[Show abstract][Hide abstract] ABSTRACT:
Recombinant cytokines are valuable tools for functional studies and candidates for vaccine additives or therapeutic use in various diseases. They can also be used to generate specific antibodies to analyze the roles of different cytokines during immune responses. We generated a mammalian expression system for recombinant cytokines using the equine IgG1 heavy chain constant region as a tag for detection and purification of the expressed cytokine, demonstrated here using equine interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL4) and transforming growth factor-beta1 (TGF-beta1). The resulting IgG1 fusion proteins were composed of the C-terminal heavy chain constant region of the IgG1 (IgGa), and the N-terminal cytokine replacing the immunoglobulin heavy chain variable domain. The fusion proteins were expressed in CHO cells as dimers and their structures had similarity to that of IgG heavy chain antibodies. In contrast to other tags, the IgG1 heavy chain constant region allowed the selection for clones secreting high levels of the recombinant protein by a sensitive ELISA. In addition, the IgG1 heavy chain constant region facilitated identification of stable transfectants by flow cytometry and the secreted recombinant fusion protein by SDS-PAGE and Western blotting. To recover the cytokine from the IgG1 fusion partner, an enterokinase cleavage site was cloned between the cytokine gene and the immunoglobulin heavy chain constant region gene. The purification of the fusion protein by protein G affinity columns, the enterokinase digestion of the cytokine from the IgG1 heavy chain region after or during purification, and the biological activity of the cytokine within the fusion protein or after its isolation was demonstrated in detail for equine IFN-gamma/IgG1 by up-regulation of major histocompatibility complex (MHC) class II expression on horse lymphocytes. Biological activity could also be confirmed for the IL-2 and IL-4/IgG1 fusion proteins. To test the crossreactivity and specificity of anti-human TGF-beta1, and anti-bovine and anti-canine IFN-gamma antibodies to respective horse cytokines, the four cytokine/IgG1 fusion proteins were successfully used in ELISA, flow cytometry and/or Western blotting. In summary, equine IgG1 fusion proteins provide a source of recombinant proteins with high structural and functional homology to their native counterparts, including a convenient system for selection of stable, high expressing transfectants, and a means for monitoring specificity of antibodies to equine cytokines.