Exploring The Mechanism Of Signaling Transduction By Interleukin-21 Receptors:Design,Production And Preliminary Analysis Of Key Proteins In This Signaling System

Interleukin 21(IL-21)is a type I cytokine produced majorly by activated T cells,regulating adaptive immune responses as well as inflammatory and autoimmune diseases effects.IL-21 performs its biological function by forming a ternary complex with its alpha and gamma receptors,leading to the activation of the JAK/STAT signaling pathway.However,the molecular mechanism of this process remains largely unknown.Structural study of IL-21 and its receptors would reveal molecular mechanism of the signal transduction in line with the notion that protein structure is critical for its function.Study of the change of relative distance between receptor proteins upon activation would also helps us understand the signal transduction as the transduction involves protein allosterical effects.In this thesis,the structure of human Interleukin 21(hIL-21)mutants and hIL-21 receptors is analyzed by solution nuclear magnetic resonance(NMR)techniques;and,the relative distance between the receptors was designed to be monitored using fluorescence resonance energy transfer(FRET)technology.Here are the details:1.Based on the requirements of NMR experiments,the variants of ? receptor were designed by truncation and segmenting.And the corresponding plasmid was constructed for subsequent study.Based on the experimental requirements of FRET technique to study the interactions between receptors in different states,we determined the positions of fusion expression between fluorescent protein and a and y receptor protein.A series of proteins were designed to replace extracellular domain proteins with leucine zipper,modeling the process of receptor protein from resting to active state.And the corresponding plasmid was constructed for subsequent studies.In addition,the conditions for polymerase chain reaction(PCR)reaction were optimized.Lastly,the mutant plasmid M-C4? was obtained,succeeding in mutating three discontinuous amino acids at the same time.2.The recombinant expression in the prokaryotic cells,purification and preliminary analysis of nuclear magnetic resonance were carried out for some of the proteins designed above.The method of methanol/chloroform extraction was used to purify the membrane protein.And the ratio of methanol/chloroform was optimized several times.Finally,the target protein band was detected in the chloroform layer.3.The expression of IL-21 mutant C4? and MUT9 was performed by prokaryotic expression system of Escherichia coli.The conditions of C4? protein solution were optimized and the protein is found to be stable for more than one week in phosphate solution at pH 4.3.And a series of spectra needed for structural analysis were collected under the condition.In collaboration with others we ultimately obtained the structure of C4? which revealed that the hypothesis previously reported that the more stable of IL-21 structure induce the stronger ability to active the signal is not established,signifying the need to be further studied.