Expression, Refolding And Purification Of Recombinant Human Interleukin-7and Its Splice Variants

Interleukin-7(IL-7) is a pluripotent cytokine, it can promote survival, differentiationand proliferation of B and T cells, and it plays a key role in regulating T-cell homeostasis,besides, it can induce cytotoxic activity in cytotoxic T-lymphocytes and macrophages, soIL-7has promising therapeutic potential for T-depleted patients (cancer, bone marrowtransplant, and AIDS). People have found IL-7splice variants with different molecular sizefrom different tumor tissues in recently years, such as IL-7-δ4, IL-7-δ4/5, IL-7-δ3/4and soon, it implicates that distinct isoforms may modulate IL-7activity. Therefore, in the presentstudy, we expressed, refolded and purified recombinant human interleukin-7(rhIL-7) andits splice variants (rhIL-7-δ4, rhIL-7-δ4/5, rhIL-7-δ3/4) with prokaryotic expression systemfor further biological activity studies, then identified it with Western Blot.Objective:In the present study, after successfully constructed IL-7and its splice variants ofprokaryotic expression vector pET21b-IL-7, pET21b-IL-7-δ4, pET21b-IL-7-δ4/5andpET21b-IL-7-δ3/4, we aimed to expressed, refolded and purified rhIL-7, rhIL-7-δ4,rhIL-7-δ4/5and rhIL-7-δ3/4for further biological activity studies.Methods:1. Expression of rhIL-7and its splice variants: After identified by PCR and DNA sequencing again, seed strains of rhIL-7and its splice variants were determine growthcurves to confirm best induction opportunities, the following was the expression andsoluble analysis of rhIL-7and its splice variants, then the concentration and time ofIPTG induction were optimized to make sure the best expression parameters,SDS-PAGE analyzed the expression products.2. Refolding of rhIL-7and its splice variants: Each recombinant protein was refolded bystepwise dialysis after expressed with the form of inclusion bodies, refolding solutionA, B, C, D, E contained4,3,2,1,0M Gu-HCl respectively, refolding auxiliaryreagents were add to the key steps of refolding to enhance the refolding efficiency, suchas L-Arg, GSSG, GSH and so on, the concentration of rhIL-7and its splice variantsbefore and after refolding were assayed by BCA Protein Assay Kit to calculaterefolding efficiency.3. Purification and Western Blot identification of rhIL-7and its splice variants: Eachrecombinant protein was purified by Ni2+affinity chromatography, Binding Buffer (30mM Iminazole) eluted non-specific binding hybrid proteins after protein was loaded,then gradient fractions were collected while each recombinant protein was eluted withElution Buffer A (60mM Iminazole), B (100mM Iminazole), C (300mM Iminazole)successively, SDS-PAGE and BandScan software analyzed the purity of eachrecombinant protein，besides, BCA Protein Assay Kit assayed the concentration of eachrecombinant protein after purification. purified recombinant proteins were identifiedwith Western Blot, primary antibodies were anti-His-tagged mAb and anti-IL-7mAbrespectively, secondary antibody was horseradish peroxidase (HRP)-labeled goatanti-mouse IgG.Results:1. Expression of rhIL-7and its splice variants: Amplified fragment sizes by PCR areequal to IL-7(531bp), IL-7-δ4(399bp), IL-7-δ4/5(345bp), IL-7-δ3/4(318bp) that weexpected, and results of DNA sequencing are consistent with reported sequences.rhIL-7, rhIL-7-δ4, rhIL-7-δ4/5, rhIL-7-δ3/4have good expression in Escherichia coli(E.coli) BL21(DE3), they have obvious bands at the place of17.4kDa,12.4kDa,10.3 kDa,9.3kDa respectively, and they are expressed with the form of inclusion bodies.Moreover, the results of growth curves analysis and optimization of express conditionsshow that the best induction opportunities of rhIL-7, rhIL-7-δ4, rhIL-7-δ4/5,rhIL-7-δ3/4are1.5h,2h,2.3h,1.8h culture at37℃,180rpm respectively; and thebest expression condition (culture at37℃,180rpm) of rhIL-7, rhIL-7-δ4, rhIL-7-δ4/5,rhIL-7-δ3/4are2h induction with1.0mM IPTG,2h induction with0.4mM IPTG,3hinduction with0.6mM IPTG,2h induction with0.8mM IPTG respectively.2. Refolding of rhIL-7and its splice variants: RhIL-7, rhIL-7-δ4, rhIL-7-δ4/5, rhIL-7-δ3/4all have good refolding efficiencies, which are about50%,48%,45%,65%respectively, moreover, the refolding method has good repeatability, so it is good forproduction of the recombinant proteins in large scale.3. Purification and Western Blot identification of rhIL-7and its splice variants: Theanalyses of SDS-PAGE and BandScan software show that all recombinant proteinshave good purification effect, their purity all are﹥95%, and their concentrationsassayed by BCA Protein Assay Kit are about0.1mg/mL after purification. The resultof Western Blot identification by anti-His-tagged mAb shows that rhIL-7, rhIL-7-δ4,rhIL-7-δ4/5, rhIL-7-δ3/4have obvious bands at the place of17.4kDa,12.4kDa,10.3kDa,9.3kDa respectively; The result of Western Blot identification by anti-IL-7mAbshows that rhIL-7, rhIL-7-δ4have obvious bands at the place of17.4kDa,12.4kDarespectively, but rhIL-7-δ4/5, rhIL-7-δ3/4have no bands at the place of10.3kDa,9.3kDa respectively. In a word, the results of Western Blot identification are consistentwith anticipated results, it demonstrates that rhIL-7and its splice variants are expressedeffectively.Conclusion:In the present study, we express rhIL-7, rhIL-7-δ4, rhIL-7-δ4/5, rhIL-7-δ3/4withprokaryotic expression system and have determined best expression parameters; analyses ofSDS-PAGE show that all recombinant proteins are expressed with the form ofinclusion bodies, and they have good refolding efficiencies, which are about50%,48%,45%,65%respectively after stepwise dialysis; all recombinant proteins have good purification effect after purified by Ni2+affinity chromatography, their purity are﹥95%,and their concentrations assayed by BCA Protein Assay Kit are about0.1mg/mL afterpurification; The results of Western Blot identification show that rhIL-7and its splicevariants are expressed effectively. Therefore, refolded rhIL-7, rhIL-7-δ4, rhIL-7-δ4/5,rhIL-7-δ3/4with high purity are obtained successfully, besides, the techniques have goodstability, they are good for production of the recombinant proteins in large scale, so it haslaid the material foundation for further biological activity studies.