Cloning And Expression Of The DCTP Deaminase Gene From Thermus Phage TSP4and The Analysis Of Its Entropy And Thermal Stability

Cytidine deaminase (Cytidine deaminase) is a cytoplasmic enzyme, which catalyzes the Hydrolysis reaction of cytidine. In gram-positive bacteria and eukaryotic organisms, dCTP Deaminases catalyze the conversion of dCMP to dUMP in the pyrimidine salvage pathway. In contrast, in the gram-negative bacteria Escherichia coli, the deamination of dCTP to dUTP is dCTP deaminase. Deoxycytidine deaminase also can get5-flucytosine to deaminate to5-fluorouracil(5-FU), a widely used chemotherapy drugs, which has a good clinical effect in malignant tumors of epithelial tissue sources such as liver, stomach and colon cancer.In this study, The gene encoding a dCTP deaminase tspdCTP from Thermus phage TSP4was cloned into pET-28a and pET-32a, generating two recombinant plasmids pET-28a-tspdCTP and pET-32a-tspdCTP which were further transformed into Escherichia coli Rosetta(DE3) for expression. Analysis of SDS-PAGE showed that target protein was highly expressed. The expressed protein was purified using Ni-NTA agarose. The enzyme characteristics including optimum catalytic temperature, pH and substrate, as well as the effects of metal ions and organic solvents on the enzyme activity were analysed. The results showed that the specific activity of the expressed tspdCTP was4.12U/mg. Its optimum catalytic temperature and pH were60℃and7.5, repectively, and its optimum substrate was dCTP. The enzyme activity was stimulated by2mM of Ca2+and Mg2+, but was inhibited by2mM of Ni2+and Cu2+. The results also showed that10%(V/V) ethyl acetate and isopropanol significantly increased the activity of the expressed tspdCTP, while10%(V/V) acetone had a significant inhibitory effect for the same enzyme.In addition, this paper explored the relationship between entropy and thermal stability of tspdCTP deaminase, the effect of functional sites and their surrounding residues structure entropy distribution characteristics for protein activity and thermal stability. According the protein transformation principle proposed by Chan et al, four mutants of dCTP deaminase of TSP4were designed, cloned, expression and their thermal stability were analysed. As a result, only two dCTP deaminase mutants were expressed in the pET-32a expression system. Thermal stability analysis of dCTP deaminase mutation showed that the higher system average structure entropy the mutate protein has, the lower thermal stability the protein may be. Their thermal stability was determined by incubation of the mutation proteins for the same time at the same temperature. This result is consistent with theoretical of lower the protein entropy value, higher the thermal stability. The specific function relationship remains to be further authentication. We also proved that transform4-9amino acids before or after the conserved residues in a protein does not lead to activity of the protein destruction, but the thermal stability change significantly.These results provide a further data of exploring the characteristics of high temperature phage life activities in high temperature environment, and will benefit to improve protein thermal stability by the use of entropy.