Prokaryotic expression, purification and identification of tetrameric-protein of methyl-CpG-binding domain
- VernacularTitle:甲基CpG结合域四聚体蛋白的原核表达、纯化及鉴定
- Author:
Xiaohua ZHU
;
Feng LI
;
Guolinag CHEN
;
Yongsheng YANG
;
Jinran LIN
;
Jinhua XU
;
Leihong XIANG
- Publication Type:Journal Article
- Keywords:
methyl-CpG-binding domain;
prokaryotic expression;
protein purification;
DNA methylation
- From:
Fudan University Journal of Medical Sciences
2009;36(4):450-453
- CountryChina
- Language:Chinese
-
Abstract:
Objective To express, purify and identify tetrameric protein of methyl-CpG-binding domain in E. coli. Methods The recombinant plasmid 4 × MBD-pET30b + were transformed into E. coli DH5a for clonal expansion and sequenced, then the tetrameric-proteins were expressed in E. coli BL21 (DE_3) under the induction of IPTG. Moreover, the expression products were purified by Ni-NTA chromatography, and were determined by SDS-PAGE and Western blot. Immunostain 293T cells with the proteins were analyzed by fluorescence microscope. Results The sequence analysis showed orientation right and was identical with the expectation. SDS-PAGE and Western blot demonstrated that the molecular weight of the tetrameric- protein was 46 0110 with the N-terminal His-tag and the C-terminal HA-tag. The MBD proteins can bind to the intracellular CpG DNA specifically.Conclusions The tetrameric-proteins of methyl-CpG-binding domain are successfully expressed and purified in E. coli. This results establish a groundwork for the further researches on DNA methylation.
