Expression Of Recombinant Thymidine Kinase And Polyphosphate Kinase From Escherichia Coli And Their Applications

Thymidine kinase (TK) is one of the key enzymes in the the thymidylate remedy metabolic pathway of E. coli. At the presence of Mg2+, thymidine (TR) was conversed to thymidylate (TMP) with adenosine triphosphate (ATP) as phosphate donor. Polyphosphate kinase (PPK) is an enzyme that catalyzes the phospho-conversion of ADP to ATP and it uses inorganic polyphosphate as a phosphate donor. According to the characteristic of polyphosphate kinase, we connected the two reactions together and constructed an ATP regeneration system. Using this system, TMP was continuously synthesized from TR by the coupling reaction of TK and PPK.The genes encoding thymidine kinase (TK) and polyphosphate kinase (PPK) from Escherichia coli K-12were cloned respectively into expression vector pET-28a. Then the recombinant plasmids were transformed into the host strain E.coli BL21(DE3) to construct recombinant strains DTK and DPK. At the same time, co-expression recombinant strain (DKK) was constructed. Enzymes were expressed after induction with IPTG, and large amount of target proteins were expressed in soluble form analyzed with SDS-PAGE. Compared with the host strain, enzyme activity of the recombinant strains had been notably improved.In the study of nucleoside transformation reaction, TR was catalyzed to TMP by recombinant strain DTK in Tris-HCl (pH8.0,45℃) and the conversion rate of TR reached60%. ADP was catalyzed to ATP by recombinant strain DPK in Tris-HCl (pH8.0,55℃) and the conversion rate of ADP reached20%. TR was catalyzed to TMP using polyphosphate and ADP as substrate by recombinant strain DKK in Tris-HCl (pH8.0,45℃) and the conversion rate of TR reached13%.