Abstract: Onconase（ONC, also known as ranpirnase or P-30 protein）has weak RNase activity and significant toxic effect on various tumor cells and solid tumors. In order to obtain ONC owing highly efficient expression, we utilized the advantage of human serum albumin（HSA）expression in Pichia pastoris, had a fusion of HSA and ONC which was expressed in P. pastoris, then separated and purified the expressed protein. We designed the fusion gene according to the amino acid sequence of HSA-ONC and synthesized it based on the codons of P. pastoris, HSA-ONC gene was inserted into vector of pPIC9, pPIC9K and pPICZα-A to form the recombinant plasmid pPIC9/HSA-ONC, pPIC9K/HSA-ONC and pPICZα-A/HSA-ONC, then the linearized recombinant plasmids were transformed into P. pastoris X-33, GSS115 and SMD1168 respectively. The expression conditions were optimized in the shake flask and 10-L bioreactor. The rHSA-ONC was purified by aqueous two-phase extraction coupling DEAE anion exchange chromatography, and the biological activity was detected by SRB assay. The highest rHSA-ONC production reached 235 mg/L in pPICZα-A/X-33/HSA-ONC combination under the condition of pH7 and 23℃in 1-L flask after induced 10 d, better than other combinations. In the 10-L bioreactor, when the induction was performed in the low basic salt medium of pH 7, and induced 10 d under 0.25% ethanol, the highest rHSA-ONC production could reach 2.02 g/L. The rHSA-ONC was purified by aqueous two-phase extraction coupling DEAE anion exchange chromatography, the purity was ≥ 95% and the yield was > 70%. The results of detecting biological activity showed that the rHSA-ONC inhibited the proliferation of various tumor cells in vitro. Conclusively, the expression level of rHSA-ONC was twice as that of rONC at the same molar dose, and rHSA-ONC inhibited the proliferation of various tumor cells in vitro.

Key words: onconase, human serum albumin, Pichia pastoris efficient expression, aqueous two-phase extraction, activity of tumor cells