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Expression and Identification of Recombinant Human Soluble GITRLaa52-177 Protein with an Isoleucine Zipper Motif

Author: CuiDaWei
Tutor: WangShengJun
School: Jiangsu University
Course: Clinical Laboratory Science
Keywords: GITRL Isoleucine zipper Trimer
CLC: R341
Type: Master's thesis
Year: 2009
Downloads: 20
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Abstract


Objective: In vitro expression isoleucine zipper (ILZ) modified hsGITRL aa52-177 recombinant protein (ILZ-hsGITRL), and a preliminary identification of the structural features and biological activity of the recombinant protein ILZ-hsGITRL. Methods: (1) Application. PCR technology to hGITRL-TA plasmid as template, amplified hGITRL aa52-177 gene fused at its N-terminal chemical synthesis of isoleucine zipper motif ( Isoleucine zipper, ILZ), the recombinant gene ILZ-hsGITRL, BamH Ⅰ, Hind Ⅲ double digested PCR products and prokaryotic expression plasmid PET32a (), connect the digestion product was transformed into competent E. coli DH5α; choose positive strains was extracted recombinant plasmid, BamH I. The Hind III restriction enzyme digestion, PCR and gene sequencing. At the same time build expression plasmid PET32a ()-hsGITRL aa52-177 expression plasmid PET32a, ()-ILZ-hsGITRL aa52-177 , were transformed into ROS expression of engineering bacteria using IPTG induction of protein expression. (2) the initial expression of recombinant proteins the Trax-hsGITRL the Trax-ILZ-hsGITRL protein were identified by SDS-PAGE and Western blot, respectively. By the Ni -IMAC column of the Trax-hsGITRL and Trax-ILZ-hsGITRL, protein purification. According to the the enterokinase operating manual of purified the Trax-hsGITRL and Trax-ILZ-hsGITRL, protein digestion, protein digestion, respectively, again by Ni -IMAC column, Trax partly due to the presence of the His tag bind to the Ni -IMAC column penetration peak protein is purified hsGITRL ILZ-hsGITRL protein. Analysis the purified hsGITRL ILZ-hsGITRL protein with anti hGITRL mAb binding specificity by ELISA. (3) the Ficoll liquid density gradient separation of human peripheral blood mononuclear cells by immunomagnetic beads separation CD4 T cells, flow cytometry CD4 T cells purity. , Respectively, will the purified hsGITRL ILZ-hsGITRL protein added in accordance with a certain concentration of human CD4 T cells cultured system in the presence of anti-CD3 mAb, and cultured for 72 hours, through the CCK-8 assay detects T cell proliferation. Collected in the same culture conditions, the cell culture supernatant by an ELISA kit for the detection of IFN-γ levels. Respectively the the purified hsGITRL and ILZ-hsGITRL adding with above human CD4 T cell culture system, cultured cells collected at the different time points specified by the Western blot assay of ERK1 / 2 protein concentration according to certain the phosphorylation level. (4) by gel filtration chromatography on Sephadex G-100 column and non-reducing SDS-PAGE analysis hsGITRL ILZ-hsGITRL protein structural features, revealed the nature of its protein. Results: (1) successfully construct the prokaryotic expression vector PET32a ()-hsGITRL aa52-177 and PET32a ()-ILZ-hsGITRL aa52-177 by PCR and restriction enzyme digestion identification visible target band of 446bp and 557bp respectively the sequencing results published hsGITRL aa52-177 genes with GenBank sequence (BC112032) completely consistent. ILZ sequence the ILZ sequence with chemically synthesized. (2) The identification of the correct expression vector into the expression of ROS engineering bacteria, to a final concentration of 0.6mmol / L IPTG, 25 ℃, induced 8h as the best protein induction conditions. The ultrasonic lysis method of cracking the cells found the inducible expression the Trax-hsGITRL and Trax-ILZ-hsGITRL, fusion protein present in the cytoplasm. By SDS-PAGE and Western blot confirmed the Trax-hsGITRL protein relative molecular weight of approximately 28 kD relative molecular weight of about to 32 kD the Trax-ILZ-hsGITRL protein. After Ni -IMAC column purification the Trax-hsGITRL, as well as the Trax-ILZ-hsGITRL fusion protein purity> 90%. Fusion protein after enterokinase digestion the Trax-hsGITRL and Trax-ILZ-hsGITRL, again purified through Ni -IMAC column the collected purified hsGITRL and ILZ-hsGITRL protein. SDS-PAGE and Western blot confirmed hsGITRL protein relative molecular weight of approximately 14 KD, ILZ hsGITRL protein relative molecular weight of about the 18 kD. The purified hsGITRL confirmed by ELISA ILZ-hsGITRL protein with anti hGITRL mAb specific binding. (3) immunomagnetic beads isolated CD4 T cells, purity> 92%. The cell proliferation assay shows alkylene concentration in the presence of anti-CD3 mAb, hsGITRL ILZ-hsGITRL protein is between 0.5 ~ 5μg/ml are able to effectively promote the proliferation of CD4 T-cells, and presence of protein concentrations dependencies ILZ-hsGITRL than stimulation of hsGITRL protein more significant. Cells detected by ELISA in the culture supernatant of IFN-γ content was found more effective in promoting the secretion of IFN-γ in ILZ-hsGITRL the ratio hsGITRL protein. Analysis of cell signaling pathways, to confirm the proteins could the hsGITRL ILZ-hsGITRL enhanced ERK1 / 2 phosphorylation levels and promote the ability of ERK1 / 2 phosphorylation is more pronounced ILZ-hsGITRL. (4) gel filtration chromatography and a non-reducing SDS-PAGE analysis shows hsGITRL in solution exists mainly in dimeric form, and monomer and trimer content is relatively small, while the ILZ-hsGITRL proteins in solution mainly in trimer content exists, the monomer and dimer content is relatively small. Conclusion: (1) successfully constructed prokaryotic expression vector PET32a ()-hsGITRL aa52-177 and PET32a ()-ILZ-hsGITRL aa52-177 , and expression in ROS engineered bacteria the expression of the target protein. By Ni -IMAC column the two purification and enterokinase digestion, obtained the soluble the hsGITRL ILZ-hsGITRL target protein. (2) hsGITRL ILZ-hsGITRL protein with anti hGITRL mAb specific binding. Compared with hsGITRL protein, ILZ-hsGITRL protein significantly promote ERK1 / 2 phosphorylation, thereby enhancing the human CD4 T cell proliferation and IFN-γ secretion. (3) hsGITRL ILZ-hsGITRL protein biological function of difference may be the their respective biological structure, have a certain relationship. ILZ-hsGITRL protein in the solution, mainly exist in the form of a trimer than dimer as the main form of existence hsGITRL protein are better able to play a biological function.

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