Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Aggregation-resistant domain antibodies selected on phage by heat denaturation

This article has been updated


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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

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


  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.


  1. Ward, E.S., Güssow, D., Griffiths, A.D., Jones, P.T. & Winter, G. Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli. Nature 341, 544–546 (1989).

    Article  CAS  Google Scholar 

  2. Ewert, S., Cambillau, C., Conrath, K. & Plückthun, A. Biophysical properties of camelid VHH domains compared to those of human VH3 domains. Biochemistry 41, 3628–3636 (2002).

    Article  CAS  Google Scholar 

  3. Dobson, C.M. Protein misfolding, evolution and disease. Trends Biochem. Sci. 24, 329–332 (1999).

    Article  CAS  Google Scholar 

  4. Rochet, J.C. & Lansbury P.T., Jr. Amyloid fibrillogenesis: themes and variations. Curr. Opin. Struct. Biol. 10, 60–68 (2000).

    Article  CAS  Google Scholar 

  5. Dumoulin, M. et al. Single-domain antibody fragments with high conformational stability. Protein Sci. 11, 500–515 (2002).

    Article  CAS  Google Scholar 

  6. Perez, J.M. et al. Thermal unfolding of a llama antibody fragment: a two-state reversible process. Biochemistry 40, 74–83 (2001).

    Article  CAS  Google Scholar 

  7. van der Linden, R.H. et al. Comparison of physical chemical properties of llama VHH antibody fragments and mouse monoclonal antibodies. Biochim. Biophys. Acta 1431, 37–46 (1999).

    Article  CAS  Google Scholar 

  8. Jespers, L., Schon, O., James, L.C., Veprintsev, D. & Winter, G. Crystal structure of HEL4, a soluble, refoldable human VH single domain with a germ-line scaffold. J. Mol. Biol. 337, 893–903 (2004).

    Article  CAS  Google Scholar 

  9. McCafferty, J., Griffiths, A.D., Winter, G. & Chiswell, D.J. Phage antibodies: filamentous phage displaying antibody variable domains. Nature 348, 552–554 (1990).

    Article  CAS  Google Scholar 

  10. Holliger, P., Riechmann, L. & Williams, R.L. Crystal structure of the two N-terminal domains of g3p from filamentous phage fd at 1.9 Å: evidence for conformational lability. J. Mol. Biol. 288, 649–657 (1999).

    Article  CAS  Google Scholar 

  11. Wilkinson, D.L. & Harrison, R.G. Predicting the solubility of recombinant proteins in Escherichia coli. Biotechnology (NY) 9, 443–448 (1991).

    CAS  Google Scholar 

  12. Chiti, F. et al. Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases. Proc. Natl. Acad. Sci. USA 99, 16419–16426 (2002).

    Article  CAS  Google Scholar 

  13. Ewert, S., Huber, T., Honegger, A. & Plückthun, A. Biophysical properties of human antibody variable domains. J. Mol. Biol. 325, 531–553 (2003).

    Article  CAS  Google Scholar 

  14. Sepulveda, J., Jin, H., Sblattero, D., Bradbury, A. & Burrone, O.R. Binders based on dimerised immunoglobulin VH domains. J. Mol. Biol. 333, 355–365 (2003).

    Article  CAS  Google Scholar 

  15. Arbabi Ghahroudi, M., Desmyter, A., Wyns, L., Hamers, R. & Muyldermans, S. Selection and identification of single domain antibody fragments from camel heavy-chain antibodies. FEBS Lett. 414, 521–526 (1997).

    Article  CAS  Google Scholar 

  16. Kristensen, P. & Winter, G. Proteolytic selection for protein folding using filamentous bacteriophages. Fold. Des. 3, 321–328 (1998).

    Article  CAS  Google Scholar 

  17. Sieber, V., Plückthun, A. & Schmid, F.X. Selecting proteins with improved stability by a phage-based method. Nat. Biotechnol. 16, 955–960 (1998).

    Article  CAS  Google Scholar 

  18. Martin, A., Sieber, V. & Schmid, F.X. In vitro selection of highly stabilized protein variants with optimized surface. J. Mol. Biol. 309, 717–726 (2001).

    Article  CAS  Google Scholar 

  19. Shusta, E.V., Holler, P.D., Kieke, M.C., Kranz, D.M. & Wittrup, K.D. Directed evolution of a stable scaffold for T-cell receptor engineering. Nat. Biotechnol. 18, 754–759 (2000).

    Article  CAS  Google Scholar 

  20. Jung, S., Honegger, A. & Plückthun, A. Selection for improved protein stability by phage display. J. Mol. Biol. 294, 163–180 (1999).

    Article  CAS  Google Scholar 

  21. Tomlinson, I.M., Walter, G., Marks, J.D., Llewelyn, M.B. & Winter, G. The repertoire of human germline VH sequences reveals about fifty groups of VH segments with different hypervariable loops. J. Mol. Biol. 227, 776–798 (1992).

    Article  CAS  Google Scholar 

  22. Wörn, A. & Plückthun, A. Stability engineering of antibody single-chain Fv fragments. J. Mol. Biol. 305, 989–1010 (2001).

    Article  Google Scholar 

  23. Muyldermans, S., Atarhouch, T., Saldanha, J., Barbosa, J.A. & Hamers, R. Sequence and structure of VH domain from naturally occurring camel heavy chain immunoglobulins lacking light chains. Protein Eng. 7, 1129–1135 (1994).

    Article  CAS  Google Scholar 

  24. Desmyter, A. et al. Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nat. Struct. Biol. 3, 803–811 (1996).

    Article  CAS  Google Scholar 

  25. Waldo, G.S. Genetic screens and directed evolution for protein solubility. Curr. Opin. Chem. Biol. 7, 33–38 (2003).

    Article  CAS  Google Scholar 

  26. Kabat, E.A. National Institutes of Health (US) & Columbia University. Sequences of proteins of immunological interest, edn. 5 (US Dept. of Health and Human Services Public Health Service, National Institutes of Health, Bethesda, MD, 1991).

    Google Scholar 

  27. Pace, C.N. & Scholtz, J.M. in Protein Structure, A Practical Approach, edn. 2 (ed. Creighton, T.E.) 299–321 (Oxford University Press, New York, 1997).

    Google Scholar 

  28. Myers, J.K., Pace, C.N. & Scholtz, J.M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 4, 2138–2148 (1995).

    Article  CAS  Google Scholar 

  29. Chothia, C. et al. Structural repertoire of the human VH segments. J. Mol. Biol. 227, 799–817 (1992).

    Article  CAS  Google Scholar 

Download references


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.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Greg Winter.

Ethics declarations

Competing interests

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

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing