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Updated: Jun 23, 2026
Subcloning Plus Insertion (SPI) - A Novel Recombineering Method for the Rapid Construction of Gene Targeting Vectors
Published on: January 8, 2015
[Cloning and expression of the novel antimicrobial target enzyme Sortase gene in two prokaryotic vectors]
Lixin Luo1, Binqiang Jiang, Moutong Chen
1School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. btlxluo@scut.edu.cn
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 Eschericheria coli BL21 (DE3) competent cells and overexpressed under 1 mmol/L IPTG (isopropy-beta-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.
