Spehr V, Schlitt A, Scheide D, Guénebaut V, Friedrich T
Institut für Biochemie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Biochemistry. 1999 Dec 7;38(49):16261-7. doi: 10.1021/bi9919605.
The proton-pumping NADH:ubiquinone oxidoreductase (complex I) of Escherichia coli is composed of 13 different subunits. The corresponding genes are organized in the nuo-operon (from NADH:ubiquinone oxidoreductase) at min 51 of the E. coli chromosome. To study the structure and function of this complex enzyme, a suitable purification protocol yielding sufficient amount of a stable protein is needed. Here, we report the overproduction of complex I in E. coli and a novel isolation procedure of the complex. Overexpression of the nuo-operon on the chromosome was achieved by replacing its 5'-promotor region with the phage-T7 RNA polymerase promotor and by expressing the genes with the T7 RNA polymerase coded on an inducible plasmid. It is shown by means of enzymatic activity and EPR spectroscopy of cytoplasmic membranes that complex I is overproduced 4-fold after induction. Complex I was isolated by chromatographic steps performed in the presence of dodecyl maltoside. The preparation comprises all subunits and known cofactors and exhibits a high enzymatic activity and inhibitor sensitivity. Due to its stability over a wide pH range and at very high salt concentrations, this preparation is well suited for structural investigations.
大肠杆菌的质子泵NADH：泛醌氧化还原酶（复合体I）由13种不同的亚基组成。相应的基因在大肠杆菌染色体51分钟处的nuo操纵子（来自NADH：泛醌氧化还原酶）中组织排列。为了研究这种复合酶的结构和功能，需要一种合适的纯化方案来获得足够量的稳定蛋白质。在此，我们报告了大肠杆菌中复合体I的过量表达以及该复合体的一种新型分离方法。通过用噬菌体T7 RNA聚合酶启动子替换其5'-启动子区域，并利用编码在诱导型质粒上的T7 RNA聚合酶表达这些基因，实现了染色体上nuo操纵子的过表达。通过细胞质膜的酶活性和电子顺磁共振光谱表明，诱导后复合体I的产量提高了4倍。在十二烷基麦芽糖苷存在的情况下，通过色谱步骤分离出复合体I。该制剂包含所有亚基和已知的辅因子，并表现出高酶活性和抑制剂敏感性。由于其在很宽的pH范围内和非常高的盐浓度下都具有稳定性，该制剂非常适合进行结构研究。
