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Expression of high-affinity human antibody fragments in bacteria

Nature Protocols volume 7, pages 364373 (2012) | Download Citation

Abstract

Here we describe protocols for the expression of human antibody fragments in Escherichia coli. Antigen-specific clones are identified by soluble fragment ELISA and concentrated by periplasmic preparation. They are then further purified by affinity chromatography. This article provides an overview of expression and purification strategies for human antibody fragments, as well as detailed protocols for the identification of high-affinity binders and for affinity maturation.

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Acknowledgements

Protocols are based on methods originally developed by D. Christ and others in G. Winter's group at the UK Medical Resarch Council (MRC) Laboratory of Molecular Biology. These were further modified at the Garvan Institute of Medical Research and at MedImmune. This work was funded by the Garvan Institute of Medical Research; MedImmune; the Australian National Health and Medical Council; the Australian Research Council; the Cancer Institute, NSW; and the UK MRC.

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Affiliations

  1. Garvan Institute of Medical Research, Darlinghurst, Sydney, New South Wales, Australia.

    • Romain Rouet
    • , Kip Dudgeon
    • , Brendan Roome
    • , Peter Schofield
    • , David Langley
    • , Peter Whitfeld
    •  & Daniel Christ
  2. The University of New South Wales, Faculty of Medicine, St. Vincent's Clinical School, Darlinghurst, Sydney, New South Wales, Australia.

    • Romain Rouet
    • , Kip Dudgeon
    • , Brendan Roome
    •  & Daniel Christ
  3. MedImmune, Cambridge, UK.

    • David Lowe
    • , John Andrews
    •  & Lutz Jermutus

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Contributions

D. Lowe, K.D., B.R., P.S., D. Langley, J.A. and P.W. wrote the paper. R.R. wrote the paper and generated figures. L.J. and D.C. wrote the paper and supervised research.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Daniel Christ.

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DOI

https://doi.org/10.1038/nprot.2011.448

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