Preparation And Purity Of CXCL14/GM-CSF Fusion Protein From Pichia Pastoris

Cytokine is a kind of low molecular weight protein with high activity and multiple functions, produced by immune cells (as mononuclear-phagocytic cell, T cell, B cell, NK cell, et al.) and non-immune cells (as vascular endothelial cell, epidermal cell, fibroblast et al.) . Cytokine can accommodate immune response, participate in the differentiation and growth of immune cells and tissue repair, mediate inflammatory reaction, and stimulate hematogenesis and so on. According to its mainfunctions, cytokine can be classified six categories—interleukin, interferon, tumornecrosis factor, colony stimulating factor, growth factor and chemokines. Preparation of cytokine fusion protein is one of the hot topics of immunology research because cytokines fusion protein constructed by genetic engineering on account of different cytokines with same or correlated functions but different action target, can elicit the biological effects of both its components cytokines and represents a better activity of delivering the two independent components though complementation and synergism effect of their biological activities. Up to now, many kinds of fusion proteins have been prepared. Some of those have been used in clinical trials such as IGF- II /IL-3 fusion protein.CXCL14 is a novel CXC chemokine from a human dentritic cell (DC) cDNA library discovered by the department immunology of Second Military MedicalUniversity. The CXC chemokine without ELR motif shares greatest homology with macrophage inflammatory protein (MIP)-2αβ, hence is designated as MIP-2γ, now is designated as CXCL14 by the International Designation Society. Previous study initially demonstrated that this novel CXC chemokine is an effective chemoattractant for some immune and hematopoietic cells, such as immature DC, neutrophils and hematopoietic stem /progenitor cell. It can also regulate the hematogenesis. In vitro colony formation essay, CXCL14 exhibits an enhancing effect on the proliferation and differentiation of the myeloid progenitor cells and the megakaryocytic progenitor cells together with many hematopoietic cell growth factors. As we all know, GM-CSF is a kind of hematopoietic growth factor with widely biologic activity, which is mainly produced by activated T cell, activated macrophage, fibroblast and endothelial cell. GM-CSF can stimulate the proliferation of hematopoietic progenitor cell and acute, chronic marrow -derived leukemic cell; promote the proliferation and differentiation of granulocyte, mononuclear cell and eosinophilic granulocyte and their function of sterilization, anti-parasite and anti-tumor; enhance phagocytosis activity of granulocyte and up-regulate the expression of its adhesion molecule; promote the cytokine release of mononuclear cell and chemotate granulocyte and mononuclear cell. Recent researches demonstrate that GM-CSF not only can induce the differentiation and growth of DC in vitro, but also can enhance the antigen presenting ability of DC and stimulate the proliferation of T cell. GM-CSF has been used in infectious diseases and malignant tumor. These data suggest that the biological functions of hCXCL14 and hGM-CSF are complementary each other. We should select hCXCL14 and hGM-CSF to construct a new fusion protein. The hCXCL14/hGM-CSF fusion protein may attract or recruit more immune cells and hematopoietic cells, and activate or enhance the functions of those cells. So the new fusion protein may have better activity than single cytokine and may regulate hematopoesis and immune response more efficiently. In addition, CXCL14, as a novel chemokine, its construction features, receptor, mechanism of action and signal transduction pathway haven't been known completely. The preparation of hCXCL14/hGM-CSF fusion protein and the research of its function and mechanism of action will be helpful to reveal the biological function andmechanism of action. Objective:To construct the yeast secretory expressive vector of pPIC9K— hCXCL14/hGM-CSF and express it in Pichia pastoris GS115.Screen the stable transformation Pichia pastoris GS115 with multiple inserts of hCXCL14/hGM-CSF fusion gene. Then optimize the expression conditions. Finally after fermentation under the induction of methanol, we gained fusion proteins with CXCL14 and GM-CSF double activities and purified this fusion protein.Method:1. Through molecular biological technique, hCXCL14/hGM-CSF fusion gene was digested by restriction enzyme EcoR I and Not I from pcDNA3.1(+)— hCXCL14/hGM-CSF and was cloned into Pichia pastories secretory expression vector pPIC9K. Then the synthetic gene was sequenced and proved to be the same as the designed. The construct was linearized with SalI and then the linearized recombinant plasmids were electrotransformed into the competent yeast strain GS115 of Pichia pastoris, the positive transformants were selected through phenotype screening and G418 resistance screening, then the transformants with multicopy and series integration of hCXCL14/hGM-CSF fusion gene were identified by PCR.2. Pichia pastoris GS115-pPIC9K—hCXCL14/hGM-CSF were cultivated under different conditions. The concentration of GM-CSF was measured by hGM-CSF ELISA quantitaty detection kit of R&D product at OD450, the content of hCXCL14/hGM-CSF fusion protein was inferred and the concentration of Pichia pastoris GS115 strain was measured by spectrophotometer at OD₆₀₀ According to these, the optimum condition was definitive. These conditions are as follows: medium(BMMY, BMGY, BMM, YPDå’ŒMM); concentration of dissolved oxygen(50%, 60%, 70%, 80%, 90%) ; cultivation time (24h, 36h, 48h, 60h, 72h, 84h, 96h,); methanol(0.05%, 0.075%, 0.1% 0.15%, 0.3%).The expressionof hCXCL14/hGM-CSF fusion protein was identified by SDS-PAGE and Western blot.3. hCXCL14/hGM-CSF fusion protein was isolated and purified through salting-out with 50% saturation of(NH4)₂SO₄; dialysis with 50mM PBS (PH7.4) ; negative ion chromatography with Hiprep 16/10 Souce30Q; molecular sieve chromatography with Sephasdex G200 from the supernatant of fermentation broth. The purity of hCXCL14/hGM-CSF fusion protein was analyzed by HPLC system.Result:1. Yeast expression vectors pPIC9K-hCXCL14/hGM-CSF was successfully gained. After electroporation of GS115,the His⁺Mut⁺, multicopy recombinants were obtained through phenotype screening and G418 concentration gradient screening and PCR identification.2. The positive transformants of Pichia pastoris strain GS115 fermentated under the induction of methanol, express the fusion protein with molecular weight of about 50kD, which have double antigenicity of hCXCL14 and hGM-CSF.3. The optimization of the expression conditions for the yeast engineering strain GS115/pPIC9K-hCXCL14/hGM-CSF was performed, the results showed that BMMY medium was the best medium for the engineering yeast strain, the concentration of dissolved oxygen and methanol was 80%～90% and 0.1% respectively, the optimal cultivation time was 3 days。 The purity of hCXCL14/hGM-CSF fusion protein analyzed by HPLC system was about 95% after the fusion protein was isolated and purified through salting-out with 50% saturation of (NH₄)₂SO₄; dialysis with 50mM PBS (PH7.4) ; negative ion chromatography with Hiprep 16/10 Souce30Q; molecular sieve chromatography with Sephasdex G200 from the supernatant of fermentation broth.Conclusion:pPIC9K-hCXCL14/hGM-CSF yeast secretory expression vector was successfully gained. The stable transformation Pichia pastoris strain GS115 which express hCXCL14/hGM-CSF fusion protein was gained. The most suitable expressionconditions—the best medium, the best concentration of dissolved oxygen andmethanol and the optimal cultivation time. The purity conditions of hCXCL14/hGM-CSF fusion protein were researched and this fusion protein has been purified, which will be useful for further research on its functions and mechanism studies.