Chen Ying, Hunsicker-Wang Laura, Pacoma Ronald L, Luna Eugene, Fee James A
Division of Biology, University of California at San Diego, 9500 Gilman Dr., La Jolla CA 92093-0116, USA.
Protein Expr Purif. 2005 Apr;40(2):299-318. doi: 10.1016/j.pep.2004.11.014.
Cytochrome ba3 is an integral membrane protein that serves as a terminal oxidase of the respiratory chain in some prokaryotes. We have cloned the complete cba operon of Thermus thermophilus HB8 in an Escherichia coli/T. thermophilus shuttle vector. The ba3-encoding operon, cba, was eliminated from the chromosome of T. thermophilus strain MT111 using the pyrE system of Yamagishi and co-workers. Expression of functional cytochrome ba3 occurred in cells grown at reduced dioxygen levels. A hepta-histidine tag was placed at the N-terminus of subunit I, and a purification method for this form of the enzyme was developed. Growth conditions were investigated for moderate sized cultures (2L) with typical yields of approximately 2 mg of highly pure enzyme per liter of culture medium. The physical properties and enzymatic activities of these recombinant enzymes were compared with those of native enzyme. Recombinant enzyme lacking the histidine tag is spectrally identical to wild-type enzyme. Histidine-tagged cytochrome ba3 shows minor differences from wild-type, and it appears be somewhat less active as a cytochrome c552 oxidase. Exemplary mutants were also produced and compared to native protein. Tyrosine I-237, previously found to be covalently bonded to I-His-233, was changed to phenylalanine (I-Y237F) and to histidine (I-Y237H) in the hepta-histidine tagged cytochrome ba3. The Y to F mutant is devoid of enzyme activity whereas the Y to H mutant possesses approximately 5% wild-type oxidase activity; their properties are compared with those of wild-type enzyme. The above versions of the histidine-tagged enzyme have been crystallized, and our analysis of a 2.3 angstrom resolution electron-density map will be discussed elsewhere.
细胞色素ba3是一种整合膜蛋白，在一些原核生物中作为呼吸链的末端氧化酶。我们已将嗜热栖热菌HB8的完整cba操纵子克隆到大肠杆菌/嗜热栖热菌穿梭载体中。利用Yamagishi及其同事的pyrE系统从嗜热栖热菌MT111菌株的染色体中消除了编码ba3的操纵子cba。在低氧水平下生长的细胞中出现了功能性细胞色素ba3的表达。在亚基I的N端放置了一个七聚组氨酸标签，并开发了这种形式酶的纯化方法。研究了中等规模培养物（2L）的生长条件，每升培养基的典型产量约为2mg高纯度酶。将这些重组酶的物理性质和酶活性与天然酶进行了比较。缺乏组氨酸标签的重组酶在光谱上与野生型酶相同。带有组氨酸标签的细胞色素ba3与野生型有细微差异，作为细胞色素c552氧化酶，其活性似乎略低。还产生了示例性突变体并与天然蛋白进行比较。在带有七聚组氨酸标签的细胞色素ba3中，先前发现与I-His-233共价结合的酪氨酸I-237被改为苯丙氨酸（I-Y237F）和组氨酸（I-Y237H）。Y到F突变体没有酶活性，而Y到H突变体具有约5%的野生型氧化酶活性；将它们的性质与野生型酶进行了比较。上述带有组氨酸标签的酶的版本已经结晶，我们对2.3埃分辨率电子密度图的分析将在其他地方讨论。
