Expression Of Recombinant Human IL-4in Pichia Pastoris And Relationship Between Its Glycosylation And Biological Activity

Background:Human interIeukin-4(hIL4) is a kind of exocrine glycoprotein, has anti-tumor and anti-inflammatory effects, in certain inflammation and autoimmune diseases has potential application value, may be the prevention and treatment of autoimmune diabetes drugs. However, the application of single dose IL4have fever, nausea and vomiting, nasal congestion, diarrhea, anorexia, weight gain and breathing difficulties and other side effects. I Phase of clinical trial show that intravenous maximum tolerated dose IL4, was not observed therapeutic effect, used alone IL4no obvious anti-tumor effect, and share other preparations, visible tumor metastasis outcome. Previous studies are almost based on source of prokaryotes expression system of recombinant IL4, no glycosylation modification after translation, whether this is the cause of limited IL4clinical treatment? In recent years, the recombinant protein technology and it clinical application widely followed, eukaryotic cell expression system such as Pichia pastoris yeast can express glycosylation modified recombinant protein, however, glycosylation effect on biological activity and function of the protein itself is uncertain in academia at present. hIL4expressed from Pichia pastoris yeast whether there is a direct relationship between glycosylation and the biological function, whether the contribution of each glycosylation sites are consistent, so far, there is no relevant research in this field in China. The purpose of this project is to study the expression of recombinant glycoprotein in Pichia pastoris yeast and the relationship between glycosylation and its biological function.Objective: To study the expression of glycosylated human IL4in Pichia pastoris yeast, a eukaryotic cells expression system, and the influence of glycosylation on protein’s biological function and to provide anew theoretical basis for the application of recombinant protein.Methods:The human IL4cDNA (153amino acids) was amplified by PCR on a plasmid encoding full-length human IL4. PCR products were purified using a PCR purification kit (Invitrogen USA). The amplified PCR DNA was analyzed and confirmed by DNA sequencing. After cloning the PCR DNA into Xhol/Xbal site of pPICZaA (Invitrogen,USA), the resulting plasmid pPICZa-IL4were transformed into E.coli(TOP10F). The recombinant plasmid was linearized by digestion with SacI and transformed into P. pastoris X-33cells,save the protein after purification. The expression of recombinant human IL4was detected by SDS-PAGE and western blotting. Glycosylation were analyzed by three methods. hIL4were digested with recombinant N-glycanase (PNGase F),then analyzed by SDS-PAGE and western blotting. Potential glycosylation sites(Asn38,Asn105) were mutated by Site-directed mutagenesis method. Recombinant plasmid was transformed into P. pastoris X-33cells. Identification of hIL4post-translational modifications was performed by mass spectrometry. Spleen cell suspensions were obtained from C57BL/6mice and isolated CD19+cells were cultured, flow cytometry analysis was performed after16h incubation of cells which treated with deglycosylated IL-4or glycosylated IL-4. The stability of hIL4in the medium and intracellular cell was determined by western blotting analysis after incubated at different temperatures (4°C, RT and37°C) for3,7, and10days. Results:Recombinant hIL4was successfully expressed in Pichia pastoris yeast, a eukaryotic cells expression system, a broad smear band was detected by SDS-PAGE and western blot analysis. The molecular mass of hIL4ranged from34to50kDa. The highest expression of hIL4occurred at96h after induction. After treated with PNGase F, compared with the control group,the molecular weight of recombinant human IL4obviously reduced, about15kDa. High purity recombinant human IL4was obtain after purification. Sequence analysis of the hIL4mutant plasmids confirmed success of the mutagenesis. hIL4with N38A mutation produced a protein completely lacking glycosylation with an expected molecular mass of15kDa, however, the protein molecular weight from N105L mutation no change,still is34-50kDa. LC-MS/MS spectrum indicated that there is not any change or modification at the Asn105. rhIL4biological activity test result showed that CD23expression were all raised in two groups,but in deglycosylated-IL4group is more obvious (p<0.01). Protein stability test result showed that deglycosylated-IL4more stable no matter in the cell and in the culture medium, The glycosylated hIL4is quite stable in the medium, but is unstable in cellsConclusion:(1) Pichia pastoris yeast, the eukaryotic cells expression system, can successfully express glycoprotein rhIL4.(2) Asn38is the single N-glycosylation site on the rhIL4protein.(3) Glycoprotein rhIL4with biological function expressed by Pichia pastoris yeast, the eukaryotic cells expression system.(4) Glycoprotein rhIL4with higher Biological activity and protein stability.