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Expression, purification, and reconstitution of the Na+/H+ exchanger sod2 in Saccharomyces cerevisiae

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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.

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Abbreviations

DDM:

n-dodecyl β-d-maltoside

DSS:

Disuccinimidyl suberate

o-PDM:

N,N′-(o-phenylene)dimaleimide

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Acknowledgments

Heng Chen was supported by the CIHR Strategic Training Initiative in Membrane Proteins and Cardiovascular Disease and by NSERC of Canada. This research was supported by NSERC of Canada and LF is supported by an AHFMR Scientist award.

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  1. Department of Biochemistry, Faculty of Medicine, University of Alberta, 347Medical Science Building, Edmonton, AB, Canada, T6G 2H7

    Heng Chen & Larry Fliegel

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  1. Heng Chen
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  2. Larry Fliegel
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Correspondence to Larry Fliegel.

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Chen, H., Fliegel, L. Expression, purification, and reconstitution of the Na+/H+ exchanger sod2 in Saccharomyces cerevisiae . Mol Cell Biochem 319, 79–86 (2008). https://doi.org/10.1007/s11010-008-9879-1

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