An improved expression plasmid for affinity purification of Staphylococcus aureus gyrase A subunit

Jacob Strahilevitz, Yoshikuni Onodera, David C. Hooper*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

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 4.3 × 105 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.

Original languageAmerican English
Pages (from-to)10-15
Number of pages6
JournalProtein Expression and Purification
Volume47
Issue number1
DOIs
StatePublished - May 2006
Externally publishedYes

Keywords

  • Expression plasmid
  • Gyrase
  • Purification
  • Solubility
  • Staphylococcus aureus
  • TEV
  • Thioredoxon

Fingerprint

Dive into the research topics of 'An improved expression plasmid for affinity purification of Staphylococcus aureus gyrase A subunit'. Together they form a unique fingerprint.

Cite this