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.