Abstract
As a source of recombinant antigen, soluble constant fragment (Fc) fusion proteins have become valuable reagents for immunotherapy and laboratory investigations. Additional applications for these reagents include flow cytometry, immunohistochemistry, and in vitro activity assays. To aid investigators in the generation of these reagents, the materials and methods required for producing Fc fusion proteins are described. The investigator’s protein moiety of interest is genetically linked to the N-terminus of murine Fc and subsequently expressed in large quantity using a mammalian cell expression system. The resulting Fc fusion proteins are purified on a protein A column and may be stored for at least one year at −20°C. The availability of easily purified, soluble Fc fusion proteins in such quantity can facilitate research in multiple fields of medicine and biotechnology.
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References
Little, M., Kipriyanov, S. M., Le Gall, F., and Moldenhauer, G. (2000) Of mice and men: hybridoma and recombinant antibodies. Immunol. Today 21, 364–370.
Kohler, G. and Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature 256, 495–497.
Kundu, P. K., Prasad, N. S., and Datta, D. (1998) Monoclonal antibody: high density culture of hybridoma cells and downstream processing for IgG recovery. Indian J. Exp. Biol. 36, 125–135.
VanAntwerp, J. J. and Wittrup, K. D. (2000) Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnol. Prog. 16, 31–37.
Feldhaus, M. J., Siegel, R. W., Opresko, L. K., et al. (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat. Biotechnol. 21, 163–170.
Tokushige, K., Moradpour, D., Wakita, T., Geissler, M., Hayashi, N., and Wands, J. R. (1997) Comparison between cytomegalovirus promoter and elongation factor-1 alpha promoter-driven constructs in the establishment of cell lines expressing hepatitis C virus core protein. J. Virol. Methods 64, 73–80.
Bebbington, C. R., Renner, G., Thomson, S., King, D., Abrams, D., and Yarranton, G.T. (1992) High-level expression of a recombinant antibody from myeloma cells using a glutamine synthetase gene as an amplifiable selectable marker. Biotechnology (N Y) 10, 169–175.
Bebbington, C. R. and Lambert, K. (1994) Genetic stability and product consistency of rDNA-derived biologicals from mammalian cells. Dev. Biol. Stand. 83, 183–184.
Birch, J. R. and Froud, S. J. (1994) Mammalian cell culture systems for recombinant protein production. Biologicals 22, 127–133.
Way, J. C., Lauder, S., Brunkhorst, B., et al. (2005) Improvement of Fc-erythropoietin structure and pharmacokinetics by modification at a disulfide bond. Protein Eng. Des. Sci. 18, 111–118.
Leenaars, M. and Hendriksen, C. F. (2005) Critical steps in the production of polyclonal and monoclonal antibodies: evaluation and recommendations. Ilar. J. 46, 269–279.
Dinnis, D. M. and James, D. C. (2005) Engineering mammalian cell factories for improved recombinant monoclonal antibody production: lessons from nature? Biotechnol. Bioeng. 91, 180–189.
Mathew, S. O., Kumaresan, P. R., Lee, J. K., Huynh, V. T., and Mathew, P. A. (2005) Mutational analysis of the human 2B4 (CD244)/CD48 interaction: Lys68 and Glu70 in the V domain of 2B4 are critical for CD48 binding and functional activation of NK cells. J. Immunol. 175, 1005–1013.g
Winter, G., Griffiths, A. D., Hawkins, R. E., and Hoogenboom, H. R. (1994) Making antibodies by phage display technology. Annu. Rev. Immunol. 12, 433–455.
Hoogenboom, H. R., Henderikx, P., and de Haard, H. (1998) Creating and engineering human antibodies for immunotherapy. Adv. Drug Deliv. Rev. 31, 5–31.
Feldhaus, J. M., Siegel, R. W., Feldhaus, J. M. and Wittrup, K. D. (2003) Yeast Display scFv Antibody Library User’s Manual Pacific Northwest National Laboratory. pp. 1–44.
Hu, P., Mizokami, M., Ruoff, G., Khawli, L. A., and Epstein, A. L. (2003) Generation of low-toxicity interleukin-2 fusion proteins devoid of vasopermeability activity. Blood 101, 4853–4861.
Ma, Q., DeMarte, L., Wang, Y., Stanners, C. P., and Junghans, R. P. (2004) Carcinoembryonic antigen-immunoglobulin Fc fusion protein (CEA-Fc) for identification and activation of anti-CEA immunoglobulin-T-cell receptor-modified T cells, representative of a new class of Ig fusion proteins. Cancer Gene. Ther. 11, 297–306.
Ruocco, A., Nicole, O., Docagne, F., et al. (1999) A transforming growth factor-beta antagonist unmasks the neuroprotective role of this endogenous cytokine in excitotoxic and ischemic brain injury. J. Cereb. Blood Flow Metab. 19, 1345–1353.
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© 2007 Humana Press Inc., Totowa, NJ
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Flanagan, M.L., Arias, R.S., Hu, P., Khawli, L.A., Epstein, A.L. (2007). Soluble Fc Fusion Proteins for Biomedical Research. In: Albitar, M. (eds) Monoclonal Antibodies. Methods in Molecular Biology, vol 378. Humana Press. https://doi.org/10.1007/978-1-59745-323-3_3
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DOI: https://doi.org/10.1007/978-1-59745-323-3_3
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