Thymidylate synthase (TS) catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMPto form dTMP. It is a primary target in the chemotherapy of colorectal cancers and some other neoplasms. Inorder to obtain pure protein for analysis of structure and biological function, an expression vector TS-pET28b(+) was constructed by inserting wild-type human thymidylate synthase (hTS) cDNA into pET28b (+). Then anexpression strain was selected after transformation of the recombined plasmid into Rosetta (DE3). Fusion proteinwith His-tag was efficiently expressed in the form of inclusion bodies after IPTG induction and the content wasapproximately 40.0% of total bacteria proteins after optimizing expression conditions. When inclusion bodieswere washed, dissolved and purified by Ni-NTA under denatured conditions, the purity was up to 90%. OnSDS-PAGE and West-blotting, the protein band was found to match well with the predicted relative molecularmass-36kDa. Bioactivity was 0.1 U/mg. The results indicated that high-level expression of wild-type hTS cDNAcan be achieved in prokaryotes with our novel method, facilitating research into related chemotherapy.
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