Expression, purification, and refolding of recombinant fusion protein hIL-2/mGM-CSF.
OBJECTIVE
To study the activities of interleukin (IL)-2 and granulocyte-macrophage colony-stimulating factor (GM-CSF) (hIL-2/mGM-CSF).


METHODS
SOE PCR was used to change the linker of the fusion protein for higher activities. The fusion protein was expressed in Escherichia coli (E. coli) BL21 (DE3) in inclusion body (IB) form. After IB was extracted and clarified, it was denatured and purified by affinity chromatography. The protein was refolded by dilution in a L-arginine refolding buffer and refined by anion chromatography. The protein activity was detected by cytokine-dependent cell proliferation assay.


RESULTS
The expression of hIL-2/mGM-CSF in E. coli yielded approximately 20 mg protein /L culture and the purity was about 90%. The specific activities of IL-2 and GM-CSF were 5.4 x 10(6) IU/mg and 7.1 x 10(6) IU/mg, respectively.


CONCLUSIONS
This research provides important information about the anti-tumor activity of hIL-2/mGM-CSF in vivo, thus facilitating future clinical research on hIL-2/mGM-CSF used in immune therapy.
Wen
Qian
Q
Institute of Molecular Immunology, Southern Medical University, Guangzhou 510515, Guangdong, China.
Ma
Li
L
Luo
Wei
W
Zhou
Ming-Qian
MQ
Wang
Xiao-Ning
XN
D000818:Animals; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D001681:Biological Assay; D049109:Cell Proliferation; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D016207:Cytokines / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D017510:Protein Folding; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D016207:Cytokines; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D001120:Arginine