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. (C) 2004 Elsevier Inc. All rights reserved.