Protocol abstract
Nature Protocols 3, - 1766 - 1777 (2008)
Published online: 23 October 2008 | doi:10.1038/nprot.2008.176
Subject Categories: Immunological techniques | Isolation purification and separation | Model organisms
E-clonal antibodies: selection of full-length IgG antibodies using bacterial periplasmic display
Yariv Mazor1,2, Thomas Van Blarcom1,2, Brent L Iverson1,3 & George Georgiou1,2
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.
- Institute for Cellular and Molecular Biology, University of Texas at Austin, Austin, Texas 78712, USA.
- Department of Chemical Engineering, University of Texas at Austin, Austin, Texas 78712, USA.
- Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
Correspondence to: George Georgiou1,2 e-mail: gg@che.utexas.edu
nature-products
A-Z product listing
- D-(+)-Glucose(MP Biomedicals)
- E. coli strains(Invitrogen)
- L-(+)-Arabinose(MP Biosciences)
- Taq DNA polymerase with ThermoPol buffer(New England Biolabs)
- 100 mm
15 mm Petri dishes(VWR) - Agarose(Sigma)
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