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Asian Pac J Cancer Prev ; 14(12): 7523-7, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24460328

RESUMO

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.


Assuntos
Escherichia coli/metabolismo , Regulação Enzimológica da Expressão Gênica/fisiologia , Timidilato Sintase/genética , Western Blotting , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Vetores Genéticos , Humanos , Proteínas Recombinantes/genética , Timidilato Sintase/isolamento & purificação
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