A novel approach to cloning and expression of human thymidylate synthase.
Asian Pac J Cancer Prev
; 14(12): 7523-7, 2013.
Article
em En
| MEDLINE
| ID: mdl-24460328
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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Base de dados:
MEDLINE
Assunto principal:
Timidilato Sintase
/
Regulação Enzimológica da Expressão Gênica
/
Escherichia coli
Idioma:
En
Ano de publicação:
2013
Tipo de documento:
Article