A novel approach to cloning and expression of human thymidylate synthase.
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.
College of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, China E-mail : cmkang@qust.edu.cn.
Ying-Tao
YT
Lv
Pei-Juan
PJ
Du
Qiao-Yan
QY
Wang
Yuan
Y
Tan
Zong-Bin
ZB
Sun
Zhong-Liang
ZL
Su
Cong-Min
CM
Kang
D015153:Blotting, Western; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D005822:Genetic Vectors; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics; D013940:Thymidylate Synthase / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D013940:Thymidylate Synthase