Skip to main content
SpringerLink
Log in

Efficient display of two enzymes on filamentous phage using an improved signal sequence

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

Abstract

Directed protein-evolution strategies generally make use of a link between a protein and the encoding DNA. In phage-display technology, this link is provided by fusion of the protein with a coat protein that is incorporated into the phage particle containing the DNA. Optimization of this link can be achieved by adjusting the signal sequence of the fusion. In a previous study, directed evolution of signal sequences for optimal display of the Taq DNA polymerase I Stoffel fragment on phage yielded signal peptides with a 50-fold higher incorporation of fusion proteins in phage particles. In this article, we show that for one of the selected signal sequences, improved display on phage can be generalized to other proteins, such as adenylate cyclases from Escherichia coli and Bordetella pertussis, and that this is highly dependent on short sequences at the C-terminus of the signal peptide. Further, the display of two enzymes on phage has been achieved and may provide a strategy for directing coevolution of the two proteins. These findings should be useful for display of large and cytoplasmic proteins on filamentous phage.

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

Similar content being viewed by others

References

  1. Bothmann, H. and Plückthun, A. (1998) Selection for a periplasmic factor improving phage display and functional periplasmic expression. Nat. Biotech. 16, 376–380.

    Article  CAS  Google Scholar 

  2. Jestin, J.L., Volioti, G. and Winter, G. (2001) Improving the display of proteins on filamentous phage. Res. Microbiol. 152, 187–191.

    Article  PubMed  CAS  Google Scholar 

  3. Holland, M.M., Leib, T.K., and Gerlt, J.A. (1988) Isolation and characterization of a small catalytic domain released from the adenylate cyclase from Escherichia coli by digestion with trypsin. J. Biol. Chem. 263, 14661–14668.

    PubMed  CAS  Google Scholar 

  4. Ladant, D., Glaser, P., and Ullmann, A. (1992) Insertional mutagenesis of Bordetella pertussis adenylate cyclase. J. Biol. Chem. 267, 2244–2250.

    PubMed  CAS  Google Scholar 

  5. Hoogenboom, H.R., Griffiths, A.D., Johnson, K.S., et al. (1991) Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody Fab heavy and light chains. Nucl. Acids Res. 19, 4133–4137.

    Article  PubMed  CAS  Google Scholar 

  6. Kristensen, P. and Winter, G. (1998) Proteolytic selection for protein folding using filamentous bacteriophages. Fold. Design 3, 321–328.

    Article  CAS  Google Scholar 

  7. Lei, S.P., Lin, H.C., Wang, S.S., Callaway, J., et al. (1987) Characterization of the Erwinia carotovora pelB gene and its product pectate lyase. J. Bacteriol. 169, 4379–4383.

    PubMed  CAS  Google Scholar 

  8. Tesar, M., Beckmann, C., Rottgen, P., et al. (1995) Monoclonal antibody against pIII of filamentous phage: an immunological tool to study pIII fusion protein expression in phage display systems. Immunotechnology 1, 53–64.

    Article  PubMed  CAS  Google Scholar 

  9. Pedersen, H., Hölder, S., Sutherlin, D.P., et al. (1998) A method for directed evolution and functional cloning of enzymes. Proc. Natl. Acad. Sci. USA 95, 10523–10528.

    Article  PubMed  CAS  Google Scholar 

  10. Jestin, J.L., Kristensen, P., and Winter, G. (1999) A method for the selection of catalytic activity using phage display and proximity coupling. Angew. Chem. Int. Ed. 38, 1124–1127.

    Article  CAS  Google Scholar 

  11. Dematris, S., Huber, A., et al. (1999) A strategy for the isolation of catalytic activities from repertoires of enzymes displayed on phage. J. Mol. Biol. 286, 617–633.

    Article  Google Scholar 

  12. Heinis, C., Huber, A. et al. (2001) Selection of catalytically active biotin ligase and trypsin mutants by phage display. Protein Eng. 14, 1043–1052.

    Article  PubMed  CAS  Google Scholar 

  13. Atwell, S. and Wells, J. A. (1999) Selection for improved subtiligases by phage display. Proc. Natl. Acad. Sci. USA 96, 9497–9502.

    Article  PubMed  CAS  Google Scholar 

  14. Ponsard, I., Galleni, M., Soumillion, P., Fastrez, J., Selection of metalloenzymes by catalytic activity using phage display and catalytic elution. Chembiochem. 2, 253–259.

  15. Lawyer, F.C., Stoffel, S., Saiki, R.K., et al. (1989) Isolation, characterisation and expression in E. coli of the DNA polymerase gene from Thermus aquaticus. J. Biol. Chem. 264, 6427–6437.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Unité de Chimie Organique, CNRS URA 2185, Département de Biologie Structurale et Chimie, Institut Pasteur, 28 rue du Dr. Roux, 75724, Paris, France

    Jean-Luc Jestin

Authors
  1. Heike Strobel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Daniel Ladant
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jean-Luc Jestin
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jean-Luc Jestin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Strobel, H., Ladant, D. & Jestin, JL. Efficient display of two enzymes on filamentous phage using an improved signal sequence. Mol Biotechnol 24, 1–9 (2003). https://doi.org/10.1385/MB:24:1:1

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1385/MB:24:1:1

Index Entries

Search

Navigation