Skip to main content

Thank you for visiting nature.com. 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.

  • Technical Report
  • Published:

A helper phage to improve single-chain antibody presentation in phage display

Nature Biotechnology volume 19, pages 75–78 (2001)Cite this article

Abstract

We show here that the number of single-chain antibody fragments (scFv) presented on filamentous phage particles generated with antibody display phagemids can be increased by more than two orders of magnitude by using a newly developed helper phage (hyperphage). Hyperphage have a wild-type pIII phenotype and are therefore able to infect F+ Escherichia coli cells with high efficiency; however, their lack of a functional pIII gene means that the phagemid-encoded pIII–antibody fusion is the sole source of pIII in phage assembly. This results in an considerable increase in the fraction of phage particles carrying an antibody fragment on their surface. Antigen-binding activity was increased about 400-fold by enforced oligovalent antibody display on every phage particle. When used for packaging a universal human scFv library, hyperphage improved the specific enrichment factor obtained when panning on tetanus toxin. After two panning rounds, more than 50% of the phage were found to bind to the antigen, compared to 3% when conventional M13KO7 helper phage was used. Thus, hyperphage is particularly useful in stoichiometric situations, when there is little chance that a single phage will locate the desired antigen.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: The concept of hyperphage: a gene pIII-deleted helper phage with wild-type infection phenotype.
Figure 2: Hyperphage increases presentation of scFv antibody fragments on filamentous phage.
Figure 3: Hyperphage increases panning efficiency from a universal library.

Similar content being viewed by others

References

  1. Hoogenboom, H.R. et al. Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains. Nucleic Acids Res. 19, 4133–4137 (1991).

    Article  CAS  Google Scholar 

  2. Breitling, F. et al. A surface expression vector antibody screening. Gene 104, 147–153 (1991).

    Article  CAS  Google Scholar 

  3. Marks, J.D. et al. By-passing immunization. Human antibodies from V-gene libraries displayed on phage. J. Mol. Biol. 222, 581–597 (1991).

    Article  CAS  Google Scholar 

  4. Barbas, C.F. et al. Assembly of combinatorial antibody libraries on phage surfaces: the gene III site. Proc. Natl. Acad. Sci. USA 88, 7978–7982 (1991).

    Article  CAS  Google Scholar 

  5. Gavilondo, J.V. & Larrick, J.W. Antibody engineering at the millennium. BioTechniques 29, 128–145 (2000).

    Article  CAS  Google Scholar 

  6. Fisch, I. et al. A strategy of exon shuffling for making large peptide repertoires displayed on filamentous bacteriophage. Proc. Natl. Acad. Sci. USA 93, 7761–7766 (1996).

    Article  CAS  Google Scholar 

  7. Knappik, A. et al. Fully synthetic human combinatorial antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides. J. Mol. Biol. 296, 57–86 (2000).

    Article  CAS  Google Scholar 

  8. Sblattero, D. & Bradburry, A. Exploiting recombination in single bacteria to make large phage antibody libraries. Nat. Biotechnol. 18, 75–80 (2000).

    Article  CAS  Google Scholar 

  9. Griffiths, A.D. et al. Human anti-self antibodies with high specificity from phage display libraries. EMBO J. 12, 725–734 (1993)

    Article  CAS  Google Scholar 

  10. Duenas, M. & Borrebaeck, C.A. Novel helper phage design: intergenic region affects the assembly of bacteriophages and the size of antibody libraries. FEMS Microbiol Lett. 125, 317–321 (1995)

    Article  CAS  Google Scholar 

  11. Rakonjac, J., Jovanovic, G. & Model, P. Filamentous phage infection-mediated gene expression: construction and propagation of the gIII deletion mutant helper phage R408d3. Gene 198, 99–103 (1997)

    Article  CAS  Google Scholar 

  12. Nelson, F.K., Friedman, S.M. & Smith, G.P. Filamentous phage DNA cloning vectors: a noninfective mutant with a nonpolar deletion in gene III. Virology 108, 338–350 (1981)

    Article  CAS  Google Scholar 

  13. Crissman, J.W. & Smith, G.P. Gene-III protein of filamentous phages: evidence for a carboxyl-terminal domain with a role in morphogenesis. Virology 132, 445–455 (1984)

    Article  CAS  Google Scholar 

  14. Dotto, G.P. & Zinder, N.D. Reduction of the minimal sequence for initiation of DNA synthesis by qualitative or quantitative changes of an initiator protein. Nature 311, 279–280 (1984).

    Article  CAS  Google Scholar 

  15. Davis, N.G., Boeke, J.D. & Model, P. Fine structure of a membrane anchor domain. J. Mol. Biol. 181, 111–121 (1985).

    Article  CAS  Google Scholar 

  16. Rondot, S. Präsentation von Peptidepitopen auf rekombinanten F-Pili für eine antigenspezifische Infektion von Bakterien (Inaugural-Dissertation). (University of Heidelberg, Germany; 1997).

  17. Dübel, S. et al. A family of vectors for surface display and production of antibodies. Gene 128, 97–101 (1993).

    Article  Google Scholar 

  18. Diederich, L., Rasmussen, L.J. & Messer, W. New cloning vectors for integration into the λ attachment site attB of the Escherichia coli chromosome. Plasmid 28, 14–24 (1992)

    Article  CAS  Google Scholar 

  19. Welschof, M. et al. The antigen-binding domain of a human IgG-anti-F(ab′)2 autoantibody. Proc. Natl. Acad. Sci. USA 94, 1902–1907 (1997).

    Article  CAS  Google Scholar 

  20. Micheel, B. et al. Production of monoclonal antibodies against epitopes of the main coat protein of filamentous fd phages. J. Immunol. Methods 171, 103–109 (1994).

    Article  CAS  Google Scholar 

  21. Koch, J. & Dübel., S. Generation of antibody libraries from human donors. In Antibody engineering, (eds Kontermann, R. & Dübel, S.) (Springer Verlag, Heidelberg/New York, in press).

  22. Yuan Q. et al. Molecular cloning, expression, and characterization of a functional single-chain Fv antibody to the mycotoxin zearalenone. Appl. Environ. Microbiol. 63, 263–269 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. Schier, R. et al. Isolation of high-affinity monomeric human anti-c-erbB-2 single chain Fv using affinity-driven selection. J. Mol. Biol. 255, 28–43 (1996).

    Article  CAS  Google Scholar 

  24. Smith, G.P. Filamentous phages as cloning vectors. Biotechnology 10, 61–83 (1988).

    CAS  PubMed  Google Scholar 

  25. Kramer, A. & Schneider-Mergener, J. Synthesis of peptide libraries on continuous cellulose membranes. Methods Mol. Biol. 87, 25–39 (1997).

    Google Scholar 

  26. Sambrook, J., Fritsch, E.F. & Maniatis, T. Molecular cloning. A laboratory manual, Edn. 2. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; 1989).

    Google Scholar 

Download references

Acknowledgements

The financial support from the Heidelberger Akademie der Wissenschaften (Germany) is gratefully acknowledged. We wish to thank M. Little for support, M. Bechtel from Genzyme Virotech GmbH for supplying the tetanus toxin, and Olaf Broders for helping with the experimental work. We are grateful for E.K.F. Bautz for his continuous support and suggestions.

Author information

Author notes

  1. Susanne Rondot and Joachim Koch: The first two authors contributed equally to this work.

Authors and Affiliations

  1. Molekulargenetisches Labor Prof. Raue, Friedrich-Ebert-Anlage 28, Heidelberg, 69117, Germany

    Susanne Rondot

  2. Forschungsstelle Hantaviren der Heidelberger Akademie der Wissenschaften, Im Neuenheimer Feld 230, Heidelberg, 69120, Germany

    Joachim Koch

  3. Abt. Genomanalyse, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, Heidelberg, 69120, Germany

    Frank Breitling

  4. Molekulare Genetik, Universität Heidelberg, Im Neuenheimer Feld 230, Heidelberg, 69120, Germany

    Stefan Dübel

Authors
  1. Susanne Rondot
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Joachim Koch
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Frank Breitling
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Stefan Dübel
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan Dübel.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rondot, S., Koch, J., Breitling, F. et al. A helper phage to improve single-chain antibody presentation in phage display. Nat Biotechnol 19, 75–78 (2001). https://doi.org/10.1038/83567

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1038/83567

This article is cited by

Search

Advanced search

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