Lipoamide脱氢酶从固氮菌vinelandii。分子克隆、组织和基因的序列分析。
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Westphal啊,德角
Lipoamide脱氢酶从固氮菌vinelandii。分子克隆、组织和基因的序列分析。
3月1。1988欧元;172 (2):299 - 305。
- PubMed ID
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2832161 (在PubMed]
- 文摘
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基因编码lipoamide脱氢酶从固氮菌vinelandii一直在大肠杆菌克隆。片段的合唱kb固氮菌vinelandii染色体DNA通过部分消化与Sau3A结扎质粒pUC9 BamHI网站的。大肠杆菌TG2细胞和由此产生的重组质粒转化。筛查克隆产生a vinelandii lipoamide脱氢酶与抗体进行了纯化酶。积极的殖民地被发现产生完整的链lipoamide脱氢酶作为结论形式SDS凝胶电泳的提取、游离蛋白质染色或用于免疫印迹。14.7 kb subcloning后插入这个质粒的结构基因可能位于3.2 kb DNA片段。这个subcloned的核苷酸序列片段(3134个基点)已经被确定。蛋白质编码序列的基因由1434个基点(478密码子,包括8月起始密码子和UAA终止密码子)。它之前是一个intracistronic地区85个基点,为succinyltransferase结构基因。一个假定的ribosome-binding网站和启动子序列。 The derived amino acid composition is in excellent agreement with that previously published for the isolated enzyme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three-dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483-496], it appears that essential residues in all four domains have been conserved. The enzyme is strongly expressed, although expression does not depend on the vector-encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.