High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid

Akihisa Takamizawa, Shigeru Miyata, Osamu Matsushita, Masato Kaji, Yuki Taniguchi, Eiji Tamai, Seiko Shimamoto, Akinobu Okabe

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

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 (Pfdx) 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 6h 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 Pfdx-based plasmid for expressing AT-rich genes in C. perfringens. The enzyme was successfully purified by two-step procedure with a specific activity of 2860U/mg using 2′-(4-methylumbelliferyl)-α-D-N- acetylneuraminic acid and a yield of 1.69mg of NanI per 100ml 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.

Original languageEnglish
Pages (from-to)70-75
Number of pages6
JournalProtein Expression and Purification
Volume36
Issue number1
DOIs
Publication statusPublished - Jul 2004
Externally publishedYes

Fingerprint

Ferredoxins
Neuraminidase
Plasmids
Genes
Clostridium perfringens
Enzymes
Purification
Clostridium
Genetic Vectors
Gene encoding
Enzyme activity
N-Acetylneuraminic Acid
Virulence Factors
Transfer RNA
Escherichia coli
Isoenzymes
Culture Media
Substrate Specificity
Codon
Productivity

Keywords

  • Clostridium perfringens
  • Expression
  • Ferredoxin
  • Sialidase
  • Transcriptional fusion

ASJC Scopus subject areas

  • Biochemistry

Cite this

High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid. / Takamizawa, Akihisa; Miyata, Shigeru; Matsushita, Osamu; Kaji, Masato; Taniguchi, Yuki; Tamai, Eiji; Shimamoto, Seiko; Okabe, Akinobu.

In: Protein Expression and Purification, Vol. 36, No. 1, 07.2004, p. 70-75.

Research output: Contribution to journalArticle

Takamizawa, Akihisa ; Miyata, Shigeru ; Matsushita, Osamu ; Kaji, Masato ; Taniguchi, Yuki ; Tamai, Eiji ; Shimamoto, Seiko ; Okabe, Akinobu. / High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid. In: Protein Expression and Purification. 2004 ; Vol. 36, No. 1. pp. 70-75.
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