Nguyen Tuan A, Lieu Samantha S, Chang Geoffrey
Department of Molecular Biology, The Scripps Research Institute, La Jolla, Calif. 92037, USA.
J Mol Microbiol Biotechnol. 2010;18(2):85-91. doi: 10.1159/000283512. Epub 2010 Feb 17.
A cell-free expression system using an Escherichia coli extract was adapted for large-scale expression and purification of mammalian membrane proteins. The system was tested with a set of human membrane proteins of different sizes, numbers of transmembrane domains, oligomeric arrangements, and native membrane locations. Tens of milligrams of protein were readily expressed and purified from an overnight cell-free reaction. Both reaction 'mode A' (proteins were expressed as precipitant) and 'mode B' (proteins were expressed in the presence of mild detergents to keep them soluble) were investigated. The combination of 'mode B' and the right detergents, used in the subsequent extraction and purification steps, is critical for obtaining properly folded proteins (CX32 and VDAC1) that can be crystallized and diffracted (VDAC1). The E. coli cell-free system is capable of efficient expression of many mammalian membrane proteins. However, fine-tuning of the system, especially to facilitate proper protein folding, will be required for each specific target.
一种使用大肠杆菌提取物的无细胞表达系统被用于大规模表达和纯化哺乳动物膜蛋白。该系统用一组不同大小、跨膜结构域数量、寡聚排列和天然膜定位的人类膜蛋白进行了测试。从过夜的无细胞反应中很容易表达和纯化出数十毫克的蛋白质。研究了反应“模式A”（蛋白质以沉淀剂形式表达）和“模式B”（蛋白质在温和去污剂存在下表达以保持其溶解性）。“模式B”与后续提取和纯化步骤中使用的合适去污剂相结合，对于获得能够结晶和衍射（VDAC1）的正确折叠蛋白（CX32和VDAC1）至关重要。大肠杆菌无细胞系统能够有效表达许多哺乳动物膜蛋白。然而，对于每个特定靶点，都需要对该系统进行微调，特别是为了促进蛋白质的正确折叠。
