Expression And Identification Of Recombinant Human Soluble GITRL_aa52-177 Protein With An Isoleucine Zipper Motif

Objective:In this study,we expressed recombinant human soluble trimeric GITRL with an isoleucine zipper motif(ILZ-hsGITRL),and identified the biological activity and biophysical characterization of ILZ-hsGITRL protein.Methods:(1) Using standard PCR and cloning techniques,the hsGITRL gene was amplified from intermediate vector PMD18-T/hGITRL.An isoleucine zipper sequence was linked to the N-terminus of human soluble GITRL (amino acids E52-S177,hsGITRL) by two leucine sequences.The recombinant sequences(ILZ-hsGITRL) were cloned into the PET32a(+) vector between the BamHâ… and Hindâ…¢sites.At the same time,the hsGITRL(amino acids E52-S177) was also cloned into the PET32a(+) vector.The PET32a(+)-hsGITRL and PET32a(+)-ILZ-hsGITRL plasmids were identified by restriction digestion,PCR amplification and nucleic acid sequence analysis.(2) The PET32a(+) vector contained Trx·Tag^TM,His·Tag,and S·Tag sequences for detection and purification.In addition,it contained cleavable enterokinase sequences for obtaining pure protein.E.coli strain Rossetta (DE3) was used as the expression host cells.The considerable soluble proteins were produced,obtained by ultrasound sonication on ice,and then identified by anti-hGITRL mAb and anti-His mAb.The Trax-hsGITRL and Trax-ILZ-hsGITRL proteins were purified by Ni⁺-IMAC column and digested by recombinant enterokinase to remove the Trx·Tag^TM,His·Tag, and S·Tag sequences.The digested soluble protein mixtures were further purified by Ni⁺-IMAC column.In the stage of the second purification,we collected filtering peak protein,because Trx·Tag and other tags were bound to the Ni⁺-IMAC column by His·Tag.The filtering peak proteins,hsGITRL or ILZ-hsGITRL,were examined by SDS-PAGE and Western blot, respectively.The purified anti-hGITRL mAb were coated onto a 96-well plate,we analyzed the binding function of hsGITRLor ILZ-hsGITRL with anti-hGITRL mAb by ELISA method.(3) Human peripheral blood CD4⁺ T cells were isolated from healthy donors by human CD4⁺ T cell Isolation Kit,the purifity of CD4⁺ T is＞92%.CD4⁺ T cells were further stained with PE conjugated anti-CD4⁺ mAb.The purity of CD4⁺ T cells population was measured by flow cytometric analysis.CD4⁺ T cells(2×10⁵/well) were cultured with hsGITRL or ILZ-hsGITRL in the presence of anti-CD3 mAb(50 ng/ml) for 72 h.In addition,different target protein concentrations were performed. The cultures were added with Cell Counting Kit-8 solution and incubated for the final 4 h,and then the O.D values were measured at 450 nm with a microplate reader.Supernatants from similar cultures were collected and IFN-γproduction was measured by ELISA Kit.CD4⁺ T cells were stimulated with hsGITRL or ILZ-hsGITRL(5.0μg/ml) in the presence of anti-CD3 mAb(50 ng/ml),harvested at the indicated time points(10,20, 40 min),washed twice with ice-cold PBS and lysed with RIPA lysis buffer, the level of ERK1/2 phosphorylation was analyzed by Western blot.(4) ILZ-hsGITRL or hsGITRL proteins were identified with non-reducing SDS-PAGE,Western blot,and Sephadex G-100 size exclusion column. ILZ-hsGITRL or hsGITRL proteins were separated from non-reducing SDS-PAGE sample buffer with 10%Bis-Tris gels.After electrophoresis, the gels were stained with Coomassie blue or blotted onto a PVDF membrane for Western blot analysis.The anti-His mAb or anti-hGITRL mAb and HRP-conjugated goat anti-mouse mAb were utilized in the analysis.Western blot was developed with ECL reagent.According to the manufacturer's protocol,the purified ILZ-hsGITRL/hsGITRL proteins were eluted from Sephadex G-100 column.The protein peaks were collected and identified with SDS-PAGE and Western blot.Result:(1) In the current study,the PET32a(+)-hsGITRL and PET32a (+)-ILZ-hsGITRL vector was successfully constructed,and then the plasmids were identified by restriction digestion,PCR amplification and nucleic acid sequence analysis,respectively,the DNA sequence showed 100%homology with the sequence for GITRLaa52-177 in GenBank (BC112032)(2) Many soluble proteins including human soluble GITRL(hsGITRL, E52-S177) without the ILZ motif or ILZ-hsGITRL expressed in E.coli strain Rossetta(DE3) were found by ultrasound sonication in solution,the expression was induced with 0.6 mM IPTG for 8 h at 25℃.Two soluble proteins have Trx·Tag^TM,His·Tag,S·Tag sequences and cleavable enterokinase sequences.We randomly selected His·Tag for purification and detection.Each protein was analyzed by Coomassie gels and Western blot. The calculated peptide molecular mass for hsGITRL and ILZ-hsGITRL was almost 28 and 32kDa,respectively.After purification,identification, and dialysis concentration,to obtain pure hsGITRL or ILZ-hsGITRL, Trx·Tag^TM,His·Tag,S·Tag sequences and enterokinase sequences were removed by recombinant enterokinase according to the instruction manual. The digested mixtures were secondly purified with Ni⁺-IMAC column.The perforation peaks were collected and identified with Coomassie gels and Western blot.The results indicated that the perforation peaks were pure hsGITRL or ILZ-hsGITRL proteins without cleavable residual protein consisting of Trx·Tag^TM,His·Tag,S·Tag,the calculated protein molecular mass for pure hsGITRL and ILZ-hsGITRL was almost 14 and 18kDa, respectively.The especial binding function of hsGITRL or ILZ-hsGITRL with anti-hGITRL mAb was certified by ELISA system.(3) The purity of human CD4⁺ T cells purified by MACS was about 92-96%.The extent of CD4⁺T cell proliferation depended on the protein concentrations in the presence of 50 ng/ml anti-CD3 mAb as a co-stimulator,although both hsGITRL and ILZ-hsGITRL induced CD4⁺T cells proliferation,ILZ-hsGITRL showed significantly higher biological activity.However,when the protein concentrations were lower,the extent of CD4⁺ T cell proliferation was not significant.There was a similar potency between the activities of the two proteins in inducing IFN-γ, secretion with the presence of 50 ng/ml anti-CD3 mAb as a co-stimulus, the observed effects were notable at higher concentrations,when the protein concentrations were lower,the effects of IFN-γsecretion were not significant.The hsGITRL induced noticeable the phosphorylation of ERK1/2 with 10 min,whereas the level of ERK1/2 phosphorylation was reduced little by little afterward.ILZ-hsGITRL induced more persistent and stronger than did hsGITRL.(4) In this study,comparing with hsGITRL,ILZ-hsGITRL profoundly increased human CD4⁺ T cells proliferation,IFN-γsecretion and ERK1/2 phosphorylation.We presumed that the ILZ motif could affect the structure and function of hsGITRL proteins.The hsGITRL was confirmed as a mixture of dimmers and trimers in solution by Sephadex G-100 filtration, the protein isolated from the dimmer and trimer peaks were hsGITRL identified by SDS-PAGE and Western blot.ILZ-hsGITRL existed as trimers in solution,and the protein isolated from the trimer peak was also ILZ-hsGITRL identified by SDS-PAGE and Western blot.The non-reducing SDS-PAGE experiments with the purified protein indicated that hsGITRL existed as a mixture of monomers and dimmers,whereas no amounts of trimer species could be observed under the condition,especially significant amounts of dimmers in solution were observed.However, ILZ-hsGITRL existed as a mixture of monomers,dimmers,trimers,and superclusters,and significant amounts of trimer species existing in solution were observed.Conclusion:(1)The PET32a(+)-hsGITRL and PET32a(+)-ILZ-hsGITRL vectors were successfully constructed.(2) The soluble Trax-hsGITRLaa_52-177 and Trax-ILZ-hsGITRLaa_52-177 proteins were successfully expressed.The purified ILZ-hsGITRL or hsGITRL proteins were obtained by recombinant enterokinase and Ni⁺-IMAC column.(3) Comparing with hsGITRL,ILZ-hsGITRL profoundly increased human CD4⁺ T cells proliferation,IFN-γsecretion and ERK1/2 phosphorylation.(4) hsGITRL existed as predominant dimmers in solution, ILZ-hsGITRL existed as predominant trimmers.