Purification of Recombinant GM-CSF/IL-3 Fusion Protein

As a new artificial haemopoietic growth factor, GM-CSF/IL-3 fusion protein appears very promi-sing to be developed as a drug in the treatment of many diseases including cancer. The purification process of the recombinant GM-CSF/IL-3 fusion protein was studied. Similar to majority of the eukaryotic proteins, GM-CSF/IL-3 was expressed as inclusion bodies in E.coli. A series of purification steps, including cell breakage, inclusion body washing, inclusion body solubilization, protein renaturation and ion-exchange chromatography, have been set up to purify the recombinant fusion protein in an active form. Experimental results showed that the inclusion body solubility increased with increasing urea concentration. During the protein renaturation process, dialysis method was chosen to remove the denaturant urea. Stepwise decrease of urea concentration in the buffer could effectively improve the protein renaturation efficiency by reducing protein aggregation. At the same time, reduced and oxidized glutathionine were added to optimize the correct disulfide bonds formation. The recombinant protein was then purified by DEAE ion-exchange chromatography. The final protein recovery was over 30%. SDS-PAGE and reverse HPLC analysis revealed over 95% purity of the final purified recombinant protein. Therefore, a laboratory-scale purification procedure of the recombinant GM-CSF/IL-3 fusion protein was basically well established.