Design And Characterization Of Long Life Recombinant Human Granulocyte Colony-stimulating Factor Fusion Protein

Human granulocyte colony-stimulating factor (hG-CSF) is a hematopoietic growth factor. It can stimulate the granulocyte progenitor cells, and promote them proliferation and differentiation to neutrophil cells, and maintain the function and survival of neutrophil cells. Clinically, hG-CSF is mainly used to increase neutrophil counts for bone marrow transplantation and for cancer chemotherapy-induced serious neutrophil deficiency. However, hG-CSF has short half-life, it should be repeadedly used in clinical application in order to maintain the effective concentration of G-CSF in serum. Repeatedly treatments result in the high cost and reduce the patient compliance.The purpose of this study is to design and characterize a new long life recombinant human G-CSF fusion protein(rhG-CSF-tag1). A 10-amino acid peptide specifically recognizing Fc fragment of human Ig was selected from the literature, and was used to fuse to the C-terminus of G-CSF to construct long life G-CSF fusion protein.First, the primers were designed, and used to amplify the cDNAs encoding hG-CSF and hG-CSF-tag1 by PCR. The PCR products were digested by EcoR I and BamH I, and then inserted into the pBV-220 vectors which were digested by the same two enzymes. The two prokaryotic expression vectors were constructed and named pBV-G-CSF and pBV-G-CSF-tag1. They were all testified by PCR amplification, restriction digestion assay, and DNA sequencing.pBV-G-CSF and pBV-G-CSF-tag1 were then transformed into E.coli DH5α. The recombinant engineering bacteria were cultured under the condition of 30℃. When OD600 reached 0.5-0.6, the cultured engineering bacteria were transferred into 42℃to induce the target protein expression. SDS-PAGE analysis showed that hG-CSF and hG-CSF-tag1 were all high expressed in E.coli and the expressed target proteins mainly existed in the form of inclusion bodies.The inclusion bodies were isolated and washed with various buffers to remove impurity proteins, then the purifed inclusion bodies were resolved in 8M urea or 7M guanidine hydrochloride. The denatured target proteins were firstly refolded, and then purified by DEAE chromatography, the eluted target proteins were collected, and were dialyzed overnight against Tris-HCl buffer(pH8.0) , and then against HAc-NaAc buffer(pH8.0).G-CSF-dependent NFS-60 cells were used to analyze the activities of the refolding hG-CSF and hG-CSF-tag1 by MTT mothed. The results showed that hG-CSF and hG-CSF-tag1 all could maintain the survival and proliferation of NFS-60 cells.BSA was used as standard protein to measure the concentration of hG-CSF and hG-CSF-tag1 protein. Cyclophosphamide was used to establish the model of neutrophil deficiency. All the BALB / C mice were randomly devided into negative control group, hG-CSF group, and hG-CSF-tag1 group. After the treatment with 10mM acetate buffer, hG-CSF or hG-CSFtag1, the leukocytes of mice were counted every day.In conclusion, hG-CSF and hG-CSF-tag1 expression vectors were constructed successfully, and they could highly express hG-CSF and hG-CSF-tag1 in E.coli. The expressed hG-CSF and hG-CSF-tag1 were refolded by dilution and dialysis, they all maintained NFS-60 cells survival and proliferation. In vivo, hG-CSF-tag1 showed long term activity compared with hG-CSF, this meaned that hG-CSF-tag1 has longer half-life in serum than hG-CSF.