Therien Alex G, Glibowicka Mira, Deber Charles M
Division of Structural Biology and Biochemistry, Research Institute, Toronto, Ontario, Canada.
Protein Expr Purif. 2002 Jun;25(1):81-6. doi: 10.1006/prep.2001.1612.
We describe a rapid method for the expression and purification of two hydrophobic protein constructs derived from the membrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR), the protein associated with cystic fibrosis. The proteins have no sequence homology but are both predicted to contain two membrane-spanning segments. The protocol involves the expression of CFTR constructs as thioredoxin fusion proteins in Escherichia coli, followed by partial purification by affinity chromatography, removal of the thioredoxin moiety by proteolytic cleavage in the presence of detergent, and final purification by reversed-phase high-performance liquid chromatography. The method yields milligram amounts of purified constructs that spontaneously insert into detergent micelles in alpha-helical conformation. We predict that this protocol will be applicable to a variety of proteins of similar size and hydrophobicity.
我们描述了一种快速表达和纯化两种源自囊性纤维化跨膜传导调节因子（CFTR）膜结构域的疏水蛋白构建体的方法，CFTR是与囊性纤维化相关的蛋白。这两种蛋白没有序列同源性，但预计都含有两个跨膜片段。该方案包括在大肠杆菌中将CFTR构建体作为硫氧还蛋白融合蛋白进行表达，然后通过亲和色谱进行部分纯化，在去污剂存在下通过蛋白酶切割去除硫氧还蛋白部分，最后通过反相高效液相色谱进行最终纯化。该方法可产生毫克量的纯化构建体，这些构建体以α-螺旋构象自发插入去污剂胶束中。我们预计该方案将适用于各种大小和疏水性相似的蛋白质。
