Cloning, Expression And Characterization Of Deoxyuridine Pyrophosphatase From Thermus Phage TSP4

Deoxyuridine pyrophosphatase dUTPase is one of the important hydrolysases in synthesis of DNA, which could hydrolyze dUTP to make dMUP and PPi, reduce the dUTP/dTTP ration in the cell and decrease the error incorporation of uracil in DNA synthesis. As a major regulator of intracellular level of dUTP, dUTPase has been regarded as many drug targets such as anti-cancer and anti-viral infectious disease. It also has been used for treatment with antagonistic thymidylate synthase inhibitors and dihydrofolate reductase agents in diagnosis and treatment.Thermus phage TSP4from Tengchong Rehai was used as a material for investigation the characterization of deoxyuridine pyrophosphatase (dUTPase) from thermophilic phage. Gene encoding TSP4dUTPase was amplified by PCR, ligated with plasmid and transformed into host cells to get a recombinant expression bacteria pET-32a-TSPdUTPase/Rosetta(DE3). Recombinant protein TSPdUTPase obtained after induced by8hours with a final concentration of1mM IPTG at16℃, The expressed protein TSPdUTPase was purified using Ni-NTA agarose and the enzyme characteristics including optimum activity temperature, pH and substrates, as well as the effects of metal ions on the enzyme activity were analyzed. The results showed that the optimum catalytic temperature and pH were65℃and7.5, respectively, The optimum catalytic substrate was dUTP, Mg2+was very important for dUTPase activity, and without Mg2+, no dUTPase activity was detected. Further more, EDTA could decrease the activity of dUTPase, for EDTA could compete Mg2+with dUTPase to decrease dUTPase activity.In order to detect the thermal stability and enzyme activity changes of recombinant protein TSPdUTPase at different temperatures, ANS fluorescent probe method was used to detect the change of its structure. Results showed that the TSPdUTPase relatively stable at below65℃, The enzyme activity will drop to about half by incubation at75℃for half an hour and none of enzyme activity was detected at80℃. In order to explore the effect of the molecular chaperone TSPcpn47on the thermal stability of recombinant protein TSPdUTPase, the molecular chaperone TSPcpn47from the phage TSP4also was cloned、expressed and interacted with TSPdUTPase。Under the condition of coexistence of both chaperone TSPcpn47and TSPdUTPase, the thermal stability of dUTPase has been greatly improved. This proved that the molecular chaperone TSPcpn47can improve thermal stability of proteins, This result also indicated that chaperone may be a import factor owning by thermophilic bacteriophages which can survive in extreme high temperature environment, and benefited for further investigation of the molecular mechanisms of heat stress tolerance in thermopilic phages.