High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid
| Autor: | Masato Kaji, Osamu Matsushita, Shigeru Miyata, Akinobu Okabe, Akihisa Takamizawa, Seiko Shimamoto, Eiji Tamai, Yuki Taniguchi |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
Clostridium perfringens
Genetic Vectors Molecular Sequence Data Neuraminidase medicine.disease_cause Sialidase Isozyme Plasmid Escherichia coli medicine Cloning Molecular Promoter Regions Genetic Gene chemistry.chemical_classification Base Sequence biology Molecular biology Recombinant Proteins Enzyme assay Enzyme chemistry Biochemistry biology.protein Ferredoxins Specific activity Plasmids Biotechnology |
| Zdroj: | Protein Expression and Purification. 36:70-75 |
| ISSN: | 1046-5928 |
| DOI: | 10.1016/j.pep.2004.03.004 |
| Popis: | 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 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 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 2860 U/mg using 2′-(4-methylumbelliferyl)-α- 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. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full Text from ScienceDirect