Preparation And Biological Function Of SA/hGM-CSF Bifunctional Funsion Protein

BackgroundMalignant tumor cause severe harm to human life, the incidence of tumor has been rising in recent years, therefore, how to overcome this difficulty is the hot spots that people concerned. Surgery, chemotherapy and radiotherapy are traditional three treatment methods in the treatment of tumors. Although some effects have been showed in tumor therapy, there are still many limitations in these methods such as lack of specificity, causing normal tissure damage and toxicity, high recurrence rate. So new therapies or new drugs with more efficiency and less toxic side effects are urgent needed to develop to solve the problems.At present, the high rate of recurrence after tumor operation has become important problem, so how to prevent or reduce the relapse rate is one focus of this study. Biological therapy for tumor in animal experiments and clinical trials have shown great potential, and is considered as another treatment which has the exact effect on tumor treatment following surgery, radiotherapy and chemotherapy. It mainly includes the tumor active specific immunotherapy, antibody targeted therapy, cytokine therapy, adoptive cellular immunotherapy and gene therapy, and cytokines is widely used in the tumor biological treatment.Cytokines are a large group of proteins, peptides or glycoproteins that are secreted by specific cells of immune system. It can regulate immune system and strengthen the lymphocyte to inhibit the tumor growth. It has been widely applied in clinic.Because the systemic administration volume is very large, so clinically, localized drug delivery methods were used to avoid the severe side effects. But because of the dispersion of the organization, the effective concentration of cytokines around the lesions in the local can’t maintain long time enough, so the clinical effects of anti-tumor limited.Streptavidin(SA), a Streptomyces avidinii-derived, non-glycosylated homotetrameric protein, can bind up to four D-biotin molecules. Biotin is also called vitamin H, the amino of cell surface proteins is biotinylated easily and biotinylated on the body and cells has no obvious side effects. Based on both the unique property of SA to bind rapidly and almost irreversibly to any biotin-linked molecule and the outstanding ability of biotin to be incorporated easily into the proteins on the cell surface, we developed a novel platform, protein anchoring technology, to display SA-tagged functional cytokines on the biotinylated surface of cancer cells for the generation of cancer cell vaccines. The first we are biotinylated the tumor cell or tissue on the surface of the membrane protein, and then we utilize streptavidin and biotin specifically combining to target protein anchored at the surface of cells or tissues, and the biotinylation reaction has no obvious adverse effects to cells, tissues and organisms. This technology can make fusion protein that is fixed in tumor lesion and its surrounding quickly and permanently, and the amount of fixed can be accurately controlled, it can maintain the concentration of cytokines in tumor cell membrane and tumor tissue in order to avoid largely cytokines get into circulation system which may cause serious side effects.GM-CSF, a prominent haematopoietic cytokine and immune regulator, is released by a myriad of cell types including macrophages, endothelial cells, mast cells and activated CD4+and CD8+T cells. GM-CSF is a significant mediator of proliferation, maturation and migration of monocytes, including macrophages and dendritic cells (DCs), it also can increase MHC molecules, co-stimulate molecule expression and enhance the capacity of tumor antigen delivery by DC. It can enhance specific immune response by promoting antigen presentation and establish the link between innate and adaptive immune.Our previous studies showed that mouse GM-CSF could be immobilized on the biotinylated surface of cancer cells so as to induce more powerful antitumor immunity in mouse models of superficial bladder cancer and prostate cancer. It alleviate the degradation of cytokines and maintain the concentration of cytokines, so it plays a very good antitumor activity. Because human and mouse are different species, it need to do further study in human GM-CSF.In this paper, SA/hGM-CSF prokaryotic expression plasmid was constructed and highly expressed in Escherichia coli, we studied purification, renaturation and the double functional activity of SA/hGM-CSF to provide substantial foundation for anti-tumor of SA/hGM-CSF in the clinical.ObjectiveTo prepare SA-tagged human GM-CSF (SA/hGM-CSF) bifunctional proteins and to study its biological functions.Methods1. Plasmid construction of SA/hGM-CSFAmplificate hGM-CSF fragment by PCR, after the double digestion, recover and purify the product, then connect it with SA-pET24a vector to get pET24a-SA-hGM-CSF and pET24a-hGM-CSF-SA prokaryotic expression plasmid. The plasmids were preliminary identified by colony PCR and double restriction enzyme digestion, and then send the plasmid to company for fully sequencing.2. Expression and purification of SA/hGM-CSF bifunctional fusion proteinExplore the best induced conditions (time, temperature, etc.) of SA/hGM-CSF bifunctional fusion protein. Collect a large number of extend bacterical strains and the supernatant and pellet were analyzed by12%SDS-PAGE. Before the purification, the protein which express in inclusion bodies required to denature (eg guanidine hydrochloride, urea, etc.) fully dissolved under the action of the denaturant. Because the fusion protein was designed to contain His tag, so it can use Ni-NTA to purify protein. Using different concentration(from low concentration to high) of immidazole phosphate buffer to elute protein and the elution peak was collected and analyzed on 12%SDS-PAGE.3. Refolding of SA/hGM-CSF bifunctional fusion proteinSA/hGM-CSF bifunctional fusion protein used the urea gradient refolding dilution method which was reduced the concentration of urea gradually. The formation of intermediates and polymers is the biggest problem for protein refolding. Reduce the protein concentration can reduce the formation of intermediates to improve the refolding yield. In order to fully open the protein disulfide bonds,50mmol/Lβ-mercaptoethanol is added into the protein. The reduced and oxidized glutathione are added in the refolding solution so as to facilitate the formation of disulfide bonds and to enhance protein refolding rates.4. Further purification of SA/hGM-CSF bifunctional fusion proteinThe refolded fusion proteins were further purified through DEAE-Sepharose chromatography, and subsequently eluted with a linear gradient of different NaCl in Tris-HCl buffer. All purification steps were monitored by SDS-PAGE.5. Western Blotting identification of SA/hGM-CSF bifunctional fusion proteinThe purified and refolded fusion protein was separated by10%SDS-PAGE and SA/hGM-CSF fusion proteins were transferred onto a poly vinylidene fluoride (PVDF) membrane by semi-dry electroblotting. The membrane was incubated with5%no-fat milk at37℃for3h, and then incubated with1:2000diluted mouse anti-hGM-CSF monoclonal antibody for12h at4℃. The membrane was washed3times with Tris-HCl buffered saline Tween-20(TBST) and then incubated by1:5000diluted horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG as a second antibody at37℃for1h. The HRP activity was demonstrated by enhanced chemiluminescence (ECL) after TBST washing.6. Activity detection of SA/hGM-CSF bifunctional fusion proteinsTF-1cells were used to evaluate the biological activity of hGM-CSF moiety of SA/hGM-CSF fusion proteins. The fusion protein activity unit was obtained through comparison with the standard hGM-CSF.7. Flow cytometry was used to detect SA/hGM-CSF bifunctional fusion proteins on the biotinylated tumor cells Biotin-binding assay of SA/hGM-CSF fusion proteins was performed as before. Cells were incubated with mouse anti-hGM-CSF monoclonal antibody (primary antibody). After the full reaction, FITC labeled goat anti-mouse IgG (second antibody) was added. After extensive washing, the quantity of SA/hGM-CSF tumor cells anchored on the cell surface was analyzed by FACS.Results1. Successfully constructed recombinant plasmid of pET24a-SA-hGM-CSF and pET24a-hGM-CSF-SA. Through the recombinant plasmid DNA sequencing, the result is the same with GenBank.2. The recombinant plasmids which were sequenced correct was transformed into E. coli Rosetta. Protein was induced at37℃,0.1mmol/LIPTG for4h. They were expressed at about20%of the total cellular protein as inclusion body. The washed inclusion body was dissolved in6M guanidine hydrochloride, and diluted10-folds with6M urea, so as to improve the recovery of fusion protein. The purity of SA/hGM-CSF was65%after washing of inclusion body. The SA-hGM-CSF fusion protein was eluted in30mmol/L imidazole concentration, and hGM-CSF-SA fusion protein was eluted in50mmol/L imidazole concentration. The purity of SA/hGM-CSF was about91%through the Ni-NTA column.3. The refolded SA/hGM-CSF fusion protein was mainly in the form of polymer, there was little precipitation during the refolding process.4. The refolded fusion proteins were further purified through DEAE-Sepharose chromatography, which was subsequently eluted with a linear gradient of0.3-1.0M NaCl in30mM Tris-HCl, pH8.0(30mmol/L,50mmol/L,100mmol/L,200mmol/L,300mmol/L,500mmol/L,1M/L NaCl). All purification steps were monitored by SDS-PAGE. The purity of the refolded SA/hGM-CSF fusion proteins which were eluted in200mmol/L NaCl reached up to96%after DEAE-Sepharose chromatography.5. SA/hGM-CSF fusion proteins were recognized specifically by a monoclonal antibody against hGM-CSF. 6. SA/hGM-CSF fusion proteins promoted the proliferation of TF-1cells in a dose-dependent manner similar to recombinant hGM-CSF. In addition, there was no significant difference between SA-hGM-CSF and hGM-CSF-SA.7. As flow cytometric analysis showed, the bifunctional SA/hGM-CSF fusion proteins were efficiently displayed with the modification rate of99%(SA-hGM-CSF99.94%, hGM-CSF-SA99.98%) on the biotinylated surface of MB49cells through the specific and tight interaction between streptavidin and biotin.Conclusion1. SA/hGM-CSF recombinant expression plasmids were constructed successfully.2. High expression quantity and high purity of SA/hGM-CSF bifunctional fusion protein were obtained successfully.3. The SA/hGM-CSF bifunctional fusion protein has a bifunctional activity, which not only has the activity of hGM-CSF but also has the biotin-binding activity of streptavidin.4. There was no significant difference between SA-hGM-CSE and hGM-CSF-SA fusion protein.5. The SA/hGM-CSF bifunctional fusion protein which we have prepared successfully may laid the foundation for futher clinical research.