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http://dx.doi.org/10.7314/APJCP.2013.14.12.7523

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)
Publication Information
Asian Pacific Journal of Cancer Prevention / v.14, no.12, 2013 , pp. 7523-7527 More about this Journal
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.
Keywords
Wild; type human thymidylate synthase cDNA; prokaryotic expression; rare codon;
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