Identification Of CHD4Interact With PTEN

【Objective】To detect the interaction of the CHD4protein and the PTEN protein in vitro andcell，further clarify the interaction structure of the domain with CHD4.【Methods】This topic is proposed on the basis of existing work, GST pull-down,co-immunoprecipitation and other methods to detect the interaction between PTEN andMi-2β/CHD4. Through constructing a truncated body to clear binding core domain. By PCRamplification the CHD4regulatory regions of chromatin (CHD4-C)，helicase motif (CHD4-H)and the DNA-binding domain (CHD4-D) gene and these fragment of the target genes wasinserted into the prokaryotic expression vector pGEX—5T with construction of a recombinantplasmid with a GST-tag, positive clones for DNA sequence determination.The prokaryotic expression of GST-CHD4-C, GST-CHD4-D and GST-CHD4-H induced bythe IPTG, glutathione agarose beads purified fusion protein, expression product wereidentified by Western blot. Using purified GST-CHD4-C、GST-CHD4-D and GST-CHD4-Hfusion protein with PTEN-HIS.GST pull-down detect the vitro-interactions between theCHD4protein and PTEN protein and the action domain. Eukaryotic plasmid of HA-PCMV,HA-CHD4transfected into293T cells, application of co-immunoprecipitation technology todetect interactions of the CHD4protein and PTEN protein at the cellular level.【Results】We successfully purified the higher purity GST, GST-CHD4-C, GST-CHD4-D andGST-CHD4-H fusion protein and GST pull-down results show that the interaction of theCHD4protein and the PTEN protein in vitro and the effect stained in CHD4binding domainend; coimmunoprecipitation technology detects the CHD4protein with PTEN proteininteractions at the cellular level.