[Cloning, expression and purification of dptC, a DNA phosphorothioate modification related gene from Salmonella...

DNA phosphorothioate modification means substituting a non-bridging oxygen with a sulfur in DNA. The modification endows DNA with such chemical property that protects the hosting bacteria against peroxide. The modification is controlled by a dnd gene cluster. Salmonella entericaserovar Cerro 87 is one of the bacteria that harbor the DNA phosphorothioate modification. The modification is carried out by dptB, C, DandE. Ourstudy is designed to clone and express dptC, to optimize the expressing condition, and then to purify the DptC.

dptC DNA fragment was amplified by KOD PCR with the special primers and S. entericaserovar Cerro 87 genomic DNA template. A fusion expression vector pJTU3622 was constructed by inserting the dptC DNA fragment into pGEX-6P-1 inSmaI and XhoI sites. The positive clone was verified by antibiotics resistance gene screening and sequenced, and then transferred into host strain E. coli BL21 (DE3) pLysS to producean engineering bacterium Anxh103. After optimizing the expression condition for dptC, we purified DptC from Anxh103 by Aikta FPLC with a GST-Trap column.

A fusion expression vector pJTU3622 and an engineering bacterium Anxh103 were produced. The optimizing expressing condition for dptC is as follows: induced at 18 degrees C for 8 - 18 h; 0.6 mmol/L IPTG, LB with 50 micromol/L Fe2+.

The anchor redeemed for high throughput expression of dptC. The TEV site in pJTU3622 made the process of purifying DptC easier and simpler. This helps lay the ground work for future study on the function of DptC. Also, the light brown color of DptC and the medium with 50 micromol/L Fe2+ showed us DptC has the same character with DndC which belongs to an iron-sufur protein with 4Fe - 4S.