Production of fusion protein IL-2/FU-MK-1-scFv by Pichia pastoris in fermenter and characterization of partially purified fusion protein

Abstract

The aim of this study was to express a fusion protein of IL-2 and humanized single chain variable fragment antibody FU-MK-1-scFv in Pichia pastoris. The FU-MK-1-scFv recognizes a cell surface glycoprotein (designated MK-1) that is overexpressed in a majority of human carcinomas. Thus, it can be used for immunotherapy of cancer. The fusion gene encoding IL-2/FU-MK-1-scFv was amplified and ligated into the expression vector pPICZαA. The expression vector pPICZαA-IL-2/FU-MK-1-scFv was successfully transformed into P. pastoris strain GS115. Next, P. pastoris strain GS115 harboring pPICZαA-IL2/FUscFv(VH-Vk) and capable of secreting fusion protein was chosen for optimizing the production of fusion protein production by examining the effect of pH, temperature, and methanol concentrations. The highest production of the fusion protein at 258±13 mg/L was obtained under pH 3 and 30 °C with a methanol concentration of 0.1% for 96 hours induction. Fed-batch cultivation in 5 L fermenter, it was found that the amount of secreted fusion protein was 109±5 mg/L when the modified basal salt medium was used whereas the amount of secreted fusion protein was increased up to 425±2 mg/L when BMMY medium was used. To investigate biding activity of the partial purified fusion protein, cell lysate ELISA method was applied in this study. Our result demonstrated that the produced fusion protein retained specific binding activity to MK-1 antigen due to it significantly bound to MK-1 expressing Chinese hamster ovary (CHO) cell, but not MK-1 non-expressing CHO cell. The results were compared using Student’s t test (p<0.05).