Reutimann H, Sjöberg B M, Holmgren A
Proc Natl Acad Sci U S A. 1985 Oct;82(20):6783-7. doi: 10.1073/pnas.82.20.6783.
Phage T7 DNA polymerase consists of a 1:1 complex of the viral T7 gene 5 protein and the host cell thioredoxin. A 3.25-kilobase T7 DNA fragment containing the complete coding sequence of gene 5, and the nearby genes 4.7 and 5.3, was cloned in the BamHI site of the plasmid pBR322. Transformation of the thioredoxin-negative (trxA-) Escherichia coli strain BH215 with the recombinant plasmid pRS101 resulted in large overproduction of gene 5 protein corresponding to a level about 60-fold higher than in T7-infected cells. Transcription of gene 5 probably originates from a previously unknown E. coli RNA polymerase promoter located immediately upstream of the structural gene. Contrary to expectation, pRS101 could be maintained also in E. coli trxA+ cells despite the in vivo formation of active T7 DNA polymerase. However, the expression of gene 5 was lower by a factor of 5-10 than in trxA- cells. Since the plasmid copy number in the two strains was the same, a gene dosage effect can be excluded. The observed difference suggests an autoregulatory interaction of T7 DNA polymerase holoenzyme on the expression of T7 gene 5. The trxA- strain BH215/pRS101 is an excellent source of gene 5 protein and T7 DNA polymerase. After in vitro reconstitution of holoenzyme by addition of excess thioredoxin, highly active T7 DNA polymerase was purified to homogeneity by a simple antithioredoxin immunoadsorbent chromatography technique.
噬菌体T7 DNA聚合酶由病毒T7基因5蛋白与宿主细胞硫氧还蛋白按1:1形成的复合物组成。一个包含基因5完整编码序列以及附近基因4.7和5.3的3.25千碱基T7 DNA片段被克隆到质粒pBR322的BamHI位点。用重组质粒pRS101转化硫氧还蛋白阴性（trxA-）的大肠杆菌菌株BH215，导致基因5蛋白大量过量表达，其水平比T7感染细胞中的水平高约60倍。基因5的转录可能源自位于结构基因紧邻上游的一个此前未知的大肠杆菌RNA聚合酶启动子。与预期相反，尽管在体内形成了活性T7 DNA聚合酶，但pRS101在大肠杆菌trxA+细胞中也能维持。然而，基因5的表达比在trxA-细胞中低5至10倍。由于两个菌株中的质粒拷贝数相同，因此可以排除基因剂量效应。观察到的差异表明T7 DNA聚合酶全酶对T7基因5的表达存在自调控相互作用。trxA-菌株BH215/pRS101是基因5蛋白和T7 DNA聚合酶极好的来源。通过添加过量硫氧还蛋白在体外重组全酶后，利用简单的抗硫氧还蛋白免疫吸附色谱技术将高活性的T7 DNA聚合酶纯化至同质。
