Fermentation And Purification Of The Fusion Protein Of Interleukin-2/Human Serum Album

Human interleukin-2 (IL-2) is an important immune factor which was produced by activated auxiliary T cells. It was applied to treat tumor, hepatitis, immunodeficiency disease in clinic. In order to prolong the half-life and improve treatment efficiency, the plasmid pPIH which harbored the encoding gene of recombinated human interleukin-2 and human serum albumin (rhIL-2-HSA) fusion protein had been constructed and expressed in P. pastoris GS115. On the basis of these results, the culture conditions of P. pastoris GS115/pPIH in shake flasks and fermentors were researched. The purification methods of rhIL-2-HSA and its biological activity were also investigated in this paper.By orthogonal experiments, medium composition was determined as 40g/L glycerol, 15g/L yeast extract, 20g/L peptone, 100mmol/L phosphate buffer of pH6.0, 0.45g/LMgSO4, 0.75g/L FeSO4, 0.75g/L MnSO4. In the induction phase, the optimal conditions was confirmed through orthogonal experiments as: 1.5%(v/v) methanol as inducer, 1:0.3volume ratio of growth phase to induction phase, induced at pH6.5 for 4 days. Under these conditions, the production of rhIL-2-HSA was 338.2 mg/L.The effects of dissolved oxygen, methanol fed manner, and pH on the production of fusion protein was studied in 5L fermentor with organic and inorganic nitrogen medium. The results showed that the production of rhIL-2-HSA was 712mg/L in organic nitrogen medium while the production was 1.46g/L in inorganic nitrogen medium. In addition, the cost was reduced greatly while the inorganic nitrogen medium was used.The purification method of rhIL-2-HSA was established. Supernatant was condensed by ultra filter after fermentation broth was centrifuged. Highly purified rhIL-2-HSA was separated by Blue Sepharose, Octyl Sepharose and DEAE ion exchange chromatography. The total recovery of rhIL-2-HSA was 16.4%. The purified product showed a single band in SDS-PAGE analysis and its purity was identified as 95% by HPLC. The purified fusion protein could react with antibodies to HSA as well as IL-2 in Western Bloting. CTLL-2 cell proliferative assay showed that the special activity of rhIL-2-HSA was 1.147×107 IU/mg.