Expression, Purification And Crystalization Of FU, SUFU-FU And SUFU-SAP18

Hedgehog signaling pathway is a quite conserved signal pathway inthe biology evolution. It is a vital regulator in embryo development ofvertebrate, including the process of cell differentiation and proliferation,tissue polarization and stem cell homeostasis, etc. A wealth of studies havefound that aberrant Hedgehog signaling is related to tumorigenesis. As aser/thr protein kinase in the Hedgehog signal pathway, FU is activated byautophosphorylation and releases transcription factor Ci155byphosphorylating Cos2and SUFU, then activated Ci155can enter nucleusand induce target genes expression. SUFU is one of conserved andnegative regulator in the Hedgehog signaling pathway. It can not onlyrestrain Gli/Ci from entering the nucleus through binding with Gli/Ci, butalso recruit the mSin3A histone deacetylase complex under the help ofadaptor protein SAP18to repress the Hh target genes transcription.Molecular cloning, prokaryotic expression and protein purificationtechnology were used to obtain human SUFU, drosophila FU, humanSAP18and human SUFU-SAP18complex. The native PAGE was used toidentify the interaction and the ratio between human SUFU and SAP18.The result demonstrated that human SUFU interacts with SAP18by amolar ratio of1:1, and by the method of gel filtration chromatography, thehigh purity and stability protein complex had been obtained afterco-purification. We have already obtained the complex crystals. In addition,a novel binding site of FU interacting with SUFU has been identified,which laid a solid foundation for the further structural biology research on the protein complex.