Cloning and expression of the novel antimicrobial target enzyme Sortase gene in two prokaryotic vectors

Abstract:

Abstract: [Objective] Sortase is a novel anti-infection target enzyme for its critical action of anchoring surface proteins to the cell wall. [Methods] We amplified the srtA gene from Staphylococcus aureus chromosomal DNA by PCR technique, and then constructed two prokaryotic expression vectors pet22-srtA and pTRX-srtA with regular molecular cloning operation. The pet22-srtA and pTRX-srtA were transformed into E. coli BL21 (DE3) competent cells and overexpressed under 1 mmol/L IPTG (isopropy-β-D-thiogalactoside) induction. [Results] SDS-PAGE and western blot results show that approximately 45 kDa and 39 kDa proteins were expressed by pet22-srtA and pTRX-srtA respectively. [Conclusion] The molecular chaperone thioredoxin was beneficial to the prokaryotic expression of srtA gene. Moreover, the experiment laid solid foundation to study sortase’s enzymatic property and inhibitors screening especially.

Key words:sortase; prokaryotic expression; Staphylococcus aureus; antimicrobial