Cloning, Expression, Purification And 3D Structure Model Construction Of TIR Domain In Human TIRAP

The next TIR domain-containing adaptor to be discovered after the finding of Myd88 was Ma1 (MyD88- adaptor like),also called TIRAP(TIR domain-containing adaptor protein). TIRAP acts as an adaptor in the MyD88-dependent pathway from TLR2 and TLR4 sigalling. TIRAP has a prominent importance and complexity in the TLRs mediated signal pathway. TIRAP can play these roles, whose reason is because it has an essential TIR domain. A principal function of TIR domains is thought to be to mediate homotypic protein-protein interactions in the signal transduction process. Homotypic and heterotypic interactions are thought to mediate receptor signaling. Activation involves liberation of NF-kappaB resulting in lymphocyte activation, immunoglobulin isotype switching and expression of cytokines and their receptors. So TIR domain is crucial in innate immune system.In this project we started with the TIR domain of TIRAP(TIRAPd), the gene sequence of TIRAP's TIR was constructed into the expression vector pET15b through DNA recombination technology. The recobinant plasmid was transformed into competent cells of E.coli and introduced by IPTG. The induced proteins TIRAPd were so many and also soluble. Then TIRAPd expressed in high yield by E.coli Rosetta (DE3) was purified. In addition, we tried to construct the 3D model about TIR domain of TIRAP based on TLR2 tructure through Modeller programme. The main results as follows:1) The gene sequence of TIRAP's TIR was constructed into the expression vector pET15b successfully.2) The proper temperature was 30°C at witch the recombinant plasmid expressed in E.coli Rosetta (DE3) .3) The expressed proteins TIRAPd were so many and also soluble.4) We obtained high-purity, high yield and stable goal protein only through two steps.5) We constructed the 3D structure model of TIR domain in TIRAP based on the known structure of TIR domain in TLR2.The TIR domain of Human TIRAP was expressed successfully in prokaryote, which decreased the experimental cost and was more quick,so there would be a wide application. It was hoped that the project start from the TIR domain of TIRAP can indicate a way to elaborates some unknown questions step by step. If TIRAP can study thoroughly, there will be not only important theory significance, but also applicaied value about the future disease treatment and medicine design in immunity system.