Expression And Purification Of Recombinant Mutant Ovine Interferon-τ In Yeast Pichia Pastoris

Interferon-Ï„(IFN-Ï„), originally known as a pregnancy recognition hormone in ruminants, has been found to be a promising new source for treating human diseases such as viral infections and autoimmune disorders, with its similar antiviral and antiproliferative activity to other type I interferons but extremely low side effects. The thesis aimed to research for new drug of the mutant P26L of ovine interferon-Ï„with genetic engineering technologies. After the mutated gene of ovine interferon-Ï„was cloned into secretive expression vector pPICZaA in Pichia pastoris, high-production strain was selected and fermented at high cell density. Then we established purification process to prepare protein sample with high-purity, measured its specific activity and tested its anti-HBV effects in cell model.The purification process was optimized in its three stages:capture, intermediate purification and polishing. In capture stage, one HIC procedure was employed instead of the original ultrafiltration and cation IXC steps to inhibit degradation, and the selection factors of salt concerntration and pH for Octyl Sepharose FastFlow" were tested. An IXC procedure using Source 30Q was supplemented as an intermediate purification stage. With the optimization of pH and efficiency factors such as column height, flow rate and capacity, the 22KD protein impurity caused by mis-cleavage of enzyme kex-2 was effectively avoided.. After including GSH in GFC equilibrating buffer on polishing stage, self-aggregation of high-purity interferon-Ï„was eliminated. At last, recovery and yield of the whole process was 46.4% and 260mg IFN-Ï„protein/L fermented supernatant respectively. The protein sample, with its purity above 95% determined by silver-stained SDS-PAGE, HPLC,SE-HPLC and molecular weight confirmed by mass spectrum, was tested to have in vitro specific activity of 1.55 X 108IU/mg, which was 21-158% higher than that of the wild type. When investigated in HBV transfected hepatoma carcinoma 2.2.1.5 cells for its Inhibition to HBV, mutant ovine interferon-Ï„showed similar anti-HBeAg secretion function, better HBV-DNA decreasing potency but significant lower cyto-toxic effects (TC50 is 75 times higher)compared to Roferon-A from Roche ltd Company, thus demonstrated its unique characteristic of high effectiveness and low toxicity.In summary, the mutant ovine interferon-Ï„was successfully expressed in Pichiapastoris, prepared to homogeneity, and proved its high effectiveness but low toxicity characteristic, which contributes to further drug research and development later.