Asian Pacific Journal of Cancer Prevention

Asian Pacific Journal of Cancer Prevention (아시아태평양암예방학회)
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A Novel Approach to Cloning and Expression of Human Thymidylate Synthase

  • Lv, Ying-Tao (College of Chemical Engineering, Qingdao University of Science and Technology) ;
  • Du, Pei-Juan (College of Chemical Engineering, Qingdao University of Science and Technology) ;
  • Wang, Qiao-Yan (College of Chemical Engineering, Qingdao University of Science and Technology) ;
  • Tan, Yuan (College of Chemical Engineering, Qingdao University of Science and Technology) ;
  • Sun, Zong-Bin (College of Chemical Engineering, Qingdao University of Science and Technology) ;
  • Su, Zhong-Liang (College of Chemical Engineering, Qingdao University of Science and Technology) ;
  • Kang, Cong-Min (College of Chemical Engineering, Qingdao University of Science and Technology)
  • Published : 2013.12.31
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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.

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References

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