Cloning,Expression And Purification Of Recombinant ONC In E.coli

In this work,the recombinant Escherichia coli BL21(DE3)-pET-22b(+)-ONC was construct-ed for producing Onconase(ONC)efficiently.After the optimization for induction conditions and fermentation medium through single-factor and Plackett-Burman methods,the expression level of ONC was improved 3?4 times.Then obtained high purity ONC protein through ion-exch-ange chromatography and gel filtration chromatography.Finally had a preliminary study of rever-se micelle extraction ONC in order to find more efficient and more economical purification method.The conditions for ONC production by the recombinant strain were optimized.The expressi-on level of ONC on shaking flask level reached 35%under the optimal conditions:concentration of the IPTG 0.2 mM,induction time 8 h,induction temperature 37?.Moreover,the expression level of ONC on 7 L fermenter level reached 55%.And the most of ONC protein was obtained as inclusion body.Denaturation and renaturation ONC in proper buffer after crushed with high pressure homo-genizer and washed the inclusion body which was obtained from recombinant E.coli fermentatio-n.Purified ONC from the renaturation buffer through Ion-Exchange Chromatography and Gel Filtration Chromatography.The ion-exchange chromatography was operated with the sample pH 7.5,cond 6.28 and 57.3 cm/h linear velocity.Then the most of target protein was gained in the elution peak which contained 0.08 M NaCl in the basal washing buffer.After this step,both purity and concentration of ONC were improve a lot.Then selected the gel filtration chromatogr-aphy for further purification,and the operation conditions was:sample volume was 5%of the column volume and 9.1 cm/h linear velocity.Finally the elution peaks have reached electrophores-is purity 100%and HPLC purity 90%.With MTT method,detected the purified ONC cytotoxici-ty to K562 cell.And its IC50=0.5?M.It declared the purified ONC have notable ability to inhibit cancer Cell.