Miroux B, Walker J E
The Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.
J Mol Biol. 1996 Jul 19;260(3):289-98. doi: 10.1006/jmbi.1996.0399.
We have investigated the over-production of seven membrane proteins in an Escherichia coli-bacteriophage T7 RNA polymerase expression system. In all seven cases, when expression of the target membrane protein was induced, most of the BL21(DE3) host cells died. Similar effects were also observed with expression vectors for ten globular proteins. Therefore, protein over-production in this expression system is either limited or prevented by bacterial cell death. From the few survivors of BL21(DE3) expressing the oxoglutarate-malate carrier protein from mitochondrial membranes, a mutant host C41(DE3) was selected that grew to high saturation cell density, and produced the protein as inclusion bodies at an elevated level without toxic effect. Some proteins that were expressed poorly in BL21(DE3), and others where the toxicity of the expression plasmids prevented transformation into this host, were also over-produced successfully in C41(DE3). The examples include globular proteins as well as membrane proteins, and therefore, strain C41(DE3) is generally superior to BL21(DE3) as a host for protein over-expression. However, the toxicity of over-expression of some of the membrane proteins persisted partially in strain C41(DE3). Therefore, a double mutant host C43(DE3) was selected from C41(DE3) cells containing the expression plasmid for subunit b of bacterial F-ATPase. In strain C43(DE3), both subunits b and c of the F-ATPase, an alanine-H(+) symporter, and the ADP/ATP and the phosphate carriers from mitochondria were all over-produced. The transcription of the gene for the OGCP and subunit b was lower in C41(DE3) and C43(DE3), respectively, than in BL21(DE3). In C43(DE3), the onset of transcription of the gene for subunit b was delayed after induction, and the over-produced protein was incorporated into the membrane. The procedure used for selection of C41(DE3) and C43(DE3) could be employed to tailor expression hosts in order to overcome other toxic effects associated with over-expression.
我们研究了在大肠杆菌 - 噬菌体T7 RNA聚合酶表达系统中七种膜蛋白的过量表达情况。在所有七种情况下，当诱导目标膜蛋白表达时，大多数BL21(DE3)宿主细胞死亡。对于十种球状蛋白的表达载体，也观察到了类似的效应。因此，在该表达系统中，细菌细胞死亡限制或阻止了蛋白质的过量表达。从表达线粒体内膜中氧代戊二酸 - 苹果酸载体蛋白的BL21(DE3)的少数存活细胞中，筛选出了一种突变宿主C41(DE3)，它能生长到高饱和细胞密度，并以较高水平产生该蛋白，且无毒性作用。一些在BL21(DE3)中表达不佳的蛋白质，以及其他因表达质粒毒性而无法转化到该宿主中的蛋白质，也在C41(DE3)中成功实现了过量表达。这些例子包括球状蛋白和膜蛋白，因此，作为蛋白质过量表达的宿主，C41(DE3)菌株通常优于BL21(DE3)。然而，一些膜蛋白过量表达的毒性在C41(DE3)菌株中仍部分存在。因此，从含有细菌F - ATPase亚基b表达质粒的C41(DE3)细胞中筛选出了双突变宿主C43(DE3)。在C43(DE3)菌株中，F - ATPase的亚基b和c、一种丙氨酸 - H⁺同向转运体以及线粒体内的ADP/ATP和磷酸载体都实现了过量表达。在C41(DE3)和C43(DE3)中，OGCP和亚基b基因的转录分别低于BL21(DE3)。在C43(DE3)中，亚基b基因的转录在诱导后延迟开始，且过量产生的蛋白整合到了膜中。用于筛选C41(DE3)和C43(DE3)的方法可用于定制表达宿主，以克服与过量表达相关的其他毒性效应。
