Jeffery Constance J
Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois.
Curr Protoc Protein Sci. 2016 Feb 2;83:29.15.1-29.15.15. doi: 10.1002/0471140864.ps2915s83.
Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins. The recombinant protein of interest with a 6His affinity tag is expressed in E. coli. After harvesting the cultures and isolating cellular membranes, mild detergents are used to solubilize the membrane proteins. Protein-detergent complexes are then purified using IMAC column chromatography. Support protocols are included to help select a detergent for protein solubilization and for use of gel filtration chromatography for further purification.
细菌整合膜蛋白发挥着许多重要作用，包括感知环境变化、将分子转运进细胞和运出细胞，并且对于共生菌或病原菌而言，还包括与宿主生物体相互作用。由于在重组表达和纯化方面存在困难，在实验室中处理膜蛋白比处理可溶性蛋白更具挑战性。本方案描述了一种表达、溶解和纯化细菌整合膜蛋白的标准方法。带有6His亲和标签的目标重组蛋白在大肠杆菌中表达。收获培养物并分离细胞膜后，使用温和的去污剂溶解膜蛋白。然后使用IMAC柱色谱法纯化蛋白质 - 去污剂复合物。还包括支持方案，以帮助选择用于蛋白质溶解的去污剂以及使用凝胶过滤色谱法进行进一步纯化。
