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


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.


  1. Ackland SP, Clarke SJ, Beale P, et al (2006). Thymidylate synthase inhibitors. Update Cancer Ther, 1, 403-27.
  2. Carrers CW, Santi DV (1995). The catalytic mechanism and structure of thymidylate synthase. Annu Rev Biochem, 64, 721-62.
  3. Davisson VJ, Sirawaraporn W, Santi DV (1989). Expression of human thymidylate synthase in Escherichia coli. J Biol Chem, 264, 9145-8.
  4. Dolnick BJ, Cheng Y (1977). Human thymidylate synthetase derived from blast cells of patients with acturemyelocyticleukemia. Purificationandchracteri-zation. J Biol Chem, 252, 7697-703.
  5. Ellis KJ, Morrison JF (1982). Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol, 87, 405-26.
  6. Fu W, Lin J, Cen P (2007). 5-Aminolevulinate production with recombinant Escherichia coli using a rare codon optimizer hoststrain. Appl Microbiol Biotechnol, 75, 777-82.
  7. Gibson LM, Celeste LR, Lovelace LL, et al (2011). Structures of human thymidylate synthase R163K with dUMP, FdUMP and glutathione show asymmetric ligand binding. Biological Crystallography, 1, 60-6.
  8. Hori T, Takahashi E, Ayusawa D, et al (1990). Regional assignment of the human thymidylate synthase (TS) gene to chromosome band by non-isotopic in situ hybridization. Hum Genet, 85, 576-80.
  9. Jarmula A (2010). Antifolate inhibitors of thymidylate synthase as anticancer drugs. Mini-Rev Med Chem, 10, 1211-22.
  10. Joan PL, Maley GF, Chu E, et al (1997). High-level expression of human thymidylate synthase. Protein Expression Purif, 10, 256-62.
  11. Lockshin A, Moran RG, Danenberg PV (1979). Thymidylate synthetase purified to homogeneity from human leukemic cells. Proc Natl Acad Sci, 76, 750-4.
  12. Liu J, Hu T, Hou X (2007). High-level expression of functional tumor suppressor LKB1 in Escherichia coli. Acta Biochim Biophys Sin, 39, 779-86.
  13. Phan J, Steadman DJ, Koli S, et al (2001). Structure of human thymidylate synthase suggests advantages of chemotherapy with noncompetitive inhibitors. J Biol Chem, 276, 14170-7.
  14. Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning: A Laboratory Manual. 2ed New York: Cold Spring Harbor Laboratory.
  15. Steadman DJ, Zhao PS, Spencer HT, et al (1998). A structural role for glutamine 214 in human thymidylate synthase. Biochemistry, 37, 7089-95.
  16. Wahba AJ, Friedkin M (1961). Direct spectrophotometric evidence for the oxidation of tetrahydrofolate during the enzymatic synthesis of thymidylate. Biol Chem, 236, 11-2.
  17. Zhang H, Cisneros RJ, Dunlap RB, et al (1989). Efficient synthesis of mouse thymidylate synthase in Escherichia coli. Gene, 84, 487-91.

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