Expression, purification, and reconstitution of the Na(+)/H (+) exchanger sod2 in Saccharomyces cerevisiae.
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.
Chen
Heng
H
Department of Biochemistry, University of Alberta, Edmonton, AB, Canada.
Fliegel
Larry
L
D015870:Gene Expression; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000235:genetics; D012568:Schizosaccharomyces / Q000201:enzymology* / Q000235:genetics; D029702:Schizosaccharomyces pombe Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D017923:Sodium-Hydrogen Exchangers / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*
D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D029702:Schizosaccharomyces pombe Proteins; C501077:Sod2 protein, S pombe; D017923:Sodium-Hydrogen Exchangers