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Construction and purification of His-tagged staphylococcal ArsB protein, an integral membrane protein that is involved in arsenical salt resistance

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Abstract

Bacterial resistance to arsenical salts encoded on plasmid pI258 occurs by active extrusion of toxic oxyanions from cells of Staphylococcus aureus. The operon encodes for three gene products: ArsR, ArsB and ArsC. The gene product of arsB is an integral membrane protein and it is sufficient to provide resistance to arsenite and antimonite. A poly His-ArsB fusion protein was generated to purify the staphylococcal ArsB protein. Cells containing the His-tagged arsB gene were resistant to arsenite and antimonite. The levels of resistance to these toxic oxyanions by the His-tagged construct were greater than the levels obtained with the wild type gene. These data would indicate that the His-tagged protein is functionally active. A new 36 kDa protein band was visualized on 10% SDS-polyacrylamide gel electrophoresis (PAGE), which was confirmed as the His-ArsB protein by immunodetection with polyclonal Hisantibodies. The His-ArsB fusion protein was purified by the use of metal-chelate affinity chromatography with a Ni+2-nitrilotriacetic acid column and size-exclusion chromatography suggests that the protein was a homodimer.

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Author notes

  1. Carmela Mascio

    Present address: Cubist Pharmaceuticals, Lexington, MA, 02421, USA

  2. Donald J. White

    Present address: Carilion Clinic, Roanoke, VA, 24014, USA

Authors and Affiliations

  1. Department of Microbiology, University of New Hampshire, Durham, NH, 03824-2617, USA

    Carmela Mascio, Donald J. White & Louis S. Tisa

Authors
  1. Carmela Mascio
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  2. Donald J. White
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  3. Louis S. Tisa
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Correspondence to Louis S. Tisa.

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Mascio, C., White, D.J. & Tisa, L.S. Construction and purification of His-tagged staphylococcal ArsB protein, an integral membrane protein that is involved in arsenical salt resistance. Indian J Microbiol 49, 212–218 (2009). https://doi.org/10.1007/s12088-009-0047-4

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  • DOI: https://doi.org/10.1007/s12088-009-0047-4

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