Fermentation,Purification,Targeting And Antitumor Activity Of Onconase

Onconase,one member of RNase A superfamily, is one ribonuclease purified from oocytes and early of Rana pipiens, which was proved that it possessed anti-tumor bioactivity in vitro and vivo. Based on experimental data from shake flask and combined with relevant parameter on fermenter, the expression of target protein was preliminary optimized in the study. Optimal inoculum size of2%,IPTG concentration to0.5mM,inducing time of3.5h were determinated.56.03g wet weight of bacteria was gained from1L fermentation broth and the recombinant protein Onconase was more than30%of the total bacterium protein on3.7L fermenter after nine hours culture. By disrupting bacteria,washing inclusion bodies, refolding and purifying inclusion,18.64g inclusion bodies were gained from100g wet weight of bacteria,and Onconase yield reached300mg in1L fermentation broth which showed significant cytotoxicity on B16cell (IC50=1.74μmol/L) and K562cell (IC50=1.74μmol/L). Final purity of Onconase was97.0%through HPLC analysis. In addition, the synergistic antitumor effect of Onconase combined with Cantharidinate on SPC-A-1and A549cells was discovered. The result expanded the application of Onconase and Cantharidinate and provided one new clue on lung cancer treatment. To enhance Onconase targeting, fusion protein (ONC-CTX) was successfully expressed in E.coil, and15mg purified protein was gained in1L culture medium. The fusion protein with dramatic antitumor capacity showed the identical specifity with Chlorotoxin,which laid a foundation on anti-tumor research in vivo.