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

Nature Biotechnology volume 25, pages 563–565 (2007)Cite this article

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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References

  1. Kohler, G. & Milstein, C. Nature 256, 495–497 (1975).

    Article  CAS  Google Scholar 

  2. Li, J. et al. Proc. Natl. Acad. Sci. USA 103, 3557–3562 (2006).

    Article  CAS  Google Scholar 

  3. Fishwild, D.M. et al. Nat. Biotechnol. 14, 845–851 (1996).

    Article  CAS  Google Scholar 

  4. Winter, G., Griffiths, A.D., Hawkins, R.E. & Hoogenboom, H.R. Annu. Rev. Immunol. 12, 433–455 (1994).

    Article  CAS  Google Scholar 

  5. Boder, E.T. & Wittrup, K.D. Nat. Biotechnol. 15, 553–557 (1997).

    Article  CAS  Google Scholar 

  6. Daugherty, P.S., Olsen, M.J., Iverson, B.L. & Georgiou, G. Protein Eng. 12, 613–621 (1999).

    Article  CAS  Google Scholar 

  7. Lipovsek, D. & Pluckthun, A. J. Immunol. Methods 290, 51–67 (2004).

    Article  CAS  Google Scholar 

  8. Harvey, B.R. et al. Proc. Natl. Acad. Sci. USA 101, 9193–9198 (2004).

    Article  CAS  Google Scholar 

  9. Hoogenboom, H.R. Nat. Biotechnol. 23, 1105–1116 (2005).

    Article  CAS  Google Scholar 

  10. Jendeberg, L. et al. J. Mol. Recognit. 8, 270–278 (1995).

    Article  CAS  Google Scholar 

  11. Simmons, L.C. et al. J. Immunol. Methods 263, 133–147 (2002).

    Article  CAS  Google Scholar 

  12. Chen, G., Dubrawsky, I., Mendez, P., Georgiou, G. & Iverson, B.L. Protein Eng. 12, 349–356 (1999).

    Article  CAS  Google Scholar 

  13. Nilsson, B. et al. Protein Eng. 1, 107–113 (1987).

    Article  CAS  Google Scholar 

  14. Maynard, J.A. et al. Nat. Biotechnol. 20, 597–601 (2002).

    Article  CAS  Google Scholar 

  15. Tao, M.H. & Morrison, S.L. J. Immunol. 143, 2595–2601 (1989).

    CAS  PubMed  Google Scholar 

Download references

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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Authors and Affiliations

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

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

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

    Robert Mabry & Brent L Iverson

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

    Yariv Mazor, Thomas Van Blarcom & George Georgiou

  4. Department of Biomedical Engineering, University of Texas at Austin, Austin, 78712, Texas, USA

    George Georgiou

  5. Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, 78712, Texas, USA

    George Georgiou

Authors
  1. Yariv Mazor
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  2. Thomas Van Blarcom
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  3. Robert Mabry
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  4. Brent L Iverson
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  5. George Georgiou
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Corresponding author

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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