Kitamura M, Koshino Y, Kamikawa Y, Kohno K, Kojima S, Miura K, Sagara T, Akutsu H, Kumagai I, Nakaya T
Department of Bioapplied Chemistry, Faculty of Engineering, Osaka City University, Osaka, Japan.
Biochim Biophys Acta. 1997 Mar 20;1351(1-2):239-47. doi: 10.1016/s0167-4781(96)00203-5.
A gene encoding rubredoxin from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 1.1-kilobase pair DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with SmaI and SalI, contained two genes, the rubredoxin gene (rub) and the desulfoferrodoxin gene (rbo) which was situated upstream of rub. The deduced amino acid sequence of desulfoferrodoxin was homologous to those from other strains and Cys residues that are responsible to bind irons were also conserved. The expression system for rub was constructed under the control of the T7 promoter in E. coli. The purified protein was soluble and had a characteristic visible absorption spectrum. Inductively coupled plasma-atomic emission analysis and electron paramagnetic resonance analysis of the recombinant rubredoxin revealed the presence of an iron ion in a distorted tetrahedral geometry that was the same as native D. vulgaris rubredoxin. In vitro NADH reduction analysis indicated that recombinant rubredoxin was active, and its redox potential was determined as -5 mV.
克隆了来自普通脱硫弧菌（宫崎F株）的编码红氧还蛋白的基因，并在大肠杆菌中进行了过表达。通过用SmaI和SalI双酶切从普通脱硫弧菌（宫崎F株）中分离出一个1.1千碱基对的DNA片段，该片段包含两个基因，即红氧还蛋白基因（rub）和位于rub上游的脱硫铁氧化还原蛋白基因（rbo）。推导的脱硫铁氧化还原蛋白氨基酸序列与其他菌株的序列同源，且负责结合铁的半胱氨酸残基也保守。在大肠杆菌中，在T7启动子的控制下构建了rub的表达系统。纯化后的蛋白可溶，具有特征性的可见吸收光谱。对重组红氧还蛋白进行电感耦合等离子体原子发射分析和电子顺磁共振分析，结果显示存在一个处于扭曲四面体几何结构的铁离子，这与天然普通脱硫弧菌红氧还蛋白相同。体外NADH还原分析表明重组红氧还蛋白具有活性，其氧化还原电位测定为-5 mV。
