中国生物工程杂志

The cDNAs encoding mouse TAp63γ and its two deletion mutants were inserted into the pGEX-2TK expression vector and transformed into competent cell of E.coli BL21(DE3), respectively. The three recombinant plasmids expressed effectively soluble GST fusion proteins in E.coli BL21(DE3) when induced by IPTG under the most optimal expression conditions. The three soluble expressed crude protein lysates were achieved by harvesting cell pellet through centrifugation, breaking the cell wall through sonication and dissolving with Triton X-100. The three near-homogeneous purified GST fusion proteins judged by SDS-PAGE were obtained by Glutathione-Sepharose affinity chromatography. Electrophoretic mobility shift assay (EMSA) indicated only wild type GST-TAp63γ protein can bind to the p63 DNA-binding consensus motif, which is also the p53 consensus sequence. Furthermore, sequence alignment and homology modeling accounted for to some content the conservation of important residues and the high similarity of three dimensional structure of the intact DNA binding domain are necessary for the DNA binding activity of mouse TAp63γ protein.