Research Of Structural Conservation Of Interleukin-21

Interleukin-21 is a four helix bundle of the type I cytokine,which is mainly secreted by CD4+ T cells and natural killer T cells,and has pleiotropic regulatory role in the innate and adaptive immune response.The biological function of IL-21 in different species is determined by its special structure.Therefore,the study on the structure evolution of interleukin 21 is helpful to explain its unique role in immune regulation mechanism.On the basis of the existing human IL-21(hIL-21)structure,we choose the mouse IL-21(mIL-21)as a breakthrough point to demonstrate the IL-21 conservation structure in experimentally.1.Expression,purification and refold of protein mIL-21When expressed in E.coli,the recombinant protein was mainly present in the form of inclusion bodies.Therefore,in the refolding process,the two step dilution dialysis method is adopted,which is to reduce the protein concentration two times,and then dialysis again to complete the refolding process.This method not only greatly reduces the time,but significantly improves the efficiency of dialysis.In addition,considering the particularity of mIL-21 of its easy degradation,we will add the appropriate EDTA and glycerol in the refolding of buffer.What is more,we also choose FPLC purification to get the purer protein and the purification effect is significantly improved.Optimized conditions above lay the foundation of 13C?15N labeled protein for nuclear magnetic resonance experiment.2.Three dimensional structure of solution protein by NMR methodUsing liquid high resolution NMR techniques to collect a series of spectra including 2D 1~H-(15)~N HSQC?3D (15)~N-edited NOESY-HSQC?3D HNCA?3D HN(CO)CA?3D NHCACB?3D NH(CO)CACB?3D H(CCCO)NH?3D(H)CCH-TOCSY?3D (13)~C-edited NOESY-HSQC and so on.Assign each atom chemical shifts of the backbone and side chain residues and analysis the NOE spectra information,then make information above as dihedral angle constraints,distance constraint and hydrogen bond constraints.Repeating rounds of CYANA calculation,then for further optimization until the structure with a satisfied assessment parameters.