Advertisement

Affitins: Ribosome Display for Selection of Aho7c-Based Affinity Proteins

  • Valentina Kalichuk
  • Stanimir Kambarev
  • Ghislaine Béhar
  • Benjamin Chalopin
  • Axelle Renodon-Cornière
  • Barbara Mouratou
  • Frédéric PecorariEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)

Abstract

Engineered protein scaffolds have made a tremendous contribution to the panel of affinity tools owing to their favorable biophysical properties that make them useful for many applications. In 2007, our group paved the way for using archaeal Sul7d proteins for the design of artificial affinity ligands, so-called Affitins. For many years, Sac7d and Sso7d have been used as molecular basis to obtain binders for various targets. Recently, we characterized their old gifted protein family and identified Aho7c, originating from Acidianus hospitalis, as the shortest member (60 amino-acids) with impressive stability (96.5 °C, pH 0–12). Here, we describe the construction of Aho7c combinatorial libraries and their use for selection of binders by ribosome display.

Key words

Ribosome display In vitro selection Sul7d Aho7c Sac7d Sso7d Affitin 

Notes

Acknowledgments

The authors thank all previous members of the laboratory who helped to develop this protocol. In memoriam of Ghislaine Béhar.

References

  1. 1.
    Mouratou B, Schaeffer F, Guilvout I, Tello-Manigne D, Pugsley AP, Alzari PM, Pecorari F (2007) Remodeling a DNA-binding protein as a specific in vivo inhibitor of bacterial secretin PulD. Proc Natl Acad Sci U S A 104(46):17983–17988CrossRefGoogle Scholar
  2. 2.
    Pecorari F, Alzari PM (2008) OB-fold used as scaffold for engineering new specific binders. Patent Publication Nos PCT/IB2007/004388Google Scholar
  3. 3.
    Béhar G, Bellinzoni M, Maillasson M, Paillard-Laurance L, Alzari PM, He X, Mouratou B, Pecorari F (2013) Tolerance of the archaeal Sac7d scaffold protein to alternative library designs: characterization of anti-immunoglobulin G Affitins. Protein Eng Des Sel 26(4):267–275CrossRefGoogle Scholar
  4. 4.
    Correa A, Pacheco S, Mechaly Ariel E, Obal G, Béhar G, Mouratou B, Oppezzo P, Alzari PM, Pecorari F (2014) Potent and specific inhibition of glycosidases by small artificial binding proteins (Affitins). PLoS One 9(5):e97438CrossRefGoogle Scholar
  5. 5.
    Krehenbrink M, Chami M, Guilvout I, Alzari PM, Pecorari F, Pugsley AP (2008) Artificial binding proteins (Affitins) as probes for conformational changes in secretin PulD. J Mol Biol 383(5):1058–1068CrossRefGoogle Scholar
  6. 6.
    Buddelmeijer N, Krehenbrink M, Pecorari F, Pugsley AP (2009) Type II secretion system secretin PulD localizes in clusters in the Escherichia coli outer membrane. J Bacteriol 191(1):161–168CrossRefGoogle Scholar
  7. 7.
    Miranda FF, Brient-Litzler E, Zidane N, Pecorari F, Bedouelle H (2011) Reagentless fluorescent biosensors from artificial families of antigen binding proteins. Biosens Bioelectron 26(10):4184–4190CrossRefGoogle Scholar
  8. 8.
    Pacheco S, Behar G, Maillasson M, Mouratou B, Pecorari F (2014) Affinity transfer to the archaeal extremophilic Sac7d protein by insertion of a CDR. Protein Eng Des Sel 27(10):431–438CrossRefGoogle Scholar
  9. 9.
    Béhar G, Pacheco S, Maillasson M, Mouratou B, Pecorari F (2014) Switching an anti-IgG binding site between archaeal extremophilic proteins results in Affitins with enhanced pH stability. J Biotechnol 192:123–129CrossRefGoogle Scholar
  10. 10.
    Kalichuk V, Renodon-Corniere A, Behar G, Carrion F, Obal G, Maillasson M, Mouratou B, Preat V, Pecorari F (2018) A novel, smaller scaffold for Affitins: showcase with binders specific for EpCAM. Biotechnol Bioeng 115(2):10CrossRefGoogle Scholar
  11. 11.
    Cinier M, Petit M, Williams MN, Fabre RM, Pecorari F, Talham DR, Bujoli B, Tellier C (2009) Bisphosphonate adaptors for specific protein binding on zirconium phosphonate-based microarrays. Bioconjug Chem 20(12):2270–2277CrossRefGoogle Scholar
  12. 12.
    Béhar G, Renodon-Cornière A, Mouratou B, Pecorari F (2016) Affitins as robust tailored reagents for affinity chromatography purification of antibodies and non-immunoglobulin proteins. J Chromatogr A 1441:44–51CrossRefGoogle Scholar
  13. 13.
    Fernandes CSM, Rd S, Ottengy S, Viecinski AC, Béhar G, Mouratou B, Pecorari F, Roque ACA (2016) Affitins for protein purification by affinity magnetic fishing. J Chromatogr A 1457:50–58CrossRefGoogle Scholar
  14. 14.
    Vukojicic P, Béhar G, Tawara MH, Fernandez-Villamarin M, Pecorari F, Fernandez-Megia E, Mouratou B (2019) Multivalent Affidendrons with High Affinity and Specificity toward as Versatile Tools for Modulating Multicellular Behaviors. ACS Appl Mater Interfaces 11(24):21391–21398Google Scholar
  15. 15.
    Kalichuk V, Behar G, Renodon-Corniere A, Danovski G, Obal G, Barbet J, Mouratou B, Pecorari F (2016) The archaeal “7 kDa DNA-binding” proteins: extended characterization of an old gifted family. Sci Rep 6:37274CrossRefGoogle Scholar
  16. 16.
    Mattheakis LC, Bhatt RR, Dower WJ (1994) An in vitro polysome display system for identifying ligands from very large peptide libraries. Proc Natl Acad Sci U S A 91(19):9022–9026CrossRefGoogle Scholar
  17. 17.
    Gera N, Hussain M, Wright RC, Rao BM (2011) Highly stable binding proteins derived from the hyperthermophilic Sso7d scaffold. J Mol Biol 409(4):601–616CrossRefGoogle Scholar
  18. 18.
    Zhao N, Schmitt MA, Fisk JD (2016) Phage display selection of tight specific binding variants from a hyperthermostable Sso7d scaffold protein library. FEBS J 283(7):1351–1367CrossRefGoogle Scholar
  19. 19.
    Hanes J, Jermutus L, Weber-Bornhauser S, Bosshard HR, Plückthun A (1998) Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries. Proc Natl Acad Sci U S A 95(24):14130–14135CrossRefGoogle Scholar
  20. 20.
    Mouratou B, Béhar G, Paillard-Laurance L, Colinet S, Pecorari F (2012) Ribosome display for the selection of Sac7d scaffolds. Methods Mol Biol 805:315–331CrossRefGoogle Scholar
  21. 21.
    Hanes J, Pluckthun A (1997) In vitro selection and evolution of functional proteins by using ribosome display. Proc Natl Acad Sci U S A 94(10):4937–4942CrossRefGoogle Scholar
  22. 22.
    Amstutz P, Binz HK, Zahnd C, Pluckthun A (2006) Ribosome display: in vitro selection of protein-protein interactions. In: Celis J (ed) Cell biology—a laboratory handbook, vol 1. Elsevier Academic Press, Cambridge, pp 497–509Google Scholar
  23. 23.
    Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5(4):725–738CrossRefGoogle Scholar
  24. 24.
    Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9:40CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  • Valentina Kalichuk
    • 1
  • Stanimir Kambarev
    • 1
  • Ghislaine Béhar
    • 1
  • Benjamin Chalopin
    • 1
  • Axelle Renodon-Cornière
    • 1
  • Barbara Mouratou
    • 1
  • Frédéric Pecorari
    • 1
    Email author
  1. 1.CRCINA, INSERM, CNRSUniversité d’Angers, Université de NantesNantesFrance

Personalised recommendations