Skip to main content

Affinity Maturation of Single-Domain Antibodies by Yeast Surface Display

  • Protocol
  • First Online:
Single Domain Antibodies

Part of the book series: Methods in Molecular Biology ((MIMB,volume 911))

Abstract

Although single-domain antibodies derived from libraries prepared either after animal immunization or naïve animals generally exhibit reasonable affinity, it is often desirable to further improve their affinity. This chapter describes protocols for improving the affinity of single-domain antibodies using quantitative library sorting by yeast surface display. An example is included where we also exploit a complementary strength of phage display in generating larger sequence diversity prior to library sorting with yeast surface display.

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

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Muyldermans S (2001) Single domain camel antibodies: current status. J Biotechnol 74:277–302

    PubMed  CAS  Google Scholar 

  2. Muyldermans S, Cambillau C, Wyns L (2001) Recognition of antigens by single-domain antibody fragments: the superfluous luxury of paired domains. Trends Biochem Sci 26:230–235

    Article  PubMed  CAS  Google Scholar 

  3. De Genst E, Silence K, Decanniere K, Conrath K, Loris R, Kinne J, Muyldermans S, Wyns L (2006) Molecular basis for the preferential cleft recognition by dromedary heavy-chain antibodies. Proc Natl Acad Sci U S A 103:4586–4591

    Article  PubMed  Google Scholar 

  4. Decanniere K, Desmyter A, Lauwereys M, Ghahroudi MA, Muyldermans S, Wyns L (1999) A single-domain antibody fragment in complex with RNase A: non-canonical loop structures and nanomolar affinity using two CDR loops. Structure 7:361–370

    Article  PubMed  CAS  Google Scholar 

  5. Desmyter A, Transue TR, Ghahroudi MA, Thi MH, Poortmans F, Hamers R, Muyldermans S, Wyns L (1996) Crystal structure of a camel single-domain VH antibody fragment in complex with lysozyme. Nat Struct Biol 3:803–811

    Article  PubMed  CAS  Google Scholar 

  6. Sidhu SS, Koide S (2007) Phage display for engineering and analyzing protein interaction interfaces. Curr Opin Struct Biol 17:481–487

    Article  PubMed  CAS  Google Scholar 

  7. 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 

  8. Boder ET, Wittrup KD (2000) Yeast surface display for directed evolution of protein expression, affinity, and stability. Methods Enzymol 328:430–444

    Article  PubMed  CAS  Google Scholar 

  9. VanAntwerp JJ, Wittrup KD (2000) Fine affinity discrimination by yeast surface display and flow cytometry. Biotechnol Prog 16:31–37

    Article  PubMed  CAS  Google Scholar 

  10. Koide A, Tereshko V, Uysal S, Margalef K, Kossiakoff AA, Koide S (2007) Exploring the capacity of minimalist protein interfaces: interface energetics and affinity maturation to picomolar KD of a single-domain antibody with a flat paratope. J Mol Biol 373:941–953

    Article  PubMed  CAS  Google Scholar 

  11. Clackson T, Wells JA (1995) A hot spot of binding energy in a hormone-receptor interface. Science 267:383–386

    Article  PubMed  CAS  Google Scholar 

  12. Weiss GA, Watanabe CK, Zhong A, Goddard A,Sidhu SS (2000) Rapid mapping of protein functional epitopes by combinatorial alanine scanning. Proc Natl Acad Sci U S A 97:8950–8954

    Article  PubMed  CAS  Google Scholar 

  13. Fellouse FA, Wiesmann C, Sidhu SS (2004) Synthetic antibodies from a four-amino-acid code: a dominant role for tyrosine in antigen recognition. Proc Natl Acad Sci U S A 101:12467–12472

    Article  PubMed  CAS  Google Scholar 

  14. Koide A, Koide S (2007) Monobodies: antibody mimics based on the scaffold of the fibronectin type III domain. Methods Mol Biol 352:95–109

    PubMed  CAS  Google Scholar 

  15. Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nat Protoc 2:31–34

    Article  PubMed  CAS  Google Scholar 

  16. Gietz RD, Woods RA (2002) Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method. Methods Enzymol 350:87–96

    Article  PubMed  CAS  Google Scholar 

  17. Ma H, Kunes S, Schatz PJ, Botstein D (1987) Plasmid construction by homologous recombination in yeast. Gene 58:201–216

    Article  PubMed  CAS  Google Scholar 

  18. Boder ET, Wittrup KD (1998) Optimal screening of surface-displayed polypeptide libraries. Biotechnol Prog 14:55–62

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported by the National Institutes of Health grants R01 GM090324 and RC1 DA028779 to SK.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Shohei Koide .

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2012 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Koide, A., Koide, S. (2012). Affinity Maturation of Single-Domain Antibodies by Yeast Surface Display. In: Saerens, D., Muyldermans, S. (eds) Single Domain Antibodies. Methods in Molecular Biology, vol 911. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-968-6_26

Download citation

  • DOI: https://doi.org/10.1007/978-1-61779-968-6_26

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-967-9

  • Online ISBN: 978-1-61779-968-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics