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- Title
Expression, purification, and reconstitution of the Na<sup>+</sup>/H<sup>+</sup> exchanger sod2 in Saccharomyces cerevisiae.
- Authors
Heng Chen; Larry Fliegel
- Abstract
Abstract 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 β-d-maltoside. Immobilized Ni-ion column affinity chromatography partially purified the protein at a yield of ~240 μg 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.
- Subjects
SACCHAROMYCES; SACCHAROMYCETACEAE; SACCHAROMYCES carlsbergensis; SACCHAROMYCES cerevisiae
- Publication
Molecular & Cellular Biochemistry, 2008, Vol 319, Issue 1/2, p79
- ISSN
0300-8177
- Publication type
Article
- DOI
10.1007/s11010-008-9879-1