Expression And Purification Of Recombinant Onconase In Escherichia Coli

Onconase, as a member of the RNase A superfamily, is endowed with antitumor activity, showing selective cytotoxicity toward several tumor cell lines. This protein has been extensively studied because of its potential as an antitumor drug which currently undergoes for phase III clinical trials. In this paper, our aim is to prepare the recombinant onconase by a genetic prokaryotic expression system.According to the amino acid sequence of the onconase,8 primers were firstly synthezed based on the prefer codon of bacterium Escherichia coli. The target gene about 350bp in length was synthezed by PCR-driven overlap extension and subsequently ligated to pGEM-T vector. After sequencing, the gene was subcloned into expression vector pET-22b(+) to obtain the recombinant expression vector, which was transformed into Rosettatm(DE3) and positive colonies were selected and confirmed by PCR and sequencing. To check the gene expression in Rosetta, the host cell was incubated for 4h at 37℃, pH7.0, IPTG was added to 0.5mmol/L and Tricine-SDS-PAGE was run with the samples. The yield of rOnconase reached 48.8% of the total proteins of the host cell. Moreover, the primary structure of expressed propose protein was proved by LC-ESI-MS/MS with the LCQ DECA XP Plus system to be correct with the coverage of 35%.After the target protein was localized in the cell, the processing of separation and purification was investigated in more detail. rOnconase fusion protein almost assembled to form inclusion body under 37℃induction. For the target proteins prepared from inclusion body, it was denatured with 7mol/L Gu-HCl and refold. After dialysis and centrifugation, the sample was loaded into SPFF column and eluted by the buffer containing 0.2mol/L NaCl. The target fractions were concentrated with 70% NH4SO4, the precipitate was dissolved and the loaded to Sephadex G-25 column, active factions were collected. With this processsing yield of the target proteins was about 255mg/L with the purity up to 97.5%.