Abstract
Thymidylate synthase (TS) catalyzes the transfer of a methyl group from methylenetetrahydrofolate to dUMP to form dTMP. It is a primary target in the chemotherapy of colorectal cancers and some other neoplasms. In order 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 an expression strain was selected after transformation of the recombined plasmid into Rosetta (DE3). Fusion protein with His-tag was efficiently expressed in the form of inclusion bodies after IPTG induction and the content was approximately 40.0% of total bacteria proteins after optimizing expression conditions. When inclusion bodies were washed, dissolved and purified by Ni-NTA under denatured conditions, the purity was up to 90%. On SDS-PAGE and West-blotting, the protein band was found to match well with the predicted relative molecular mass-36kDa. Bioactivity was 0.1 U/mg. The results indicated that high-level expression of wild-type hTS cDNA can be achieved in prokaryotes with our novel method, facilitating research into related chemotherapy.
Highlights
Thymidylate synthase (TS, E 2.1.1.45), consisting of two identical subunits with molecular weight of 36.0kDa, catalyzes the reductive methylation of dUMP to produce dTMP (Carrers et al, 1995)
The results indicated that high-level expression of wild-type human thymidylate synthase (hTS) cDNA can be achieved in prokaryotes with our novel method, facilitating research into related chemotherapy
We describe a novel method for improving the expression of wild-tpye human TS cDNA in E. coli
Summary
Thymidylate synthase (TS, E 2.1.1.45), consisting of two identical subunits with molecular weight of 36.0kDa, catalyzes the reductive methylation of dUMP to produce dTMP (Carrers et al, 1995). It is reported that a new crystal form of human TS (hTS) which allows binding studies by soaking crystals in artificial mother liquors containing ligands that bind in the active site. Using this approach, crystal structures of hTS complexes with FdUMP and dUMP were obtained, indicating that this form should facilitate in high-throughput analysis of hTS complexes with drug candidates. Crystal soaking experiments using oxidized glutathione revealed that hTS binds this ligand (Jarmula 2010)
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