Comparison of four methods for the purification and refolding of human interleukin-2-mouse granulocyte/macrophage colony-stimulating factor fusion protein.
The combination of IL-2 (interleukin-2) and GM-CSF (granulocyte/macrophage colony-stimulating factor) has been broadly studied in antitumour immune therapy, but its efficacy is uncertain. To better exert the activities of the two cytokines and study them in a mouse model, we have constructed a bifunctional protein, hIL-2-mGM-CSF (human IL-2-mouse GM-CSF), fused to a C-terminal tag of six histidine residues (His(6)). The fusion protein was expressed in Escherichia coli as IBs (inclusion bodies). After extracting and clarifying the IBs, four methods of protein purification and refolding were compared in order to optimize the preparation technique. Of these methods, the best result was obtained with a four-step process consisting of (1) purification with denaturing affinity chromatography, (2) followed by fully denaturing the protein with system conversion, (3) then refolding by isovolumetric ultrafiltration and (4) finally, purification by anion-exchange chromatography. The purity of the hIL-2-mGM-CSF was approx. 95%, yielding approx. 20 mg of protein/l of culture. The fusion protein retained the natural activities of IL-2 and GM-CSF, with specific activities of 8.7 x 10(6) and 1.1 x 10(7) i.u./mg respectively. Flow-cytometric analysis indicated that hIL-2-mGM-CSF could specifically bind to the corresponding receptor-positive cells. The present study provides important preliminary information for studying the antitumour activity of hIL-2-mGM-CSF in vivo, which will facilitate future clinical research into the use of hIL-2/hGM-CSF in immune therapy.
Wen
Qian
Q
Institute of Molecular Immunology, Southern Medical University, Guangzhou 510515, People's Republic of China.
Ma
Li
L
Luo
Wei
W
Zhou
Ming-Qian
MQ
He
Dong
D
Lin
Ying
Y
Wu
Zhen-Qiang
ZQ
He
Xiao-Wei
XW
Wang
Ju-Fang
JF
Wang
Xiao-Ning
XN
D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D015964:Gene Expression Regulation, Bacterial; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D006801:Humans; D007376:Interleukin-2 / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D051379:Mice; D020673:Protein Renaturation; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor / Q000378:metabolism; D015375:Receptors, Interleukin-2 / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014461:Ultracentrifugation
D007376:Interleukin-2; D016187:Receptors, Granulocyte-Macrophage Colony-Stimulating Factor; D015375:Receptors, Interleukin-2; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor