title: Construction and purification of His-tagged staphylococcal ArsB protein, an integral membrane protein that is involved in arsenical salt resistance.
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.
authors.affiliation: Department of Microbiology, University of New Hampshire, Durham, NH 03824-2617 USA.
authors.forename: Carmela
authors.initials: C
authors.lastname: Mascio
authors.forename: Donald J
authors.initials: DJ
authors.lastname: White
authors.forename: Louis S
authors.initials: LS
authors.lastname: Tisa
keywords: Affinity chromatography; Arsenic resistance; Arsenite; Membrane protein; Transport