Lama J, Carrasco L
Centro de Biología Molecular, Universidad Autónoma de Madrid, Canto Blanco.
Biochem Biophys Res Commun. 1992 Nov 16;188(3):972-81. doi: 10.1016/0006-291x(92)91327-m.
The poliovirus 3AB gene has been cloned and overproduced in T7 expression vectors using different approaches to allow reduction of basal levels of expression. Expression of the poliovirus 3AB gene is highly toxic for E. coli cells, due to drastic changes induced in membrane permeability of the bacteria that lead to cell lysis when the T7 lysozyme is present. The best production of 3AB was achieved with the T7/lac system in cells lacking T7 lysozyme, where this toxic protein was synthesized to high levels and during several hours in the absence of cell lysis. These results show the efficient synthesis of a highly damaging membrane protein and open the possibility to apply heterologous gene expression in E. coli to other lytic proteins.
脊髓灰质炎病毒3AB基因已被克隆,并使用不同方法在T7表达载体中过量表达,以降低基础表达水平。脊髓灰质炎病毒3AB基因的表达对大肠杆菌细胞具有高度毒性,这是由于当存在T7溶菌酶时,细菌膜通透性发生剧烈变化,导致细胞裂解。在缺乏T7溶菌酶的细胞中,利用T7/乳糖系统实现了3AB的最佳表达,在这种情况下,这种有毒蛋白在数小时内以高水平合成,且未发生细胞裂解。这些结果表明高效合成了一种具有高度破坏性的膜蛋白,并为将大肠杆菌中的异源基因表达应用于其他裂解蛋白开辟了可能性。