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
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
复制标题
DOI：
10.1016/0014-5793(94)01274-1
复制
发表时间：
1994-12-19
期刊：
影响因子：
3.5
通讯作者：
UNEMOTO, T
中科院分区：
文献类型：
--
作者：
HAYASHI, M;HIRAI, K;UNEMOTO, T
从海洋细菌溶藻弧菌（Vibrio alginolyticus）中纯化的Na+转运NADH-醌还原酶由α、β和γ三个亚基组成。从每个亚基的N-末端氨基酸序列和通过用V8蛋白酶部分消化获得的其多肽片段，合成分别对应于编码α、β和γ亚基的每个基因（NQR A、B和C）的正向和反向引物的寡核苷酸。使用这些引物，通过PCR方法从溶藻弧菌的染色体DNA扩增每个基因的一部分，并将PCR产物用于在λ噬菌体中克隆NQR基因。在通过探针C选择的亚克隆中，通过活性染色和Western印迹法在大肠杆菌膜中检测到作为基因产物的β-亚基的表达。
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.