Davison J, Chevalier N, Brunel F
Unit of Molecular Biology, International Institute of Cellular and Molecular Pathology, Brussels, Belgium.
Gene. 1989 Nov 30;83(2):371-5. doi: 10.1016/0378-1119(89)90124-8.
The rifampicin (Rif)-resistant RNA polymerase of phage T7 has proved invaluable for the exclusive over-expression, in Escherichia coli, of genes cloned downstream from the T7 phi 10 promoter [Tabor and Richardson, Proc. Natl. Acad. Sci. USA 82 (1985) 1074-1078]. Here, we demonstrate that the system can be extended to Gram-negative bacteria other than E. coli, by the use of compatible wide host range plasmids. As an example, the Rif-resistant in vivo synthesis and specific radiolabelling of E. coli galactokinase in Pseudomonas ATCC19151, is demonstrated. The incidental observation that 30 min after treatment with Rif, two polypeptides continue to be synthesized in plasmid-free Pseudomonas ATCC19151, indicates that these proteins are produced by very stable mRNA species.
噬菌体T7的利福平（Rif）抗性RNA聚合酶已被证明对于在大肠杆菌中特异性过表达克隆于T7 phi 10启动子下游的基因非常有价值[Tabor和Richardson，美国国家科学院院刊82（1985）1074 - 1078]。在此，我们证明通过使用兼容的广宿主范围质粒，该系统可扩展至除大肠杆菌外的革兰氏阴性菌。例如，证明了在假单胞菌ATCC19151中利福平抗性的体内合成及大肠杆菌半乳糖激酶的特异性放射性标记。偶然观察到，在用利福平处理30分钟后，无质粒的假单胞菌ATCC19151中仍有两种多肽在继续合成，这表明这些蛋白质是由非常稳定的mRNA种类产生的。
