895:caseinase