Optimization of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR
Optimization of Recombinant Membrane Protein Production in the Engineered Escherichia coli Strains SuptoxD and SuptoxR
复制标题
DOI：
10.1021/acssynbio.9b00120
复制
发表时间：
2019-07-01
影响因子：
4.7
通讯作者：
Skretas, Georgios
中科院分区：
文献类型：
--
作者：
Michou, Myrsini;Kapsalis, Charalampos;Skretas, Georgios
膜蛋白(MPS)在所有生物体中执行多种重要的生物学功能，约占目前药物发现靶点的一半。与可溶性蛋白质的情况一样，大肠杆菌也是膜蛋白生化/结构研究中非常受欢迎的过表达宿主。然而，细菌重组膜蛋白的生产通常受到细胞积累不足和对宿主的严重毒性的阻碍，这导致最终生物量和微小体积产率的水平较低。在以前的工作中，我们培育了工程菌株SupoxD和SuptoxR，它们分别通过共表达效应基因djlA或rraA，可以抑制MP过表达所引起的细胞毒性，并提高MP产量。在这里，我们系统地寻找在这些菌株中最大限度地积累膜整合和折叠良好的重组MPS的基因过表达和培养条件。我们发现，在最佳条件下，SupoxD和SuptoxR可以极大地提高各种MP的重组产量，无论它们来自古细菌、真细菌还是真核细胞。此外，我们还证明，使用这些工程菌株可以高质量和高产量地生产折叠良好的重组MPS，这适用于功能和结构研究。我们预计SupoxD和SuptoxR将成为在细菌中生产重组MP的广泛使用的表达宿主。
Membrane proteins (MPs) execute a wide variety of critical biological functions in all living organisms and constitute approximately half of current targets for drug discovery. As in the case of soluble proteins, the bacterium Escherichia coli has served as a very popular overexpression host for biochemical/structural studies of membrane proteins as well. Bacterial recombinant membrane protein production, however, is typically hampered by poor cellular accumulation and severe toxicity for the host, which leads to low levels of final biomass and minute volumetric yields. In previous work, we generated the engineered E. coli strains SuptoxD and SuptoxR, which upon coexpression of the effector genes djlA or rraA, respectively, can suppress the cytotoxicity caused by MP overexpression and produce enhanced MP yields. Here, we systematically looked for gene overexpression and culturing conditions that maximize the accumulation of membrane-integrated and well-folded recombinant MPs in these strains. We have found that, under optimal conditions, SuptoxD and SuptoxR achieve greatly enhanced recombinant production for a variety of MP, irrespective of their archaeal, eubacterial, or eukaryotic origin. Furthermore, we demonstrate that the use of these engineered strains enables the production of well-folded recombinant MPs of high quality and at high yields, which are suitable for functional and structural studies. We anticipate that SuptoxD and SuptoxR will become broadly utilized expression hosts for recombinant MP production in bacteria.