title: Expression, refolding, purification, and bioactivity of recombinant bifunctional protein, hIL-2/GM-CSF.
abstract: 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.
authors.affiliation: Institute of Molecular Immunology, Southern Medical University, Guangzhou 510515, People's Republic of China. wqrv@hotmail.com
authors.forename: Qi-Rui
authors.initials: QR
authors.lastname: Wang
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Ming-Qian
authors.initials: MQ
authors.lastname: Zhou
authors.forename: Nu-Yun
authors.initials: NY
authors.lastname: Liu
authors.forename: Shen-Rong
authors.initials: SR
authors.lastname: Jing
authors.forename: Quan-Ming
authors.initials: QM
authors.lastname: Zou
authors.forename: Xiao-Ning
authors.initials: XN
authors.lastname: Wang
mesh_terms: 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
chemical_list: D004731:Endotoxins; D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D007544:Isopropyl Thiogalactoside; D006639:Histidine; D016178:Granulocyte-Macrophage Colony-Stimulating Factor