Skip to main content
SpringerLink
Log in

Oligovalent Fab Display on M13 Phage Improved by Directed Evolution

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Efficient display of antibody on filamentous phage M13 coat is crucial for successful biopanning selections. We applied a directed evolution strategy to improve the oligovalent display of a poorly behaving Fab fragment fused to phage gene-3 for minor coat protein (g3p). The Fab displaying clones were enriched from a randomly mutated Fab gene library with polyclonal anti-mouse IgG antibodies. Contribution of each mutation to the improved phenotype of one selected mutant was studied. It was found out that two point mutations had significant contribution to the display efficiency of Fab clones superinfected with hyperphage. The most dramatic effect was connected to a start codon mutation, from AUG to GUG, of the PelB signal sequence preceding the heavy chain. The clone carrying this mutation, FabMGUG, displayed Fab 19-fold better and yielded twofold higher phage titers than the original Fab.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  1. Bradbury, A., & Marks, J. (2004). Antibodies from phage antibody libraries. Journal of Immunological Methods, 290, 29–49.

    Article  CAS  Google Scholar 

  2. Malone, J., & Sullivan, M. (1996). Analysis of antibody selection by phage display utilizing anti-phenobarbital antibodies. Journal of Molecular Recognition, 9, 738–745.

    Article  CAS  Google Scholar 

  3. Röthlisberger, D., Honegger, A., & Plückthun, A. (2005). Domain interactions in the Fab fragment: A comparative evaluation of the single-chain Fv and Fab format engineered with variable domains of different stability. Journal of Molecular Biology, 347, 773–789.

    Article  Google Scholar 

  4. Kortt, A., Lah, M., Oddie, G., Gruen, C., Burns, J., Pearce, L., et al. (1997). Single-chain Fv fragments of anti-neuraminidase antibody NC10 containing five- and ten-residue linkers form dimers and with zero-residue linker a trimer. Protein Engineering, 10, 423–433.

    Article  CAS  Google Scholar 

  5. Hust, M., & Dübel, S. (2004). Mating antibody phage display with proteomics. Trends in Biotechnology, 22, 8–14.

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  7. Kirsch, M., Zaman, M., Meier, D., Dübel, S., & Hust, M. (2005). Parameters affecting the display of antibodies on phage. Journal of Immunological Methods, 301, 173–185.

    Article  CAS  Google Scholar 

  8. Lee, C., Sidhu, S., & Fuh, G. (2004). Bivalent antibody phage display mimics natural immunoglobulin. Journal of Immunological Methods, 284, 119–132.

    Article  CAS  Google Scholar 

  9. Hust, M., Jostock, T., Menzel, C., Voedisch, B., Mohr, A., Brenneis, M., et al. (2007). Single chain Fab (scFab) fragment. BMC Biotechnology, 7, 14.

    Article  Google Scholar 

  10. Rondot, S., Koch, J., Breitling, F., & Dübel, S. (2001). A helper phage to improve single-chain antibody presentation in phage display. Nature Biotechnology, 19, 75–78.

    Article  CAS  Google Scholar 

  11. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning a laboratory manual (2nd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.

    Google Scholar 

  12. Korpimäki, T., Brockmann, E., Kuronen, O., Saraste, M., Lamminmäki, U., & Tuomola, M. (2004). Engineering of a broad specificity antibody for simultaneous detection of 13 sulfonamides at the maximum residue level. Journal of Agriculture and Food Chemistry, 52, 40–47.

    Article  Google Scholar 

  13. Deyev, S., Waibel, R., Lebedenko, E., Schubiger, A., & Plückthun, A. (2003). Design of multivalent complexes using the barnase*barstar module. Nature Biotechnology, 21, 1486–1492.

    Article  CAS  Google Scholar 

  14. Jung, S., & Plückthun, A. (1997). Improving in vivo folding and stability of a single-chain Fv antibody fragment by loop grafting. Protein Engineering, 10, 959–966.

    Article  CAS  Google Scholar 

  15. Lefranc, M., Pommié, C., Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., et al. (2003). IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Developmental and Comparative Immunology, 27, 55–77.

    Article  CAS  Google Scholar 

  16. Lefranc, M., Pommié, C., Kaas, Q., Duprat, E., Bosc, N., Guiraudou, D., et al. (2005). IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains. Developmental and Comparative Immunology, 29, 185–203.

    Article  CAS  Google Scholar 

  17. Miller, J., & Albertini, A. (1983). Effects of surrounding sequence on the suppression of nonsense codons. Journal of Molecular Biology, 164, 59–71.

    Article  CAS  Google Scholar 

  18. Løset, G., Kristinsson, S., & Sandlie, I. (2008). Reliable titration of filamentous bacteriophages independent of pIII fusion moiety and genome size by using trypsin to restore wild-type pIII phenotype. Biotechniques 44, 551–552, 554.

    Google Scholar 

  19. Goldsmith, M., & Konigsberg, W. (1977). Adsorption protein of the bacteriophage fd: Isolation, molecular properties, and location in the virus. Biochemistry, 16, 2686–2694.

    Article  CAS  Google Scholar 

  20. Knappik, A., & Plückthun, A. (1995). Engineered turns of a recombinant antibody improve its in vivo folding. Protein Engineering, 8, 81–89.

    Article  CAS  Google Scholar 

  21. Kay, B. K. (1996). Phage display of peptides and proteins a laboratory manual. San Diego, CA: Academic Press.

  22. Ewert, S., Huber, T., Honegger, A., & Plückthun, A. (2003). Biophysical properties of human antibody variable domains. Journal of Molecular Biology, 325, 531–553.

    Article  CAS  Google Scholar 

  23. O’Donnell, S., & Janssen, G. (2001). The initiation codon affects ribosome binding and translational efficiency in Escherichia coli of cI mRNA with or without the 5′ untranslated leader. Journal of Bacteriology, 183, 1277–1283.

    Article  Google Scholar 

  24. Clark, B., & Marcker, K. (1966). The role of N-formyl-methionyl-sRNA in protein biosynthesis. Journal of Molecular Biology, 17, 394–406.

    Article  CAS  Google Scholar 

  25. Jestin, J., Volioti, G., & Winter, G. (2001). Improving the display of proteins on filamentous phage. Research in Microbiology, 152, 187–191.

    Article  CAS  Google Scholar 

  26. Lin, B., Renshaw, M., Autote, K., Smith, L., Calveley, P., Bowdish, K., et al. (2008). A step-wise approach significantly enhances protein yield of a rationally-designed agonist antibody fragment in E. coli. Protein Expression and Purification, 59, 55–63.

    Article  CAS  Google Scholar 

Download references

Acknowledgment

This study was supported by the Finnish Funding Agency for Technology and Innovation (Tekes).

Author information

Authors and Affiliations

  1. Department of Biochemistry and Food Chemistry, University of Turku, 20520, Turku, Finland

    Tuomas Huovinen, Hanna Sanmark, Jani Ylä-Pelto, Markus Vehniäinen & Urpo Lamminmäki

Authors
  1. Tuomas Huovinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Hanna Sanmark
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jani Ylä-Pelto
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Markus Vehniäinen
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Urpo Lamminmäki
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Urpo Lamminmäki.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Huovinen, T., Sanmark, H., Ylä-Pelto, J. et al. Oligovalent Fab Display on M13 Phage Improved by Directed Evolution. Mol Biotechnol 44, 221–231 (2010). https://doi.org/10.1007/s12033-009-9231-3

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-009-9231-3

Keywords

Search

Navigation