High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid.
A "large" sialidase isozyme (NanI) from Clostridium perfringens is a representative microbial sialidase with broad substrate specificity, being used for the analysis of sialoglycoconjugates. It is also a possible virulence factor. However, purification of the native enzyme in a large quantity is not practical due to its low productivity. To obtain the enzyme in a satisfactory yield, a gene encoding the NanI was transcriptionally fused to the fdx gene promoter (P(fdx)) in a shuttle-vector, pFF, and transformed into C. perfringens 13. The resultant strain released the enzyme into the culture medium, as the original strain does. The enzyme activity increased during the first 6 h of culture and thereafter remained at maximal levels. The maximal activity was approximately 3000-fold compared with that of the original strain, and 15-fold compared with that of recombinant Escherichia coli, which possesses extra copies of the tRNA gene for selected rare codons. This suggests the usefulness of a P(fdx)-based plasmid for expressing AT-rich genes in C. perfringens. The enzyme was successfully purified by two-step procedure with a specific activity of 2860 U/mg using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and a yield of 1.69 mg of NanI per 100 ml of culture. The method described here can facilitate purification of NanI in enough quality and quantity to analyze the role of sialoglycoconjugates in cells and the pathogenic importance of NanI sialidase.
Takamizawa
Akihisa
A
Department of Microbiology, Faculty of Medicine, Kagawa Medical University, 1750-1, Miki-cho, Kita-gun, Kagawa 761-0793, Japan.
Miyata
Shigeru
S
Matsushita
Osamu
O
Kaji
Masato
M
Taniguchi
Yuki
Y
Tamai
Eiji
E
Shimamoto
Seiko
S
Okabe
Akinobu
A
D001483:Base Sequence; D003001:Cloning, Molecular; D003016:Clostridium perfringens / Q000201:enzymology*; D004926:Escherichia coli / Q000201:enzymology; D005288:Ferredoxins / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification
D005288:Ferredoxins; D011994:Recombinant Proteins; D009439:Neuraminidase