High Level Expression Of Recombinant Human GST-PD-1 Fusion Protein And Study On Its Biological Effects

The B7-CD28 superfamily, one of the best-characterized costimulatory family, not only provides critical positive second signals to initiate and sustain T cell response, but also contributes key negative second signals to downregulate and terminate T cell response. The PD-1 receptor, which belongs to CD28 family, is a 55KDa type I transmembrane protein of Ig superfamily with an extracellular IgV-like domain. The domain plays an important role in binding to ligands. Ligation PD-1 with its two ligands, PD-L1 and PD-L2, triggers inhibitory signals to inhibit T and B cell activation and production of cytokines. PD-L and PD-1 interaction involves in maintenance of peripheral tolerance. In addition, the PD-1 expression and biological effects are closely relatesd to a series of diseases including autoimmune diseases, tumor and trasplantation rejection. Studies on mouse models showed that PD-1 receptor was a promising target in the intervention of human autoimmune disease, transplantation rejection and malignant cancers. Otherwise, human PD-1 recombinant protein is not available so far, which restricts its studies. In our studies, the cDNA fragments edcoding full-length and extra-cellular region of PD-1 were obtained and extra-cellular fragment was cloned into GST fusion protein expression vector. The target protein was expressed in E coli and then purified with higher purity. Furthermore, a method for characterizing its biological activity was set up.1. Cloning the cDNA ecoding PD-1 full-length and extra-cellular fragmentsThe published cDNA sequence for PD-1 was used for specific pimer design. Extra-cellular fragment of PD-1 gene was amplified through RT-PCR from PHA-activated human peripheral blood T cells and was cloned into T-vector plasmid. The derived plasmid was verified by DNA sequence analysis, and then cloned into GST fusion protein expression vector (pGEX-5X-3). Expression vector named pGEX-5X-3-PD-l was transformed into BL21 (DE3) strain of Escherichia coli for further expression of fusion protein. Full-length fragment of PD-1 gene including 5'UTR was amplified at the same time. Recombinant PCR was carried out, and then aquired fragment was cloned into pcDNA3.1 vector. The derived recombinant plasmid was certified by DNA sequence analysis.2. Expression, purification and characterization of GST-PD-1 fusion proteinBL21 expression strain was propagated in lura broth. A series of methods (low temperature induction, shorten of induction time, etc) was tried to induce soluble GST-PD-1 fusion protein but without sucesses. Fusion protein was mainly expressed as inclusion body. Dynamics study indicated that the most high-level protein expression was after 5 hour IPTG induction at Immol/L. The cell slurry was disrupted by sonication, and then fusion protein was purified through GST affinity chromatography column after denaturation, refolding and dialysis. Purity of protein is above 90% with 2mg/ml concentration. Western blotting showed a single band of molecular mass 46KDa.3. Setting up a way to characterize biological activity of GST-PD-1 fusion proteinHuman peripheral blood T cells were cocultured with PD-L1 transfected L929 fibroblast cell strain at different PHA concentration. IL-2 and IFN- Y concentration of supernatant were determined by ELISA after 48 hours. Compared with mock tansfected L929 cells, PD-L1 transfected L929 cells inhibited significantly production of these cytokines of activated T cells. GST-PD-1 fusion protein reversed the inhibitory effects at a higher concentration.These results suggest GST-PD-1 fusion protein can be expressed in BL21 Escherichia coli strain at a high yield. GST-PD-1 fusion protein with higher purity has inhibitory biological effects. Further studies should be concentrated to optimize the process of refolding in order to obtain a nature equal activity of PD-1 molecule.