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


CONCLUSIONS
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.
Luo
Lixin
L
School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, China. btlxluo@scut.edu.cn
Jiang
Binqiang
B
Chen
Moutong
M
D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics*; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000737:chemistry / Q000201:enzymology*
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases