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Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli

Abstract

We describe facile isolation of full-length IgG antibodies from combinatorial libraries expressed in E. coli. Full-length heavy and light chains are secreted into the periplasm, where they assemble into aglycosylated IgGs that are captured by an Fc-binding protein that is tethered to the inner membrane. After permeabilizing the outer membrane, spheroplast clones expressing so-called E-clonal antibodies, which specifically recognize fluorescently labeled antigen, are selected using flow cytometry. Screening of a library constructed from an immunized animal yielded several antibodies with nanomolar affinities toward the protective antigen of Bacillus anthracis.

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Figure 1: The E-clonal technology.
Figure 2: Analysis of IgG clones selected from the anti-PA library.

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Acknowledgements

We thank Clinton E. Leysath for preparation of the cDNA from splenocytes of PA-immunized mice. This work was supported by the Foundation for Research.

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Correspondence to George Georgiou.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Expression and purification of intact IgGs in the bacterial periplasm. (PDF 463 kb)

Supplementary Fig. 2

Binding of IgG to spheroplasts displaying the protein ZZ domain. (PDF 63 kb)

Supplementary Fig. 3

Evaluation of the NlpA-ZZ-IgG complex stability. (PDF 48 kb)

Supplementary Fig. 4

Construction of immunized anti-PA IgG Library. (PDF 30 kb)

Supplementary Fig. 5

Amino acid sequence analysis of the isolated anti-PA IgG individual clones. (PDF 15 kb)

Supplementary Fig. 6

An example of kinetic analysis of YMF10 IgG toward PA-63 using an IgG capture method. (PDF 46 kb)

Supplementary Methods (PDF 48 kb)

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Mazor, Y., Van Blarcom, T., Mabry, R. et al. Isolation of engineered, full-length antibodies from libraries expressed in Escherichia coli. Nat Biotechnol 25, 563–565 (2007). https://doi.org/10.1038/nbt1296

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