Human Serum Albumin - Human Granulocyte Colony Stimulating Factor (Ⅰ) Fusion Protein Expression And Purification And GMP Production

Human granulocyte colony-stimulating factor (hG-CSF) has an important role in tumor radiotherapy.chemotherapy-induced neutropenia, aplastic anemia, other blood diseases and bone marrow transplantation, has been applied in clinical, is the largest annual sales of several biological products of the drugs.hG-CSF, its biggest drawback is the short half-life in plasma. In order to extend the half-life of hG-CSF, I took advantage of a methanol yeast expression system, developed a human serum albumin and granulocyte colony-stimulating factor fusion protein (rHSA-G-CSF) as a long-term genetic engineering drugs successfully. First, the fragments of HSA and hG-CSF connected by PCR method, and then cloned into the yeast secretory type expression vector pPIC9, integration was linearized after protoplast transformed into yeast cells. Expressed the fusion protein in the fermenter, purified by chromatography to separate the fusion protein. The purified fusion protein tested by western blot analysis (Western-Blot) showed that also has the immunogenicity of HSA and G-CSF, and the stability is increased, half-life is greatly extended, reaching25-30h, is the G-CSF monomer plasma half-life of15to20times. Vitro biological activity analysis showed that the activity of G-CSF is modified by fusion still retain more than50%of the G-CSF monomer. Compared with the Amgen company’s product, they used polyethylene glycol(PEG) covalently-modified protein of the G-CSF, the fusion protein has a similar half-life and biological activity.Further, the production process of the present method is more simple, and lower production costs. In clinical applications, such products are likely instead of the monomers G-CSF.