The Expression And Activity Assay Of Human Thymylate Synthase In E. Coli

Thymidylate synthase(TS) catalyzes the reductive methylaton of dUMP by CH2H4folate to produce dTMP. This process is the only de novo source of dTMP, which is subsequently metabolised to thymidine triphosphate (dTTP), exclusively for incorporation into DNA during synthesis and repair. For these reason, TS is one of the most important target in cancer therapy for many years. it need massive enzyme to study the mechanism of TS with its inhibitors, but there is no enough from bio-extraction. With the development of gene recombination and prokaryotic expression system, it is feasible to expression human TS in E.coli.In the experiment, the plasmid--TS-pcDNA3.1-zeo(+) carrying wild-type TS cDNA was uesed as PCR template. Target gene was amplified by PCR and TA cloning, then connected with the expression vector--pET-28b(+) to construct recombination expression vector--TS-pET28b(+); TS-pET28b(+) was transformed into E.coli Rosetta(DE3) by CaCl2 means. Positive transformant were screened through kanamycin resistance plate, further identified by PCR as well as double digestion, and finally to obtain the positive ones--TS-pET28b(+)/Rosetta(DE3). TS-pET28b(+)/ Rosetta(DE3) was induced by IPTG to produce the recombinant human TS with 6*His-tag, which was identified with SDS-PAGE and West-blotting. Then the optimal conditions was confirmed through orthogonal test, inducing temperature: 37℃, IPTG concentration: 1.5mM, inducing time: 8h. Under the optimal conditions, the yield of human TS in Rosseta(DE3) was up to about 43.5% of the E. coli protein, and human TS expressing in Rosetta(DE3) was mainly on inclusion body.In order to obtain the high purity and natural bioactive human TS, the recombinant human TS with 6*His-tag was purified by Ni-NTA affinity chromatography, which purity was up to 90%. The elution buffer was refolded by dialysis, which refolding yield was 86.67%. its enzyme activity determined by spectrophotometry was 0.012U, and specific activity was 0.1U/mg. The inhibitory concentration 50% (IC50) of LY231514 and Tomudex to refolding TS was respectively 1.0×10-4M,1.0×10-6M. The results indicated wild-type hTS cDNA had achieved high-level expression in prokaryote with a novel method.