Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)
Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)
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DOI：
10.1016/j.jmb.2012.07.019
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发表时间：
2012-11-02
影响因子：
5.6
通讯作者：
de Gier, Jan-Willem
中科院分区：
文献类型：
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作者：
Schlegel, Susan;Lofblom, John;de Gier, Jan-Willem
大肠杆菌BL 21（DE 3）被广泛用于过表达蛋白质。在这种过表达宿主中，编码靶蛋白的基因位于质粒上，并在T7启动子的控制下，T7启动子仅被T7 RNA聚合酶（RNAP）识别。17 RNAP基因位于染色体上，其表达由不可滴定的IPTG诱导型lacUV 5启动子控制。最近，我们构建了Lemo 21（DE 3）菌株，其允许改进对来自17启动子的基因表达的控制。Lemo 21（DE 3）是一种BL 21（DE 3）菌株，其配备有携带编码T7溶菌酶的基因的质粒，T7溶菌酶是T7 RNAP的抑制剂，在非常良好可滴定的鼠李糖启动子的控制下。Lemo 21（DE 3）中大量膜蛋白在不同浓度的鼠李糖下的过表达产量表明，该菌株可能非常适合于优化膜蛋白的生产。然而，缺乏对Lemo 21（DE 3）中实现优化表达产量的机制的了解。此外，过表达的蛋白质是否适合功能和结构研究仍有待测试。在这里，我们表明，在Lemo 21（DE 3）中，（i）17溶菌酶对17 RNAP活性的调节是优化正确插入细胞质膜的膜蛋白与非插入蛋白的比例的关键;（ii）最大化膜蛋白的产量伴随着膜蛋白过表达的不利影响的减少，导致稳定的过表达;和（iii）产生的膜蛋白可用于功能和结构研究。(C)2012爱思唯尔有限公司保留所有权利。
Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The 17 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the 17 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the 17 RNAP by the 17 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies. (C) 2012 Elsevier Ltd. All rights reserved.