Chen Heng, Fliegel Larry
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Mol Cell Biochem. 2008 Dec;319(1-2):79-86. doi: 10.1007/s11010-008-9879-1. Epub 2008 Jul 16.
Sod2, is a Na(+)/H(+) exchanger present on the cytoplasmic membrane of the fission yeast Schizosaccharomyces pombe. It expels toxic Na(+) from the cytosol. Sod2 was expressed in Saccharomyces cerevisiae with a C-terminal histidine tag under control of the GAL1 promoter. Western blots using anti-V5 antibodies identified the tagged protein. Solubilization of the protein was by n-dodecyl beta-D: -maltoside. Immobilized Ni-ion column affinity chromatography partially purified the protein at a yield of ~240 microg per liter of culture. Sod2 was present as a 40-kDa and an 80-kDa protein, however, it co-purified with a number of other proteins. Cross linking of sod2 with N,N'-(o-phenylene)dimaleimide showed that sod2 was present in association with a number of other proteins as a larger molecular weight complex. Partially purified sod2 protein was reconstituted in proteoliposomes and functionally active. Our results suggest that the sod2 protein associates with a number of other proteins and can be expressed in S. cerevisiae in active form.
Sod2是裂殖酵母粟酒裂殖酵母细胞质膜上存在的一种Na(+)/H(+)交换体。它从细胞质中排出有毒的Na(+)。Sod2在GAL1启动子的控制下，在带有C末端组氨酸标签的酿酒酵母中表达。使用抗V5抗体的蛋白质印迹法鉴定了带标签的蛋白质。蛋白质的溶解使用正十二烷基-β-D-麦芽糖苷。固定化镍离子柱亲和色谱法对蛋白质进行了部分纯化，每升培养物的产量约为240微克。Sod2以40 kDa和80 kDa的蛋白质形式存在，然而，它与许多其他蛋白质一起被共纯化。用N,N'-(邻亚苯基)双马来酰亚胺对sod2进行交联表明，sod2以更大分子量的复合物形式与许多其他蛋白质结合存在。部分纯化的sod2蛋白被重组到蛋白脂质体中且具有功能活性。我们的结果表明，sod2蛋白与许多其他蛋白质结合，并且可以在酿酒酵母中以活性形式表达。
