Cloning of the Na(+)-translocating NADH-quinone reductase gene from the marine bacterium Vibrio alginolyticus and the expression of the beta-subunit in Escherichia coli.
The Na(+)-translocating NADH-quinone reductase purified from the marine bacterium Vibrio alginolyticus is composed of three subunits, alpha, beta and gamma. 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 alpha, beta and gamma 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 lambda phage. Among the subclones selected by probe C, the expression of the beta-subunit as a gene product was detected in Escherichia coli membranes by activity staining and Western blotting.
Hayashi
M
M
Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
Hirai
K
K
Unemoto
T
T
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D015870:Gene Expression; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D011808:Quinone Reductases / Q000096:biosynthesis* / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C084142:sodium-translocating NADH-quinone reductase; D011808:Quinone Reductases