Expression, purification and identification of CHD4/Mi-2β truncated proteins.

Objective To construct chromodomain helicase DNA-bingding protein 4 (CHD4/Mi-2β) truncated recombinant plasmids, induce the expression of GST fusion proteins in E.coli, and purify and identify GST fusion proteins of CHD4/Mi-2β. Methods The gene fragments of chromodomains (CHD4-C), SWI2/SNF2-related ATPase/helicase region (CHD4-H) and DNA-binding domain (CHD4-D) were amplified by PCR and cloned into pGEX-5T vector. After the target genes were sequenced, the plasmids were transformed into E.coli BL21 (DE3) respectively, and induced with 0.8mmol/L IPTG to express fusion proteins. The GST-fusioned recombined proteins were purified with glutathione-agarose resin, and identified by SDS-PAGE and Western blotting. Results Three CHD4 truncated recombinant plasmids were constructed. The recombinant fusion proteins of GST-CHD4-C, GST-CHD4-D and GST-CHD4-H were expressed in E.coli BL21 (DE3) and existed mostly in the form of soluble protein with expected relative molecular weight of 130, 110 and 90ku. The purity of the fusion proteins reached 94.5% after purification by high-affinity glutathione-agarose resin. Conclusion GST-CHD4-C, GST-CHD4-D and GST-CHD4-H have been successfully purified, which lays an experimental foundation for further study of CHD4/Mi-2β in chromatin remodeling. [ABSTRACT FROM AUTHOR]