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Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display

Nature Biotechnology volume 24, pages 823–831 (2006)Cite this article

Abstract

Even proteins that fold well in bacteria are frequently displayed poorly on filamentous phages. Low protein presentation on phage might be caused by premature cytoplasmic folding, leading to inefficient translocation into the periplasm. As translocation is an intermediate step in phage assembly, we tested the display levels of a range of proteins using different translocation pathways by employing different signal sequences. Directing proteins to the cotranslational signal recognition particle (SRP) translocation pathway resulted in much higher display levels than directing them to the conventional post-translational Sec translocation pathway. For example, the display levels of designed ankyrin-repeat proteins (DARPins) were improved up to 700-fold by simply exchanging Sec- for SRP-dependent signal sequences. In model experiments this exchange of signal sequences improved phage display from tenfold enrichment to >1,000-fold enrichment per phage display selection round. We named this method 'SRP phage display' and envision broad applicability, especially when displaying cDNA libraries or very stable and fast-folding proteins from libraries of alternative scaffolds.

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Figure 1: Scheme of the expression cassette of the pDST phagemid vector series.
Figure 2: Display yields of the various POIs on phage particles.
Figure 3: Increased display yields using SRP-dependent signal sequences.
Figure 4: Protein translation and translocation of the various unfused POIs to the periplasm.
Figure 5: Display yields and periplasmic translocation of DARPin E3_5 and TrxA using various Sec- and SRP-dependent signal sequences.

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References

  1. Smith, G.P. & Petrenko, V.A. Phage Display. Chem. Rev. 97, 391–410 (1997).

    Article  CAS  PubMed  Google Scholar 

  2. Russel, M., Lowman, H.B. & Clackson, T. Introduction to phage biology and phage display. in Phage Display: A Practical Approach (eds. Lowman, H.B. & Clackson, T.) 1–26, (Oxford University Press, New York, USA, 2004).

    Google Scholar 

  3. Sidhu, S.S., Lowman, H.B., Cunningham, B.C. & Wells, J.A. Phage display for selection of novel binding peptides. Methods Enzymol. 328, 333–363 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Bradbury, A.R. & Marks, J.D. Antibodies from phage antibody libraries. J. Immunol. Methods 290, 29–49 (2004).

    Article  CAS  PubMed  Google Scholar 

  5. Rakonjac, J., Feng, J. & Model, P. Filamentous phage are released from the bacterial membrane by a two-step mechanism involving a short C-terminal fragment of pIII. J. Mol. Biol. 289, 1253–1265 (1999).

    Article  CAS  PubMed  Google Scholar 

  6. Wilson, D.R. & Finlay, B.B. Phage display: applications, innovations, and issues in phage and host biology. Can. J. Microbiol. 44, 313–329 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Deng, S.J. et al. Selection of antibody single-chain variable fragments with improved carbohydrate binding by phage display. J. Biol. Chem. 269, 9533–9538 (1994).

    CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  9. Krebber, A., Burmester, J. & Plückthun, A. Inclusion of an upstream transcriptional terminator in phage display vectors abolishes background expression of toxic fusions with coat protein g3p. Gene 178, 71–74 (1996).

    Article  CAS  PubMed  Google Scholar 

  10. Brinkmann, U., Chowdhury, P.S., Roscoe, D.M. & Pastan, I. Phage display of disulfide-stabilized Fv fragments. J. Immunol. Methods 182, 41–50 (1995).

    Article  CAS  PubMed  Google Scholar 

  11. Rodi, D.J., Soares, A.S. & Makowski, L. Quantitative assessment of peptide sequence diversity in M13 combinatorial peptide phage display libraries. J. Mol. Biol. 322, 1039–1052 (2002).

    Article  CAS  PubMed  Google Scholar 

  12. Krebber, A. et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing a reengineered phage display system. J. Immunol. Methods 201, 35–55 (1997).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  14. Bothmann, H. & Plückthun, A. The periplasmic Escherichia coli peptidylprolyl cis,trans-isomerase FkpA. I. Increased functional expression of antibody fragments with and without cis-prolines. J. Biol. Chem. 275, 17100–17105 (2000).

    Article  CAS  PubMed  Google Scholar 

  15. Kramer, R.A. et al. A novel helper phage that improves phage display selection efficiency by preventing the amplification of phages without recombinant protein. Nucleic Acids Res. 31, e59 (2003).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Baek, H., Suk, K.H., Kim, Y.H. & Cha, S. An improved helper phage system for efficient isolation of specific antibody molecules in phage display. Nucleic Acids Res. 30, e18 (2002).

    Article  PubMed  PubMed Central  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  19. Forrer, P., Stumpp, M.T., Binz, H.K. & Plückthun, A. A novel strategy to design binding molecules harnessing the modular nature of repeat proteins. FEBS Lett. 539, 2–6 (2003).

    Article  CAS  PubMed  Google Scholar 

  20. Binz, H.K., Stumpp, M.T., Forrer, P., Amstutz, P. & Plückthun, A. Designing repeat proteins: well-expressed, soluble and stable proteins from combinatorial libraries of consensus ankyrin repeat proteins. J. Mol. Biol. 332, 489–503 (2003).

    Article  CAS  PubMed  Google Scholar 

  21. Binz, H.K. et al. High-affinity binders selected from designed ankyrin repeat protein libraries. Nat. Biotechnol. 22, 575–582 (2004).

    Article  CAS  PubMed  Google Scholar 

  22. Amstutz, P. et al. Intracellular kinase inhibitors selected from combinatorial libraries of designed ankyrin repeat proteins. J. Biol. Chem. 280, 24715–24722 (2005).

    Article  CAS  PubMed  Google Scholar 

  23. Fekkes, P. & Driessen, A.J. Protein targeting to the bacterial cytoplasmic membrane. Microbiol. Mol. Biol. Rev. 63, 161–173 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Koch, H.G., Moser, M. & Müller, M. Signal recognition particle-dependent protein targeting, universal to all kingdoms of life. Rev. Physiol. Biochem. Pharmacol. 146, 55–94 (2003).

    Article  CAS  PubMed  Google Scholar 

  25. Valent, Q.A. Signal recognition particle mediated protein targeting in Escherichia coli . Antonie Van Leeuwenhoek 79, 17–31 (2001).

    Article  CAS  PubMed  Google Scholar 

  26. Luirink, J. & Sinning, I. SRP-mediated protein targeting: structure and function revisited. Biochim. Biophys. Acta 1694, 17–35 (2004).

    CAS  PubMed  Google Scholar 

  27. Fisher, A.C. & DeLisa, M.P. A little help from my friends: quality control of presecretory proteins in bacteria. J. Bacteriol. 186, 7467–7473 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Robinson, C. & Bolhuis, A. Tat-dependent protein targeting in prokaryotes and chloroplasts. Biochim. Biophys. Acta 1694, 135–147 (2004).

    Article  CAS  PubMed  Google Scholar 

  29. Rapoza, M.P. & Webster, R.E. The filamentous bacteriophage assembly proteins require the bacterial SecA protein for correct localization to the membrane. J. Bacteriol. 175, 1856–1859 (1993).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Plückthun, A. et al. in Antibody Engineering, edn. 1 (eds. McCafferty, J., Hoogenboom, H.R. & Chiswell, D.J.) 203–252, (IRL Press, Oxford, 1996).

    Google Scholar 

  31. Debarbieux, L. & Beckwith, J. The reductive enzyme thioredoxin 1 acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc. Natl. Acad. Sci. USA 95, 10751–10756 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Jonda, S., Huber-Wunderlich, M., Glockshuber, R. & Mössner, E. Complementation of DsbA deficiency with secreted thioredoxin variants reveals the crucial role of an efficient dithiol oxidant for catalyzed protein folding in the bacterial periplasm. EMBO J. 18, 3271–3281 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Schierle, C.F. et al. The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the Escherichia coli periplasm via the signal recognition particle pathway. J. Bacteriol. 185, 5706–5713 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yang, F. et al. Novel fold and capsid-binding properties of the lambda-phage display platform protein gpD. Nat. Struct. Biol. 7, 230–237 (2000).

    Article  CAS  PubMed  Google Scholar 

  35. Huber, D. et al. Use of thioredoxin as a reporter to identify a subset of Escherichia coli signal sequences that promote signal recognition particle-dependent translocation. J. Bacteriol. 187, 2983–2991 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Slootstra, J.W., Kuperus, D., Plückthun, A. & Meloen, R.H. Identification of new tag sequences with differential and selective recognition properties for the anti-FLAG monoclonal antibodies M1, M2 and M5. Mol. Divers. 2, 156–164 (1997).

    Article  CAS  PubMed  Google Scholar 

  37. Georgescu, R.E., Li, J.H., Goldberg, M.E., Tasayco, M.L. & Chaffotte, A.F. Proline isomerization-independent accumulation of an early intermediate and heterogeneity of the folding pathways of a mixed alpha/beta protein, Escherichia coli thioredoxin. Biochemistry 37, 10286–10297 (1998).

    Article  CAS  PubMed  Google Scholar 

  38. Huber, D. et al. A selection for mutants that interfere with folding of Escherichia coli thioredoxin-1 in vivo. Proc. Natl. Acad. Sci. USA 102, 18872–18877 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Forrer, P., Chang, C., Ott, D., Wlodawer, A. & Plückthun, A. Kinetic stability and crystal structure of the viral capsid protein SHP. J. Mol. Biol. 344, 179–193 (2004).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  41. Jäger, M., Gehrig, P. & Plückthun, A. The scFv fragment of the antibody hu4D5–8: evidence for early premature domain interaction in refolding. J. Mol. Biol. 305, 1111–1129 (2001).

    Article  PubMed  Google Scholar 

  42. Paschke, M. & Höhne, W. A twin-arginine translocation (Tat)-mediated phage display system. Gene 350, 79–88 (2005).

    Article  CAS  PubMed  Google Scholar 

  43. DeLisa, M.P., Tullman, D. & Georgiou, G. Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway. Proc. Natl. Acad. Sci. USA 100, 6115–6120 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Crameri, R., Hemmann, S. & Blaser, K. PJuFo: a phagemid for display of cDNA libraries on phage surface suitable for selective isolation of clones expressing allergens. Adv. Exp. Med. Biol. 409, 103–110 (1996).

    Article  CAS  PubMed  Google Scholar 

  45. Löhning, C., Urban, M. & Knappik, A. Novel methods for displaying (poly) peptides/proteins on bacteriophage particles via disulfide bonds Patent WO0105950 (2001).

  46. Sambrook, J. & Russell David, W. (eds.). Molecular Cloning: A Laboratory Manual, edn. 3. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

    Google Scholar 

  47. Clackson, T. & Lowman, H.B. . Phage Display: A Practical Approach (Oxford University Press, New York, 2004).

    Google Scholar 

  48. Barbas, C.F., III, Burton, D.R., Scott, J.K. & Silvermann, G.J. . Phage Display: A Laboratory Manual. (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY 2001).

    Google Scholar 

  49. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng. 10, 1–6 (1997).

    Article  CAS  PubMed  Google Scholar 

  50. Gailus, V. & Rasched, I. The adsorption protein of bacteriophage fd and its neighbour minor coat protein build a structural entity. Eur. J. Biochem. 222, 927–931 (1994).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank Patrick Amstutz and H. Kaspar Binz for valuable discussions and J. Beckwith and D. Huber for advice on experimental procedures. This work was supported by the Swiss National Center of Competence in Research (NCCR) in Structural Biology and by KTI Discovery.

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Authors and Affiliations

  1. Department of Biochemistry, University of Zürich, Winterthurerstrasse 190, Zürich, 8057, Switzerland

    Daniel Steiner, Patrik Forrer, Michael T Stumpp & Andreas Plückthun

  2. c/o Department of Biochemistry, Molecular Partners AG, University of Zürich, Winterthurerstrasse 190, 8057, Zürich, Switzerland

    Patrik Forrer & Michael T Stumpp

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Contributions

A.P. conceived the project; D.S., P.F., M.T.S. and A.P. designed experiments; D.S. performed the experiments; D.S., P.F., M.T.S. and A.P. analyzed the data; D.S., P.F., M.T.S. and A.P. wrote the manuscript.

Note: Supplementary information is available on the Nature Biotechnology website.

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Correspondence to Andreas Plückthun.

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The technology in this paper has been patented by D.S., M.T.S., P.T. and A.P.

Supplementary information

Supplementary Figure 1

Schematic representation of the phagemid pDST22. (PDF 306 kb)

Supplementary Table 1

E. coli strains. (PDF 109 kb)

Supplementary Table 2

Signal sequences. (PDF 84 kb)

Supplementary Methods

Phagemid cloning. (PDF 111 kb)

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Steiner, D., Forrer, P., Stumpp, M. et al. Signal sequences directing cotranslational translocation expand the range of proteins amenable to phage display. Nat Biotechnol 24, 823–831 (2006). https://doi.org/10.1038/nbt1218

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