Purification of
Recombinant GM-CSF/IL-3 Fusion Protein
ZHANG Yi*, QU Xian-Ming, LU Jian-Feng, YANG
Sheng-Li
( Shanghai Research Center of Biotechnology, the Chinese Academy of Sciences,
Shanghai 200233, China )
Abstract 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.
Key words inclusion body； protein renaturation； protein purification； fusion protein； haemopoietic growth factor
Received: November 3,
1999 Accepted: December 14, 1999
This work was supported by a grant from the Key Research Program of the Chinese
Academy of sciences, No. KY95-J1-302
* Corresponding author: Tel, 86-21-64700892-364; Fax, 86-21-64700244; e-mail, [email protected]