Gebreselassie D, Rajarathnam K, Fliegel L
Department of Biochemistry, University of Alberta, Edmonton, Canada.
Biochem Cell Biol. 1998;76(5):837-42. doi: 10.1139/bcb-76-5-837.
The Na+/H+ exchanger is a pH regulatory protein that is responsible for removal of excess intracellular protons in exchange for extracellular Na+. It is a plasma membrane protein with a large cytoplasmic carboxyl terminal domain that regulates activity of the membrane domain. We overexpressed and purified the cytoplasmic domain that was produced in Escherichia coli. This region (516-815 amino acids) was under control of the tac promoter from the plasmid pGEX-KG and was fused with glutathione S-transferase. Upon induction, the fusion protein was principally found in inclusion bodies. Purified inclusion bodies were solubilized and fractionated using preparative SDS polyacrylamide gel electrophoresis. To obtain free Na+/H+ exchanger protein the fusion protein was dialyzed against cleavage buffer and cleaved at the thrombin cleavage site between glutathione S-transferase and the Na+/H+ exchanger domain. Free Na+/H+ exchanger protein was obtained by rerunning the sample on preparative gel electrophoresis. The final yield of the purified protein was 2.15 mg protein/L of cell culture. After exhaustive dialysis the secondary structure of the purified protein was assessed using circular dichroism spectroscopy. The results indicated that the protein was 35% alpha-helix, 17% beta-turn, and 48% random coil. They suggest that the cytoplasmic domain is structured and some regions may be compact in nature.
钠氢交换体是一种pH调节蛋白，负责将细胞内多余的质子排出，以交换细胞外的钠离子。它是一种质膜蛋白，具有一个大的胞质羧基末端结构域，可调节膜结构域的活性。我们对在大肠杆菌中产生的胞质结构域进行了过表达和纯化。该区域（516 - 815个氨基酸）受质粒pGEX-KG中tac启动子的控制，并与谷胱甘肽S-转移酶融合。诱导后，融合蛋白主要存在于包涵体中。纯化的包涵体用制备型SDS聚丙烯酰胺凝胶电泳进行溶解和分级分离。为了获得游离的钠氢交换体蛋白，将融合蛋白用裂解缓冲液进行透析，并在谷胱甘肽S-转移酶和钠氢交换体结构域之间的凝血酶切割位点进行切割。通过在制备型凝胶电泳上重新运行样品获得游离的钠氢交换体蛋白。纯化蛋白的最终产量为每升细胞培养物2.15毫克蛋白。经过充分透析后，使用圆二色光谱法评估纯化蛋白的二级结构。结果表明，该蛋白含有35%的α-螺旋、17%的β-转角和48%的无规卷曲。结果提示胞质结构域具有一定结构，某些区域可能本质上是紧密的。
