Studies On Prokaryotic Expression, Purification, Conformation And Function Of Mouse TAp63γ Gene

The p53 tumor suppressor gene is one of the most frequently mutated genes in human cancers, and its constitutional mutation or loss is associated with more than 50% tumorigenesis. The p53 protein functions primarily as a transcription factor regulating the expression of genes involved in cell cycle arrest, cellular senescence, anti-angiogenesis, and apoptosis. Recently, two homologous genes, p63 and p73, were identified, and both p63 and p73 share high homology with p53 in DNA sequence. The p63 gene contains two transcriptional start sites encoding proteins with and without N-terminal transactivation domain termed TAp63 and ANp63, respectively. Both TAp63 and ANp63 isoforms can undergo alternative splicing to yield three different C-terminal tails, termed TAp63α, TAp63β, TAp63γ, ANp63α,â–³Np63βandâ–³Np63γ. The p63 isoforms function differently as transactivation factors, the TAp63 mainly acts as tumor suppressors to promote cell apoptosis, while theâ–³Np63 plays a central role in inhibiting growth arrest and cell apoptosis, and possesses oncogenic property. In particular, p63 appears to be essential in epithelial and limb development as demonstrated by the mouse models and is a key regulator of ectodermal, orofacial and limb development. Mutations in the p63 gene can lead to at least five different syndromes: ectrodactyly, ectodermal dysplasia and cleft lip/palate syndrome (EEC, OMIM 604292), ankyloblepharon-ectodermal defects-cleft lip/palate syndrome (AEC, OMIM 106260), limb lammary syndrome (LMS, OMIM 603543), acro-dermato-ungual-lacrimal-tooth syndrome (ADULT, OMIM 103285) and Rapp-Hodgkin syndrome (RHS, OMIM 129400). Furthermore, two non-syndromic human disorders are caused by p63 mutations: isolated split hand/foot malformation (SHFM4, OMIM 605289) and non-syndromic cleft lip. Because of its significant role as the regulator in development and tumorigenesis, the study about p63 is one of the research hotspots recently. So far, little is known about the biochemical and biological properties of the different isoforms of p63, and no all-sided report about its prokaryotic expression has been found. Here we overexpressed and purified mouse TAp63γ, and its two deletion mutants in Escherichia coli, then the research about the relationship between TAp63γstructure and function were carried out in the following four aspects and the corresponding results were obtained.First, we cloned the cDNA coding sequence of TAp63γand constructed its prokaryotic expression recombinant plasmid termed pGEX-2TK-TAp63γ. Then the two deletion mutants of TAp63γ, namedas pGEX-2TK-TAp63γDM1 and pGEX-2TK-TAp63γDM2, were obtained by the method of gene deletion with the pGEX-2TK-TAp63γplasmid as the female parent. The three recombinant plasmids, pGEX-2TK-TAp63γ, pGEX-2TK-TAp63γ(DM1) and .pGEX-2TK-TAp63γ(DM2), were transformed into E.coli BL21(DE3) competent cells, respectively. The positive colonies expressed effectively soluble GST-TAp63γ, GST-TAp63γDM1, GST-TAp63γDM2 and Sj26GST protein in E. coli BL21 (DE3) when induced by IPTG under the optimal condition. The heterogeneous expression condition, including the expression monocolony, time, concentration of IPTG, temperature and the effect of some small molecules on expression, was explored further. The soluble expressed crude protein lysate was achieved by harvesting cell pellet through centrifugation, breaking the cell wall through sonication and dissolving with Triton X-100. Near-homogeneous purified GST-TAp63γfusion protein judged by SDS-PAGE was obtained by one-step Glutathione-Sepharose affinity chromatography. According to the similar methods above, GST-TAp63γDM1, GST-TAp63γDM2 and Sj26GST protein were expressed in E. coli BL21 (DE3) and purified by one-step Glutathione-Sepharose affinity chromatography. GST-TAp63γfusion protein was cut with thrombin under the optimal condition, then TAp63γprotein without GST tag was obtained. All proteins were quantified with the Bradford method, detected by SDS-PAGE and preserved in a small quantity at -80℃.Second, the determination of structure and physical and chemical properties of the protein purified above. UV spectra show a UV absorption characteristic peak of TAp63γat 275 nm; fluorescence spectra indicate that TAp63γ, is of typical B-type protein; 3-D scanning fluorescence shows a fluorescence emission characteristic peak of TAp63γ, at 340 nm; far-UV circular dichroism reveals that TAp63γis rich in alpha-helix and beta-sheet and near-UV circular dichroism analysis shows that TAp63γ, contains many disulfide bonds. Research of flaermal stability suggests that TAp63γis not sensitive to heat: when incubated at 75℃for 30 min, the majority of protein remain soluble; fluorescence quenching shows that the characteristic fluorescence emission intensity of TAp63γhas sustained decrease as temperature rises; at 65℃or below, fluorescence quenching is not obvious; fluorescence quenching is significantly strengthened when temperature is higher than 65℃. Thermo-induced far-UV circular dichroism shows that the higher the temperature, the less the content of theα-helix and the more the content of the random coil in TAp63γmolecular. Binding activity of TAp63γwith target DNA measured at different temperature shows that its DNA binding activity is weakened as temperature rises, but still performed a detectable DNA-binding activity at 50℃. Chemical cross-linking analysis of TAp63γpolymerization shows that the active state in vivo may be dimer or tetramer, and Sj26GST mainly function as dimeric conformation. At the same time, protein conformational changes before and after the induction of urea detected by tryptophan fluorescence emission spectra shows the conformational change of Sj26GST induced by urea is reversible; its conformational change belongs to one-step model, denaturing curve analysis shows the unfolding/refolding parameters of urea-induced equilibrium of Sj26GST, with aâ–³G=34.46±0.16 kcal/mol and a m=5.95±0.02 kcal/mol/M. The interaction of heavy metal cations Hg²⁺ ion with Sj26GST was intensively studied by means of spectroscopy. UV difference spectra show Hg²⁺ ion induced the occurrence of LMCT band of Sj26GST in UV range and with the increasing concentration of Hg²⁺ ion, LMCT intensity increases. Fluorescence quenching shows a marked quenching effect of Hg²⁺ ion on Sj26GST characteristic fluorescence emission peak. Circular dichroism reveals that Hg²⁺ ion lightly perturbs the structure of Sj26GST: theα-helix content decreased slightly and the random coil increase lightly, while little influence on the higher structure. Enzyme inhibition kinetics measurement shows the inhibition of Hg²⁺ ion to Sj26GST is of noncompetitive inhibition, with a inhibition constant Ki of 1.64 mmol/L.Third, the purified GST-TAp63γand Sj26GST were used as antigen to immunize New Zealand rabbits for the preparation of polyclonal antibody. Double-immunodiffusion, western blotting and immunoprecipitation all confirmed the preparation of anti-rabbit GST-TAp63γand Sj26GST Polyclonal antibody of high specificity. These assays also show that anti-rabbit GST-TAp63γpolyclonal antibody is not only able to identify specific GST-TAp63γantigen, but also identify specific TAp63γantigen and Sj26GST antigen; anti-rabbit Sj26GST polyclonal antibodies is not only capable of identifying specific Sj26GST antigens, but also identifying GST-TAp63γspecific antigen, suggesting that anti-rabbit Sj26GST polyclonal antibody can be used as standard antibody of GST-tagged fusion protein in genetic engineering and that thus there are certain commercial prospects. Anti-rabbit GST-TAp63γand Sj26GST polyclonal antibodies have achieved good results in the study of the TAp63γwith target DNA and protein interaction. These obtained antibodies have paved the way for the further study of TAp63γin the next step. Finally, we explore the interaction of mice TAp63γwith target DNA and other relevant proteins by means of experiment and bioinformatics. Electrophoretic mobility shift assay (EMSA) confirmed that only wild-type TAp63γprotein can bind to p53 target sequence, and can also maintain certain binding activity even at 50℃, while the deletion mutants can not bind to the p53 target sequence. To explain this phenomenon, bioinformatics has been employed to perform sequence alignment and homology modeling at the molecular level. Analysis shows that the integrity of TAp63γDNA binding domain, the high conservative of the key amino acid and the three-dimensional structure similarity may be necessary for its DNA binding activity. Further verification of DNA binding activity with antibody supershift assay also shows that a antibody-protein-probe ternary complex is formed. Analysis of GST pull-down assay of the interaction between TAp63γand p19ARF shows the formation of Glutathione Sepharose—GST-TAp63γ—p19ARF complex instead of Glutathione Sepharose—GST—p19-ARF complex. Co-immunoprecipitation studies also showed a antibody—GST-TAp63γ—p19ARF complex can formed, while antibody—GST—p19ARF complex can not be formed. These two assays reveal that TAp63γcan interact with p19ARF, suggesting p19ARF and TAp63γmight be in the same signal-transduction pathway, as trans-acting factors in the regulation of target genes or as cell cycle regulators.