Duvoisin R M, Belin D, Krisch H M
Gene. 1986;45(2):193-201. doi: 10.1016/0378-1119(86)90254-4.
Two new expression vectors have been constructed to take advantage of several useful properties of bacteriophage T4-infected Escherichia coli. These plasmids, pRDB8 and pRDB9, contain the promoter region and start codon of T4 gene 32, a contiguous multiple cloning site (MCS), and translation and transcription termination signals. DNA fragments inserted into the MCS are transcribed and translated at a high level in both uninfected and phage T4-infected cells. Furthermore, the extreme stability of the hybrid mRNA after infection permits the specific biosynthetic labeling of the protein encoded by the cloned gene. In addition, the cloned gene product is stabilized, since the host-mediated degradation of foreign proteins is inhibited by phage infection. The properties of this expression system were demonstrated with the constant region of a rabbit immunoglobulin lambda light chain (C lambda) gene. Although proteolytic degradation of the C lambda fusion protein was rapid in uninfected cells, degradation was blocked in phage-infected cells and the protein accumulated in greater amounts.
构建了两种新的表达载体,以利用噬菌体T4感染的大肠杆菌的几种有用特性。这些质粒pRDB8和pRDB9包含T4基因32的启动子区域和起始密码子、一个连续的多克隆位点(MCS)以及翻译和转录终止信号。插入到MCS中的DNA片段在未感染和噬菌体T4感染的细胞中均能高水平转录和翻译。此外,感染后杂交mRNA的极高稳定性允许对克隆基因编码的蛋白质进行特异性生物合成标记。另外,由于噬菌体感染抑制了宿主介导的外源蛋白质降解,克隆的基因产物得以稳定。用兔免疫球蛋白λ轻链(Cλ)基因的恒定区证明了该表达系统的特性。尽管Cλ融合蛋白在未感染细胞中的蛋白水解降解很快,但在噬菌体感染的细胞中降解被阻断,并且蛋白大量积累。