Cloning of the Na+‐translocating NADH‐quinone reductase gene from the marine bacterium Vibrio alginolyticus and the expression of the β‐subunit in Escherichia coli

FEBS Letters

Hayashi, Maki¹  Unemoto, Tsutomu¹  Hirai, Kouji¹ 

[1] Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263, Japan

关键词: Na+ pump;    NADH-quinone reductase;    Cloning of NQR gene;    Expression of β-subunit;    Vibrio alginolyticus;   

DOI  :  10.1016/0014-5793(94)01274-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

The Na⁺-translocating NADH-quinone reductase purified from the marine bacterium Vibrio alginolyticus is composed of three subunits, α, β and γ. From the N-terminal amino acid sequences of each subunit and its polypeptide fragment obtained by partial digestion with V8 protease, oligonucleotides corresponding to forward and reverse primers for each gene (NQR A, B and C) encoding the α, β and γ subunit, respectively, were synthesized. Using these primers, a part of each gene was amplified from the chromosomal DNA of V. alginolyticus by a PCR method, and the PCR products were used for the cloning of the NQR gene in λ phage. Among the subclones selected by probe C, the expression of the β-subunit as a gene product was detected in Escherichia coli membranes by activity staining and Western blotting.

【 授权许可】

Unknown