Hayashi M, Hirai K, Unemoto T
Laboratory of Membrane Biochemistry, Faculty of Pharmaceutical Sciences, Chiba University, Japan.
FEBS Lett. 1994 Dec 19;356(2-3):330-2. doi: 10.1016/0014-5793(94)01274-1.
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.
从海洋细菌溶藻弧菌中纯化得到的Na(+)-转运NADH-醌还原酶由α、β和γ三个亚基组成。根据每个亚基的N端氨基酸序列及其经V8蛋白酶部分消化得到的多肽片段，分别合成了与编码α、β和γ亚基的每个基因（NQR A、B和C）的正向和反向引物对应的寡核苷酸。使用这些引物，通过PCR方法从溶藻弧菌的染色体DNA中扩增出每个基因的一部分，PCR产物用于将NQR基因克隆到λ噬菌体中。在探针C筛选出的亚克隆中，通过活性染色和蛋白质免疫印迹法在大肠杆菌膜中检测到了作为基因产物的β亚基的表达。
