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Aggregation-resistant domain antibodies selected on phage by heat denaturation

Nature Biotechnology volume 22, pages 1161–1165 (2004)Cite this article

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Abstract

We describe a method for selecting aggregation-resistant proteins by heat denaturation. This is illustrated with antibody heavy chain variable domains (dAbs), which are prone to aggregate1,2. The dAbs were displayed multivalently at the infective tip of filamentous bacteriophage, and heated transiently to induce unfolding and to promote aggregation of the dAbs. After cooling, the dAbs were selected for binding to protein A (a ligand common to these folded dAbs). Phage displaying dAbs that unfold reversibly were thereby enriched with respect to those that do not. From a repertoire of phage dAbs, six dAbs were characterized after selection; they all resisted aggregation, and were soluble, well expressed in bacteria and could be purified in good yields. The method should be useful for making aggregation-resistant proteins and for helping to identify features that promote or prevent protein aggregation, including those responsible for misfolding diseases3,4.

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Figure 1: Effect of heat treatment of phage (80 °C for 10 min then cooled to 4 °C) on binding of displayed dAbs, phage tips and infectivity.
Figure 2: Biophysical properties of selected human VH3 dAbs.

Change history

  • 15 August 2004

    appended corrigendum pdf to AOP PDF; corrected online date will appear in print

Notes

  1. *Note: In the PDF of the version of this article originally published online, approximately 600 words of text was omitted, including the end of paragraph three through the first half of paragraph eight. This mistake has been corrected for the print version of this article.

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Acknowledgements

We thank John Berriman for expert assistance with the electron microscopy. While working at the Laboratory of Molecular Biology, L.J. and O.S. were funded by Domantis Limited (Cambridge, UK) under a collaborative research program with the Medical Research Council.

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Author notes

  1. Laurent Jespers & Oliver Schon

    Present address: Domantis Limited, 315 Cambridge Science Park, Cambridge, CB4 0WG, England, UK

Authors and Affiliations

  1. Laboratory of Molecular Biology, Medical Research Council Centre, Hills Road, Cambridge, CB2 2QH, England, UK

    Laurent Jespers, Oliver Schon & Greg Winter

  2. Centre for Protein Engineering, Medical Research Council Centre, Hills Road, Cambridge, CB2 2QH, England, UK

    Kristoffer Famm & Greg Winter

  3. Domantis Limited, 315 Cambridge Science Park, Cambridge, CB4 0WG, England, UK

    Laurent Jespers & Oliver Schon

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Correspondence to Greg Winter.

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G.W. is a founder, shareholder and director of Domantis.

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Jespers, L., Schon, O., Famm, K. et al. Aggregation-resistant domain antibodies selected on phage by heat denaturation. Nat Biotechnol 22, 1161–1165 (2004). https://doi.org/10.1038/nbt1000

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