Construction of prokaryotic expression vector of Id3 and expression of fusion protein in E. coli
- VernacularTitle:人分化抑制因子3基因原核表达载体的构建及融合蛋白的表达
- Author:
Li JIA
;
Xiaojun LI
- Publication Type:Journal Article
- Keywords:
Inhibitor of differentiation 3;
Prokaryotic expression;
Fusion protein
- From:
Journal of Medical Postgraduates
2003;0(10):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective: To express and purify the inhibitor of differentiation(Id3) in E.coli.Methods: The DNA fragment in the coding region of the human Id3 gene was amplified by PCR and cloned into the pGEM-T Easy vector for sequencing.The Id3 cDNA fragment was then subcloned into the prokaryotic expression vector(pET-32a)(+) and transformed into E.coli BL 21(DE3).The expression of the histidine-tagged(His-Tag) fusion protein was induced with isopropy-?-D-thiogalactoside(IPTG),confirmed by Western blot and purified by the immobilized Ni2+ absorption chromatographic column.Results: The prokaryotic expression vector of Id3 was successfully constructed.Western blot confirmed the expression of the His-Tag fusion protein in E.coli BL 21(DE3) and that of the Id3 fusion protein with the relative molecular mass size of 33 000 after purified by the affinity chromatographic column. Conclusion: Recombinant Id3 can be expressed in E.coli BL 21(DE3) and the obtained fusion protein can be purified by the Ni-affinity chromatography column.
