| Cytokines IL-2,IL-4,IL-7,IL-9,IL-15 and IL-21 are known as ?C family cytokines because they share ?C receptors.They play an important role in immunity.The function of cytokines depends on the receptors on the membrane surface.However,the molecular mechanism of the common receptor ?C is still unclear.At present,the structure of the extracellular domain of the receptor complexes of IL-2/IL-2Ra/IL-2R?/?C and IL-4/IL-4Ra/?C has been reported,but the structure of the transmembrane and intracellular domain of ?C is rarely studied However,as ?C receptors are single transmembrane proteins with a large freedom degree of transmembrane domain and loose folding of intracellular domain,which make it difficult to study.In this paper,we successfully obtained the transmembrane domain protein samples and preliminarily studied the structure of natural intracellular domain protein in living cells.The study of these two parts can provide information for the structure of receptors in resting and activating states,and help to understand the signal transduction process of ?C.For transmembrane proteins,solid-state nuclear magnetic resonance spectroscopy is the most suitable method to study their structure.ssNMR requires that membrane proteins are unifor in the detergent and lipid environment and can better inhibit the"swing" of protein molecules.Therefore,we constructed three prokaryotic expression plasmids and explored the preliminary conditions to meet the requirements of ssNMR.We expressed and purified ?C-(179)in M9 medium and break the intermolecular S-S bonds with 5mM DTT,and then purified the protein by gel exclusion chromatography.It was found that the target protein was easy to form various aggregation forms,in order to solve the problem that target proteins are easy to form polymers and to restrict the"swing" of proteins,we use the idea of charge interaction,we truncate the receptor,and add KKR and EED to the static end of ?C and IL-21Ra sequences,and so that the expressed proteins ?C-TMD(80)and IL-21R?TMD is positive and negative charges respectively.The two proteins may conform to each other due to electrostatic interaction in detergents or lipids.We successfully expressed and purified the corresponding proteins in M9 medium,and studied the circular dichroism spectra structure and liquid magnetic resonance of ?C-TMD(80)protein in four detergents DPC,DM,DDM,OG.The most suitable detergent for ?C-TMD(80)was DPC,in which the two-dimensional helix structure of ?C-TMD(80)could be maintained.Intracellular domain proteins are loosely folded,so it is unsuitable for liquid nuclear magnetic resonance,cryo-electron microscopy or X-ray.Our team has obtained some structural information by chemical cross-linking mass spectrometry,but the proteins are derived from prokaryotic expression,but the prokaryotic system lacks post-modification of proteins,so there may be some differences in the structure and properties of proteins.Therefore,we constructed a full-length eukaryotic expression plasmid of ?C-(369)and expressed it in HEK293T cells.The target proteins were crosslinked in vivo by membrane permeable crosslinker DSS.Several methods were tried to improve the solubility of membrane proteins,The cross-links were analyzed by mass spectrometry after a certain amount of ?C-(369)was obtained.Finally,two pairs of cross-linking sites were obtained... |
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