Abstract
We have developed a technology for the facile isolation of full-length IgG antibodies with desired specificity from combinatorial libraries expressed in Escherichia coli. Full-length heavy and light chains are expressed from a bicistronic operon and are secreted into the periplasm where they assemble into aglycosylated IgGs that are fully functional for antigen binding. Expression of an inner membrane-tethered Fc-binding protein is used to capture the IgG molecules and anchor them to the cell. Following outer membrane disruption, clones expressing IgGs that specifically recognize fluorescently labeled antigen are selected by flow cytometry. This technique was used for the isolation of several IgGs with nanomolar affinities toward the protective antigen of Bacillus anthracis from immune libraries. High-throughput isolation of E. coli-derived full-length IgG can greatly expedite the discovery and production of antibodies for therapeutic and diagnostic applications.
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References
Imai, K., and Takaoka, A. (2006) Comparing antibody and small-molecule therapies for cancer. Nat. Rev. Cancer 6, 714–27.
Hoogenboom, H. R. (2005) Selecting and screening recombinant antibody libraries. Nat. Biotechnol. 23, 1105–16.
Milenic, D. E., Yokota, T., Filpula, D. R., Finkelman, M. A., Dodd, S. W., Wood, J. F., Whitlow, M., Snoy, P., and Schlom, J. (1991) Construction, binding properties, metabolism, and tumor targeting of a single-chain Fv derived from the pancarcinoma monoclonal antibody CC49. Cancer Res. 51, 6363–71.
Pini, A., and Bracci, L. (2000) Phage display of antibody fragments. Curr. Protein Pept. Sci. 1, 155–69.
Worn, A., and Pluckthun, A. (2001) Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305, 989–1010.
Mazor, Y., Blarcom, T. V., Mabry, R., Iverson, B. L., and Georgiou, G. (2007) Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli. Nat. Biotechnol. 25, 563–5.
Krebber, A., Bornhauser, S., Burmester, J., Honegger, A., Willuda, J., Bosshard, H. R., and Pluckthun, A. (1997) Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods 201, 35–55.
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© 2009 Humana Press, a part of Springer Science+Business Media, LLC
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Mazor, Y., Van Blarcom, T., Iverson, B.L., Georgiou, G. (2009). Isolation of Full-Length IgG Antibodies from Combinatorial Libraries Expressed in Escherichia coli . In: Dimitrov, A. (eds) Therapeutic Antibodies. Methods in Molecular Biology™, vol 525. Humana Press. https://doi.org/10.1007/978-1-59745-554-1_11
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DOI: https://doi.org/10.1007/978-1-59745-554-1_11
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