Skip to main content
SpringerLink
Log in

Surface display of a single-domain antibody library on Gram-positive bacteria

  • Research article
  • Published:
Cellular and Molecular Life Sciences Aims and scope Submit manuscript

Abstract

Combinatorial protein engineering for selection of proteins with novel functions, such as enzymes and affinity reagents, is an important tool in biotechnology, drug discovery, and other biochemical fields. Bacterial display is an emerging technology for isolation of new affinity proteins from such combinatorial libraries. Cells have certain properties that are attractive for directed evolution purposes, in particular the option to use quantitative flow-cytometric cell sorting for selection of binders. Here, an immune library of around 107 camelid single-domain antibody fragments (Nanobodies) was displayed on both the Gram-positive bacterium Staphylococcus carnosus and on phage. As demonstrated for the first time, the antibody repertoire was found to be well expressed on the bacterial surface and flow-cytometric sorting yielded a number of Nanobodies with subnanomolar affinity for the target protein, green fluorescent protein (GFP). Interestingly, the staphylococcal output repertoire and the binders from the phage display selection contained two slightly different sets of clones, containing both unique as well as several similar variants. All of the Nanobodies from the staphylococcal selection were also shown to enhance the fluorescence of GFP upon binding, potentially due to the fluorescence-based sorting principle. Our study highlights the impact of the chosen display technology on the variety of selected binders and thus the value of having alternative methods available, and demonstrates in addition that the staphylococcal system is suitable for generation of high-affinity antibody fragments.

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

Similar content being viewed by others

References

  1. Bradbury ARM, Marks JD (2004) Antibodies from phage antibody libraries. J Immunol Methods 290(1–2):29–49. doi:10.1016/j.jim.2004.04.007

    Article  PubMed  CAS  Google Scholar 

  2. Lofblom J, Frejd FY, Stahl S (2011) Non-immunoglobulin based protein scaffolds. Curr Opin Biotechnol 22(6):843–848. doi:10.1016/j.copbio.2011.06.002

    Article  PubMed  Google Scholar 

  3. Lofblom J (2011) Bacterial display in combinatorial protein engineering. Biotechnol J 6(9):1115–1129. doi:10.1002/biot.201100129

    Article  PubMed  Google Scholar 

  4. Gai SA, Wittrup KD (2007) Yeast surface display for protein engineering and characterization. Curr Opin Struct Biol 17(4):467–473. doi:10.1016/J.Sbi.2007.08.012

    Article  PubMed  CAS  Google Scholar 

  5. Lipovsek D, Pluckthun A (2004) In vitro protein evolution by ribosome display and mRNA display. J Immunol Methods 290(1–2):51–67. doi:10.1016/j.jim.2004.04.008

    Article  PubMed  CAS  Google Scholar 

  6. Boder ET, Wittrup KD (1997) Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol 15(6):553–557

    Article  PubMed  CAS  Google Scholar 

  7. Daugherty PS, Chen G, Olsen MJ, Iverson BL, Georgiou G (1998) Antibody affinity maturation using bacterial surface display. Protein Eng 11(9):825–832. doi:10.1093/protein/11.9.825

    Article  PubMed  CAS  Google Scholar 

  8. VanAntwerp JJ, Wittrup KD (2000) Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnol Prog 16(1):31–37. doi:10.1021/bp990133s

    Article  PubMed  CAS  Google Scholar 

  9. Lofblom J, Wernerus H, Stahl S (2005) Fine affinity discrimination by normalized fluorescence activated cell sorting in staphylococcal surface display. FEMS Microbiol Lett 248(2):189–198. doi:10.1016/j.femsle.2005.05.040

    Article  PubMed  Google Scholar 

  10. Nilvebrant J, Alm T, Hober S, Lofblom J (2011) Engineering bispecificity into a single albumin-binding domain. PLoS ONE 6(10):e25791. doi:10.1371/journal.pone.0025791

    Article  PubMed  CAS  Google Scholar 

  11. Garcia-Rodriguez C, Levy R, Arndt JW, Forsyth CM, Razai A, Lou J, Geren I, Stevens RC, Marks JD (2007) Molecular evolution of antibody cross-reactivity for two subtypes of type A botulinum neurotoxin. Nat Biotechnol 25(1):107–116. doi:10.1038/nbt1269

    Article  PubMed  CAS  Google Scholar 

  12. Ho M, Nagata S, Pastan I (2006) Isolation of anti-CD22 Fv with high affinity by Fv display on human cells. P Natl Acad Sci USA 103(25):9637–9642. doi:10.1073/pnas.0603653103

    Article  CAS  Google Scholar 

  13. Beerli RR, Bauer M, Buser RB, Gwerder M, Muntwiler S, Maurer P, Saudan P, Bachmann MF (2008) Isolation of human monoclonal antibodies by mammalian cell display. Proc Natl Acad Sci USA 105(38):14336–14341. doi:10.1073/pnas.0805942105

    Article  PubMed  CAS  Google Scholar 

  14. Bowers PM, Horlick RA, Neben TY, Toobian RM, Tomlinson GL, Dalton JL, Jones HA, Chen A, Altobell L 3rd, Zhang X, Macomber JL, Krapf IP, Wu BF, McConnell A, Chau B, Holland T, Berkebile AD, Neben SS, Boyle WJ, King DJ (2011) Coupling mammalian cell surface display with somatic hypermutation for the discovery and maturation of human antibodies. Proc Natl Acad Sci USA 108(51):20455–20460. doi:10.1073/pnas.1114010108

    Article  PubMed  CAS  Google Scholar 

  15. Daugherty PS (2007) Protein engineering with bacterial display. Curr Opin Struct Biol 17(4):474–480. doi:10.1016/j.sbi.2007.07.004

    Article  PubMed  CAS  Google Scholar 

  16. Harvey BR, Georgiou G, Hayhurst A, Jeong KJ, Iverson BL, Rogers GK (2004) Anchored periplasmic expression, a versatile technology for the isolation of high-affinity antibodies from Escherichia coli-expressed libraries. Proc Natl Acad Sci USA 101(25):9193–9198. doi:10.1073/Pnas.0400187101

    Article  PubMed  CAS  Google Scholar 

  17. Kronqvist N, Lofblom J, Jonsson A, Wernerus H, Stahl S (2008) A novel affinity protein selection system based on staphylococcal cell surface display and flow cytometry. Protein Eng Des Sel 21(4):247–255. doi:10.1093/protein/gzm090

    Article  PubMed  CAS  Google Scholar 

  18. Feldhaus MJ, Siegel RW, Opresko LK, Coleman JR, Feldhaus JMW, Yeung YA, Cochran JR, Heinzelman P, Colby D, Swers J, Graff C, Wiley HS, Wittrup KD (2003) Flow-cytometric isolation of human antibodies from a nonimmune Saccharomyces cerevisiae surface display library. Nat Biotechnol 21(2):163–170. doi:10.1038/nbt785

    Article  PubMed  CAS  Google Scholar 

  19. Kronqvist N, Malm M, Rockberg J, Hjelm B, Uhlen M, Stahl S, Lofblom J (2010) Staphylococcal surface display in combinatorial protein engineering and epitope mapping of antibodies. Recent Pat Biotechnol 4(3):171–182

    Article  PubMed  CAS  Google Scholar 

  20. Gotz F (1990) Staphylococcus carnosus: a new host organism for gene cloning and protein production. Soc Appl Bacteriol Symp Ser 19:49S–53S. doi:10.1111/j.1365-2672.1990.tb01797.x

    Article  PubMed  CAS  Google Scholar 

  21. Lofblom J, Feldwisch J, Tolmachev V, Carlsson J, Stahl S, Frejd FY (2010) Affibody molecules: engineered proteins for therapeutic, diagnostic and biotechnological applications. FEBS Lett 584(12):2670–2680. doi:10.1016/j.febslet.2010.04.014

    Article  PubMed  CAS  Google Scholar 

  22. Hjelm B, Fernandez CD, Lofblom J, Stahl S, Johannesson H, Rockberg J, Uhlen M (2010) Exploring epitopes of antibodies toward the human tryptophanyl-tRNA synthetase. N Biotechnol 27(2):129–137. doi:10.1016/j.nbt.2009.11.001

    Article  PubMed  CAS  Google Scholar 

  23. Rockberg J, Lofblom J, Hjelm B, Uhlén M, Stahl S (2008) Epitope mapping of antibodies using bacterial surface display. Nat Methods 5(12):1039–1045. doi:10.1038/nmeth.1272

    Article  PubMed  CAS  Google Scholar 

  24. Kronqvist N, Malm M, Gostring L, Gunneriusson E, Nilsson M, Hoiden Guthenberg I, Gedda L, Frejd FY, Stahl S, Lofblom J (2011) Combining phage and staphylococcal surface display for generation of ErbB3-specific Affibody molecules. Protein Eng Des Sel 24(4):385–396. doi:10.1093/protein/gzq118

    Article  PubMed  CAS  Google Scholar 

  25. Conrath KE, Wernery U, Muyldermans S, Nguyen VK (2003) Emergence and evolution of functional heavy-chain antibodies in Camelidae. Dev Comp Immunol 27(2):87–103

    Article  PubMed  CAS  Google Scholar 

  26. van der Linden RH, de Geus B, Frenken GJ, Peters H, Verrips CT (2000) Improved production and function of llama heavy-chain antibody fragments by molecular evolution. J Biotechnol 80(3):261–270

    Article  PubMed  Google Scholar 

  27. Kirchhofer A, Helma J, Schmidthals K, Frauer C, Cui S, Karcher A, Pellis M, Muyldermans S, Casas-Delucchi CS, Cardoso MC, Leonhardt H, Hopfner KP, Rothbauer U (2010) Modulation of protein properties in living cells using nanobodies. Nat Struct Mol Biol 17(1):133–138. doi:10.1038/nsmb.1727

    Article  PubMed  CAS  Google Scholar 

  28. Harmsen MM, Haard HJ (2007) Properties, production, and applications of camelid single-domain antibody fragments. Appl Microbiol Biot 77(1):13–22. doi:10.1007/s00253-007-1142-2

    Article  CAS  Google Scholar 

  29. Conrath KE, Lauwereys M, Galleni M, Matagne A, Frere JM, Kinne J, Wyns L, Muyldermans S (2001) Beta-lactamase inhibitors derived from single-domain antibody fragments elicited in the Camelidae. Antimicrob Agents Chemother 45(10):2807–2812. doi:10.1128/AAC.45.10.2807-2812.2001

    Article  PubMed  CAS  Google Scholar 

  30. Yau KYF, Groves MAT, Li SH, Sheedy C, Lee H, Tanha J, MacKenzie CR, Jermutus L, Hall JC (2003) Selection of hapten-specific single-domain antibodies from a non-immunized llama ribosome display library. J Immunol Methods 281(1–2):161–175. doi:10.1016/J.Jim.2003.07.011

    Article  PubMed  CAS  Google Scholar 

  31. Ryckaert S, Pardon E, Steyaert J, Callewaert N (2010) Isolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of Pichia pastoris. J Biotechnol 145(2):93–98. doi:10.1016/j.jbiotec.2009.10.010

    Article  PubMed  CAS  Google Scholar 

  32. Pellis M, Pardon E, Zolghadr K, Rothbauer U, Vincke C, Kinne J, Dierynck I, Hertogs K, Leonhardt H, Messens J, Muyldermans S, Conrath K (2012) A bacterial-two-hybrid selection system for one-step isolation of intracellularly functional Nanobodies. Arch Biochem Biophys. doi:10.1016/j.abb.2012.04.023

  33. Deschamps JR, Miller CE, Ward KB (1995) Rapid purification of recombinant green fluorescent protein using the hydrophobic properties of an HPLC size-exclusion column. Protein Expr Purif 6(4):555–558. doi:10.1006/prep.1995.1073

    Article  PubMed  CAS  Google Scholar 

  34. Ruther U (1982) pUR 250 allows rapid chemical sequencing of both DNA strands of its inserts. Nucleic Acids Res 10(19):5765–5772

    Article  PubMed  CAS  Google Scholar 

  35. Lofblom J, Kronqvist N, Uhlén M, Stahl S, Wernerus H (2007) Optimization of electroporation-mediated transformation: Staphylococcus carnosus as model organism. J Appl Microbiol 102(3):736–747. doi:10.1111/j.1365-2672.2006.03127.x

    Article  PubMed  CAS  Google Scholar 

  36. Sidhu SS, Lowman HB, Cunningham BC, Wells JA (2000) Phage display for selection of novel binding peptides. Methods Enzymol 328:333–363

    Article  PubMed  CAS  Google Scholar 

  37. de Genst E (2005) Strong in vivo maturation compensates for structurally restricted H3 loops in antibody repertoires. J Biol Chem 280(14):14114–14121. doi:10.1074/jbc.M413011200

    Article  PubMed  Google Scholar 

  38. Nguyen VK, Hamers R, Wyns L, Muyldermans S (2000) Camel heavy-chain antibodies: diverse germline V(H)H and specific mechanisms enlarge the antigen-binding repertoire. EMBO J 19(5):921–930. doi:10.1093/emboj/19.5.921

    Article  PubMed  CAS  Google Scholar 

  39. Riechmann L, Muyldermans S (1999) Single-domain antibodies: comparison of camel VH and camelised human VH domains. J Immunol Methods 231(1–2):25–38

    Article  PubMed  CAS  Google Scholar 

  40. Nilsson B, Moks T, Jansson B, Abrahmsen L, Elmblad A, Holmgren E, Henrichson C, Jones TA, Uhlen M (1987) A synthetic IgG-binding domain based on staphylococcal protein A. Protein Eng 1(2):107–113. doi:10.1093/protein/1.2.107

    Article  PubMed  CAS  Google Scholar 

  41. Gronwall C, Sjoberg A, Ramstrom M, Hoidén Guthenberg I, Hober S, Jonasson P, Stahl S (2007) Affibody-mediated transferrin depletion for proteomics applications. Biotechnol J 2(11):1389–1398. doi:10.1002/biot.200700053

    Article  PubMed  Google Scholar 

  42. Bowley DR, Labrijn AF, Zwick MB, Burton DR (2007) Antigen selection from an HIV-1 immune antibody library displayed on yeast yields many novel antibodies compared to selection from the same library displayed on phage. Protein Eng Des Sel 20(2):81–90. doi:10.1093/protein/gzl057

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the Swedish Research Council (VR) [2009-5758], Affinomics (EU-collaborative Project) and the VINNOVA excellence center for protein technology (ProNova). The VIB laboratory was financially supported by Affinomics (EU-collaborative Project, 241481).

Author information

Authors and Affiliations

  1. Division of Molecular Biotechnology, School of Biotechnology, KTH, Royal Institute of Technology, AlbaNova University Center, 106 91, Stockholm, Sweden

    Filippa Fleetwood, Stefan Ståhl & John Löfblom

  2. Laboratory of Cellular and Molecular Immunology, Vrije Universiteit Brussel, Pleinlaan 2, 1050, Brussels, Belgium

    Nick Devoogdt, Mireille Pellis & Serge Muyldermans

  3. Laboratory of In Vivo Cellular and Molecular Imaging, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium

    Nick Devoogdt

  4. Department of Structural Biology, VIB, Pleinlaan 2, 1050, Brussels, Belgium

    Mireille Pellis & Serge Muyldermans

  5. Central Veterinary Research Laboratory (CVRL), PO Box 597, Dubai, United Arab Emirates

    Ulrich Wernery

Authors
  1. Filippa Fleetwood
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nick Devoogdt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Mireille Pellis
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ulrich Wernery
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Serge Muyldermans
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Stefan Ståhl
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. John Löfblom
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to John Löfblom.

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Fleetwood, F., Devoogdt, N., Pellis, M. et al. Surface display of a single-domain antibody library on Gram-positive bacteria. Cell. Mol. Life Sci. 70, 1081–1093 (2013). https://doi.org/10.1007/s00018-012-1179-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00018-012-1179-y

Keywords

Search

Navigation