Horse cytokine/IgG fusion proteins–mammalian expression of biologically active cytokines and a system to verify antibody specificity to equine cytokines.

This research outlines a new system for creating and studying recombinant cytokines, proteins known for their immune response triggers, with a specific focus on equine cytokines. This system utilized the equine IgG1 heavy chain constant region as a tag for expressed cytokines to aid in detection, purification, and functional studies.

Generation of a Mammalian Expression System for Recombinant Cytokines

• The researchers created a mammalian expression system for recombinant cytokines. They utilized the equine IgG1 heavy chain constant region as a tag for detection and purification of the cytokine. This system was demonstrated using equine interferon-gamma (IFN-gamma), interleukin-2 (IL-2), interleukin-4 (IL-4), and transforming growth factor-beta1 (TGF-beta1).
• The resulting IgG1 fusion proteins consisted of the C-terminal heavy chain constant region of the IgG1 (IgGa), and the N-terminal cytokine replacing the immunoglobulin heavy chain variable domain.

Fusion Proteins

• These fusion proteins were expressed in CHO cells, presenting as dimers. The structures of these proteins were found to have similarity to that of IgG heavy chain antibodies.
• The IgG1 heavy chain constant region facilitated identifying stable transfectants through flow cytometry, and the secreted recombinant fusion protein via SDS-PAGE and Western blotting.

Recovery and Verification of Cytokines

• For the cytokine recovery from the IgG1 fusion partner, an enterokinase cleavage site was included between the cytokine gene and the immunoglobulin heavy chain constant region gene.
• The fusion protein’s purification via protein G affinity columns, the entrance of the cytokine from the IgG1 heavy chain region during or after purification, and the cytokine’s biological activity within the fusion protein or after its isolation were all demonstrated in detail.

Biological Activity Verification

• They were able to confirm biological activity in the IL-2 and IL-4/IgG1 fusion proteins, as well as the equine IFN-gamma/IgG1 fusion protein.
• To test for the crossreactivity and specificity of anti-human TGF-beta1, and anti-bovine and anti-canine IFN-gamma antibodies to corresponding horse cytokines, the four cytokine/IgG1 fusion proteins were successfully applied in ELISA, flow cytometry, and/or Western blotting.

Conclusion

• In conclusion, researchers confirmed that equine IgG1 fusion proteins offer a source of recombinant proteins with high structural and functional homology to their native counterparts. This includes a useful system for selection of stable, high expressing transfectants, and a means for monitoring specificity of antibodies to equine cytokines.