Expression, refolding, purification, and bioactivity of recombinant bifunctional protein, hIL-2/GM-CSF.
Interleukin-2 (IL-2) can stimulate T cell proliferation and differentiation when binding to its receptor on T cells. It produces a marked effect by enhancing the cytotoxicity of CD8+ T cells and natural killer cells. Granulocyte-macrophage colony stimulating factor (GM-CSF) is associated with many cells proliferation, such as dendritic cells, macrophages. Here, we report the construction, expression and purification of a bifunctional protein, hIL-2/GM-CSF, which may facilitate interaction between T cells and the antigen presentation cells and improve the efficiency of antigen presentation. We found that the use of chemicals and temperature shift is a peculiar system for induction of the Escherichia coli transformed with an IPTG-regulated hIL-2/GM-CSF expression vector in this research. After renaturation, anion exchange chromatography, metal affinity chromatography, and strict endotoxin-free cation exchange chromatography, the fusion protein devoid of endotoxin showed high purity. Cell proliferation experiments proved that this bifunctional protein retains both hIL-2 and GM-CSF biological activities. These results will facilitate the numerous subsequent studies on this bifunctional molecule.
Wang
Qi-Rui
QR
Institute of Molecular Immunology, Southern Medical University, Guangzhou 510515, People's Republic of China. wqrv@hotmail.com
Ma
Li
L
Zhou
Ming-Qian
MQ
Liu
Nu-Yun
NY
Jing
Shen-Rong
SR
Zou
Quan-Ming
QM
Wang
Xiao-Ning
XN
D015153:Blotting, Western; D049109:Cell Proliferation / Q000187:drug effects; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development; D005822:Genetic Vectors; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology*; D006639:Histidine / Q000737:chemistry; D002479:Inclusion Bodies / Q000378:metabolism; D007376:Interleukin-2 / Q000235:genetics / Q000494:pharmacology*; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008033:Limulus Test; D010957:Plasmids; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology
D004731:Endotoxins; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor