Cloning of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and determination of the NH2-terminal sequence.
Cloning of the gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and determination of the NH2-terminal sequence.
复制标题
克隆来自普通脱硫弧菌 (Hildenborough) 的氢化酶编码基因并测定 NH2 末端序列。
DOI：
10.1111/j.1432-1033.1985.tb08868.x
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发表时间：
1985
期刊：
影响因子：
--
通讯作者：
Sydney Brenner
中科院分区：
文献类型：
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作者：
G. Voordouw;J. E. Walker;Sydney Brenner
普通脱硫弧菌(Hildenborough)的氢酶编码基因已在大肠杆菌中克隆。用限制性内切酶EcoRI和SalI双酶切小球藻DNA，将其连接到载体pUC9[Vieira，J.&Msing，J.(1982)gene 19,259-268]。通过转化E.coliJM101，然后在富平板上生长，氨苄西林筛选，异丙基-β-D-硫代半乳糖苷和5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside存在下检测重组子，获得了约9000个重组克隆。用纯化的氢酶兔抗血清和125I标记的蛋白A对重组克隆进行免疫活性蛋白的筛选，初步筛选到28个阳性克隆。进一步的免疫竞争实验表明，来自一个克隆的蛋白质产物表现出与纯化的氢酶相同的行为。从该克隆中分离到的pHV15含有4.7×10(3)碱基对的SalI/EcoRI插入片段。经Western blotting检测，转化PHV-15的大肠杆菌JM-101细胞产生一种相对分子质量为46 kDa的氢酶多肽。大肠杆菌产生的多肽的质量和克利夫兰图谱与从普通金黄色葡萄球菌(Hildenborough)分离的氢酶相同。以缺口翻译的4.7×10(3)碱基对SalI/EcoRI片段为探针，对限制性内切酶消化的寻常型假丝酵母菌DNA进行Southern blotting，表明存在一个带有内部PstI位点的单基因。氢酶的NH2末端序列被确定为：(序列在文本中)。这一信息应该可以明确地识别氢酶基因。
The gene encoding the hydrogenase from Desulfovibrio vulgaris (Hildenborough) has been cloned in Escherichia coli. D. vulgaris DNA was digested with the restriction endonucleases EcoRI and SalI and ligated into the vector pUC9 [Vieira, J. & Messing, J. (1982) Gene 19, 259-268], which had been cut with these same enzymes. Approximately 9000 recombinant clones were obtained by transformation of E. coli JM 101 followed by growth on rich plates with ampicillin for selection and isopropyl-beta-D-thiogalactoside and 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside present for detection of recombinants. The recombinant clones were then screened for production of immunoreactive proteins with rabbit antisera against purified hydrogenase and 125I-labelled protein A. 28 positive clones were found in this initial screening. These were further tested in an immunocompetition experiment, which showed that the protein product from one clone behaved identically to purified hydrogenase. The plasmid pHV15 isolated from this clone has a 4.7 X 10(3)-base-pair SalI/EcoRI insert. Cells of E. coli JM 101 transformed with pHV 15 produce a hydrogenase polypeptide of molecular mass 46 kDa as detected by Western blotting. The mass, as well as the Cleveland mapping pattern of the polypeptide produced by E. coli, are identical with those of the hydrogenase isolated from D. vulgaris (Hildenborough). Southern blotting of restriction-enzyme-digested D. vulgaris DNA, using the nick-translated 4.7 X 10(3)-base-pair SalI/EcoRI fragment as a probe, indicates the presence of a single gene with an internal PstI site. The NH2-terminal sequence of the hydrogenase was determined to be: (sequence in text). This information should allow an unambiguous identification of the hydrogenase gene.