The Study On The Dimerization Mechanism Of Chicken Interleukin-2

IL-2is a cytokine, which plays an important role in the proliferation anddifferentiation of T-cells, B-cells,Monocytes and Natural killer cells, so do it in theactivation and maintenance of both acquired and innate immune defenses. Thehomodimers of IL-6、IL-8or IL-12have been found to present different Bioactivityfrom each monomer. IL-2homodimer also was found in human, fish, hamsters, andmice, etc.. These IL2dimer showed novel bioactivities in comparsion with monomer,respectively. Previous studies have shown that two elution peaks presented in HPLCduring the purification of endogenous Chicken IL-2, and the molecular weight oflarge one was twice as much as short one in SDS-PAGE. The mechnism of IL2dimerization still has been illustrated so far.To investigate how chIL2dimerized in our purified chIL2from prekaryotic aswell as eukaryotic expression system,366bp chIL-2gene without N-terminal signalsequence was cloned into pET-28a vector to fused a C-terminal His-tag.This His-tagfused IL2ORF was subcloned into pFastBac-Dual vector for expression in sf9insectcells.366bp chIL-2gene without N-Terminal signal sequence also was cloned intopTYB1vector to fused with Intein gene (1362bp) in the same ORF which encoding a71kDa fusion protein of chIL-2-Intein. chIL-2-Intein was eluted with100mmol/LNaOH from Chitin column and then used as antigen to immunized rabbits for thegeneration of polyclonal antibodies against chIL2and intein proteins. UntaggedchIL-2protein was eluted from Chitin column post incubation with the cleavagebuffer containing β-ME. The results suggested polyclonal antibodies against chIL-2were successfully generated with a high titer of4.096million,and could detect chIL2monomer and dimer of His-tagged chIL-2protein purified from E.coli andBaculovirus Insect Cells Expression System as well as non-tagged chIL-2protein withWestern blot.The results indicate that chIL-2protein dimerized in both of E.coli and insect cells.TOur study also explored the role of temperature on chIL-2dimerization.Purified chIL-2-His-tag proteins from Sf9and E.coli were incubated at4℃and37℃for24,48,72h followed by Western blot analysis. The resultsshowed the dimer/monomer ratio of chIL-2-His-tag proteins purified from Sf9andE.coli presented no significant changes after incubation at4℃, while all of thechIL-2protein presented as dimer after incubation at37℃for1d,.But it is differentthat the ratio of the two forms ofthe E.coli expressed fusion protein withoutsignificant changes when incubated at37℃for different times. The resultssuggested that the dimerization of purified chIL-2-His-tag proteins from sf9issensitive to temperature, but the one from E.coli showed a slow dimerization, and itmay indicated the post-translational modification of sf9Expression System and E.coliExpression System infulunced chIL2dimerization.To identify the interaction of two chIL-2moleculess, thiocyanate (SCN-), bilesalts, Triton X-100, urea, guanidine hydrochloride, EDTA, dithiothreitol (DTT) and β-mercaptoethanol (β-ME) were used to dissociate chIL2homodimer. Thereducingagents DTT and β-ME could completely dissociate the chIL-2dimer into monomericprotein. The results suggested that the dimerization between chIL-2molecules islikely to be a result of covalent disulfide bond formation.Site-directed mutagenesis of pET-28a-chIL-2was carried out to determine thefunction of the four Cys-residues in chIL-2protein for dimerization.The mutant ofchIL2with1,2,3or4Cys residues mutations were obtained with E.coli ExpressionSystem.Western blot was carried out for the detecting of mutant proteins dimer. ThechIL2mutants with1,2and3Cys mutations still showed dimer but4Cys mutationstotally disrupted chIL-2dimer. The results suggested all Cys residues contribute todisulfide bond formation for chIL2dimerization.