Expression, refolding, purification, and bioactivity of recombinant bifunctional protein, hIL-2/GM-CSF

• Published in: Protein expression and purification 2005-02, Vol.39 (2), p.131-136
• Main Authors: Wang, Qi-Rui, Ma, Li, Zhou, Ming-Qian, Liu, Nu-Yun, Jing, Shen-Rong, Zou, Quan-Ming, Wang, Xiao-Ning
• Format: Article
• Language: English
• Subjects: Bifunctional molecule; Bioactivity; Blotting, Western; Cell Proliferation - drug effects; Chromatography, Ion Exchange; Electrophoresis, Polyacrylamide Gel; Endotoxins - metabolism; Escherichia coli - genetics; Escherichia coli - growth & development; Expression; Genetic Vectors; Granulocyte-macrophage colony stimulating factor; Granulocyte-Macrophage Colony-Stimulating Factor - genetics; Granulocyte-Macrophage Colony-Stimulating Factor - isolation & purification; Granulocyte-Macrophage Colony-Stimulating Factor - metabolism; Granulocyte-Macrophage Colony-Stimulating Factor - pharmacology; Histidine - chemistry; Inclusion Bodies - metabolism; Interleukin-2; Interleukin-2 - genetics; Interleukin-2 - pharmacology; Isopropyl Thiogalactoside - pharmacology; Limulus Test; Plasmids; Protein Renaturation; Purification; Recombinant Fusion Proteins - isolation & purification; Recombinant Fusion Proteins - metabolism; Recombinant Fusion Proteins - pharmacology; Renaturation
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2004.09.017

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.