Molecular Cloning Of Oxc Gene Of Oxalobacter Formigenes And Prokaryotic Expression Of It's Center Domain

[Objective] It is to construct the recombinant expression plasmids about the OXC-1 protein of Oxalyl-CoAdecarboxylase(OXC) from Oxalobacter formigenes, which are pET-OXC-1(202～351aa), and theirrecombinant E.coli His-OXC-1, thus active recombinant center domain of OXC can be obtained. It will beused in the detection about the content of OXC in the body and will be the foundation for forecasting the riskof stone disease as well as preventing the stone disease.[Method] Using the polymerase chain reaction (PCR) technology, the 1704bp of oxc gene from Oxalobacterformigenes(ATCC 35274) was amplified. After ligating the product of PCR to pGEM-T easy vector, therecombinant plasmids pGEM-T-oxc was obtained. Adopting the recombinant DNA technology, theprokaryotic recombinant expression plasmids of pET-OXC-1(202～351aa) was constructed. Then it wastransformed into E.coli BL21(DE3), and the recombinant bacterium E.coli His-OXC-1 was obtained. Afteroptimizing the expression conditions and inducing the recombinant bacterium, recombinant His-OXC-1 wasexpressed. After collecting the bacterial cells and breaking the cell by means of ultrasonic, the inclusion bodieswere extracted. The extracted products were denatured with 8 mol/L urea and purified through Ni-NTAcolumn. The purified products were renatured by dialyzation, and then were analyzed by SDS-PAGE.[Results] The recombinant E.coli His-OXC-1(202～351aa) had been constructed successfully, and theycould produce the expectant molecular size about 19.8ku of fusion proteins by inducing the recombinantbacterium. The expressed expectant proteins were inclusion bodies in bacteria. Under the optimized expressioncondition(37℃, 1mmol/L IPTG, induced for 6 h), the recombinant expression product was account for 30%～45% of overall bacterium proteins. After spliting the bacteria, the molecular size about 19.8 ku of inclusionprotein had been obtained. After being purified through Ni-NTA column, the contaminative proteins about31～40 ku were removed and the high purity His-OXC-1 protein was the molecular size about 19.8 ku ofprotein. The renatured products had a higher purity and the only band of 19.8 ku of protein could be seen inSDS-PAGE.[Conclusion] Cloning the oxc gene from Oxalobacter formigenes (ATCC 35274) and successfullyconstructing the recombinant plasmid pET-OXC-1 (202～351aa) about the OXC-1 protein, the recombinantE.coli His-OXC-1 as well as the recombinant active center domain of OXC protein have been obtained.