title: An improved expression plasmid for affinity purification of Staphylococcus aureus gyrase A subunit.
abstract: Of the bacterial topoisomerases, the gyrase A subunit (GyrA) of Staphylococcus aureus is particularly difficult to purify because of its tendency to form inclusion bodies. Previous attempts at purification yielded low concentrations of protein with reduced specific activity. To overcome this problem, we modified the commercially available plasmid expression vector, pBAD/Thio-TOPO, via the addition of DNA sequences encoding a hexahistidine tag upstream and a cleavage site for tobacco etch virus protease downstream of the gene encoding thioredoxin. The resulting expression system consisting of the modified plasmid, pSAGA7, and the recommended host strain, Escherichia coli TOP 10, facilitated high level expression of soluble GyrA and its affinity purification to over 95% homogeneity. Purified GyrA had high biological activity as evidenced by a specific activity of 4.3x10(5)U/mg. The pSAGA7/TOP10 expression system also facilitated the expression and purification of a subunit of S. aureus topoisomerase IV, ParE, and a recently discovered protein unrelated to topoisomerases, QnrB, two "hard to purify" proteins. We conclude that pSAGA7 might be useful for high-level soluble expression and purification of diverse microbial proteins.
authors.affiliation: Division of Infectious Diseases, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA.
authors.forename: Jacob
authors.initials: J
authors.lastname: Strahilevitz
authors.forename: Yoshikuni
authors.initials: Y
authors.lastname: Onodera
authors.forename: David C
authors.initials: DC
authors.lastname: Hooper
mesh_terms: D002846:Chromatography, Affinity; D027081:DNA Gyrase / Q000235:genetics / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000302:isolation & purification; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000201:enzymology* / Q000235:genetics
chemical_list: D010450:Endopeptidases; C099488:TEV protease; D027081:DNA Gyrase