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E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display

Nature Protocols volume 3, pages 1766–1777 (2008)Cite this article

Abstract

Here we describe a protocol for the selection of full-length IgG antibodies from repertoires displayed on Escherichia coli. In the method described here, full-length heavy and light chains are assembled in the periplasm 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 permeabilization, fluorescently labeled ligand-binding library clones are selected by multiple rounds of fluorescence-activated cell sorting. Selection of a comprehensive set of IgG clones can typically be obtained within 3–4 weeks, a timescale that is comparable with most prevalent antibody display technologies. The isolated antibodies are well expressed in bacteria and exhibit affinities per binding site in the nanomolar range.

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Figure 1: The E-clonal technology: libraries of full-length IgG antibodies expressed in the periplasm of E. coli are captured by an inner-membrane-tethered NlpA-ZZ fusion protein.
Figure 2: Map of plasmid pMAZ360-IgG for expression of soluble intact IgGs in the E. coli periplasm.
Figure 3: Representative flow cytometry data.

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Authors and Affiliations

  1. Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, 78712, Texas, USA

    Yariv Mazor, Thomas Van Blarcom, Brent L Iverson & George Georgiou

  2. Department of Chemical Engineering, University of Texas at Austin, Austin, 78712, Texas, USA

    Yariv Mazor, Thomas Van Blarcom & George Georgiou

  3. Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, 78712, Texas, USA

    Brent L Iverson

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Correspondence to George Georgiou.

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Mazor, Y., Van Blarcom, T., Iverson, B. et al. E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display. Nat Protoc 3, 1766–1777 (2008). https://doi.org/10.1038/nprot.2008.176

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