Institute of Biochemistry, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, 40225, Duesseldorf, Germany.
CNRS, UMR5086 "Molecular Microbiology and Structural Biochemistry", Université de Lyon, 7 Passage du vercors, 69007, Lyon, France.
Microb Cell Fact. 2019 Aug 10;18(1):131. doi: 10.1186/s12934-019-1182-1.
The overexpression and purification of membrane proteins is a bottleneck in biotechnology and structural biology. E. coli remains the host of choice for membrane protein production. To date, most of the efforts have focused on genetically tuning of expression systems and shaping membrane composition to improve membrane protein production remained largely unexplored.
In E. coli C41(DE3) strain, we deleted two transporters involved in fatty acid metabolism (OmpF and AcrB), which are also recalcitrant contaminants crystallizing even at low concentration. Engineered expression hosts presented an enhanced fitness and improved folding of target membrane proteins, which correlated with an altered membrane fluidity. We demonstrated the scope of this approach by overproducing several membrane proteins (4 different ABC transporters, YidC and SecYEG).
In summary, E. coli membrane engineering unprecedentedly increases the quality and yield of membrane protein preparations. This strategy opens a new field for membrane protein production, complementary to gene expression tuning.
膜蛋白的过表达和纯化是生物技术和结构生物学的一个瓶颈。大肠杆菌仍然是生产膜蛋白的首选宿主。迄今为止，大多数努力都集中在遗传调控表达系统和塑造膜组成上，以提高膜蛋白的产量，但这方面的研究仍在很大程度上尚未探索。
在大肠杆菌 C41(DE3)菌株中，我们删除了两个参与脂肪酸代谢的转运蛋白（OmpF 和 AcrB），即使在低浓度下，它们也是难以结晶的顽固污染物。经过工程改造的表达宿主表现出增强的适应性和目标膜蛋白的折叠改善，这与膜流动性的改变相关。我们通过过量表达几种膜蛋白（4 种不同的 ABC 转运蛋白、YidC 和 SecYEG）证明了这种方法的范围。
总之，大肠杆菌膜工程以前所未有的方式提高了膜蛋白制剂的质量和产量。该策略为膜蛋白生产开辟了一个新的领域，与基因表达调控相辅相成。
