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. 2009 Aug 22;49(3):212–218. doi: 10.1007/s12088-009-0047-4

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

Carmela Mascio 1, Donald J White 1, Louis S Tisa 1,✉
PMCID: PMC3450018  PMID: 23100771

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.

Keywords: Arsenite, Membrane protein, Transport, Affinity chromatography, Arsenic resistance

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