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Yeast Display of Engineered Antibody Domains

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Therapeutic Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 899))

Abstract

Yeast display is an efficient technology for selection of antibodies and other proteins with high affinity and thermal stability. Here, we describe a method for affinity maturation of engineered antibody domains (eAds) using yeast display. EAd yeast libraries of relatively large size (∼109) were generated and subjected to alternating rounds of magnetic-activated cell sorting (MACS), fluorescent-activated cell sorting (FACS), and random mutagenesis. The highest affinity clones from the final round of maturation were identified and analyzed. We discuss extensively each key step, and provide detailed protocols and helpful notes.

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References

  1. Chao G, Lau WL, Hackel BJ, Sazinsky SL, Lippow SM, Wittrup KD (2006) Isolating and engineering human antibodies using yeast surface display. Nat Protoc 1:755–768

    Article  PubMed  CAS  Google Scholar 

  2. 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 25:107–116

    Article  PubMed  CAS  Google Scholar 

  3. Walker LM, Bowley DR, Burton DR (2009) Efficient recovery of high-affinity antibodies from a single-chain Fab yeast display library. J Mol Biol 389:365–375

    Article  PubMed  CAS  Google Scholar 

  4. Wang Z, Kim GB, Woo JH, Liu YY, Mathias A, Stavrou S, Neville DM Jr (2007) Improvement of a recombinant anti-monkey anti-CD3 diphtheria toxin based immunotoxin by yeast display affinity maturation of the scFv. Bioconjug Chem 18:947–955

    Article  PubMed  CAS  Google Scholar 

  5. Zhao Q, Feng Y, Zhu Z, Dimitrov DS (2011) Human monoclonal antibody fragments binding to insulin-like growth factors 1 and 2 with picomolar affinity. Mol Cancer Ther 10:1677–1685

    Google Scholar 

  6. Hackel BJ, Kapila A, Wittrup KD (2008) Picomolar affinity fibronectin domains engineered utilizing loop length diversity, recursive mutagenesis, and loop shuffling. J Mol Biol 381:1238–1252

    Article  PubMed  CAS  Google Scholar 

  7. Lipovsek D (2011) Adnectins: engineered target-binding protein therapeutics. Protein Eng Des Sel 24:3–9

    Article  PubMed  CAS  Google Scholar 

  8. Richman SA, Healan SJ, Weber KS, Donermeyer DL, Dossett ML, Greenberg PD, Allen PM, Kranz DM (2006) Development of a novel strategy for engineering high-affinity proteins by yeast display. Protein Eng Des Sel 19:255–264

    Article  PubMed  CAS  Google Scholar 

  9. Dam J, Guan R, Natarajan K, Dimasi N, Chlewicki LK, Kranz DM, Schuck P, Margulies DH, Mariuzza RA (2003) Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2K(b). Nat Immunol 4:1213–1222

    Article  PubMed  CAS  Google Scholar 

  10. Esteban O, Zhao H (2004) Directed evolution of soluble single-chain human class II MHC molecules. J Mol Biol 340:81–95

    Article  PubMed  CAS  Google Scholar 

  11. Benatuil L, Perez JM, Belk J, Hsieh CM (2010) An improved yeast transformation method for the generation of very large human antibody libraries. Protein Eng Des Sel 23:155–159

    Article  PubMed  CAS  Google Scholar 

  12. Dimitrov DS (2009) Engineered CH2 domains (nanoantibodies). MAbs 1:26–28

    Article  PubMed  Google Scholar 

  13. Xiao X, Feng Y, Vu BK, Ishima R, Dimitrov DS (2009) A large library based on a novel (CH2) scaffold: identification of HIV-1 inhibitors. Biochem Biophys Res Commun 387:387–392

    Article  PubMed  CAS  Google Scholar 

  14. Gong R, Vu BK, Feng Y, Prieto DA, Dyba MA, Walsh JD, Prabakaran P, Veenstra TD, Tarasov SG, Ishima R, Dimitrov DS (2009) Engineered human antibody constant domains with increased stability. J Biol Chem 284:14203–14210

    Article  PubMed  CAS  Google Scholar 

  15. Gong R, Wang Y, Feng Y, Zhao Q, Dimitrov DS (2011) Shortened engineered human antibody CH2 domains: increased stability and binding to the human neonatal receptor. J Biol Chem 286:27288–27293

    Google Scholar 

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Acknowledgments

We thank Professor Dane Wittrup for providing the yeast display vector pCTCON2 and yeast strain EBY100 and members of our group for helpful discussions. This project was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research and by the Gates Foundation (DSD).

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Authors and Affiliations

  1. Protein Interactions Group, Center for Cancer Research Nanobiology Program, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

    Qi Zhao & Zhongyu Zhu

  2. Protein Interactions Group, Frederick National Laboratory for Cancer Research, National Cancer Institute, National Institutes of Health, Frederick, MD, USA

    Dimiter S. Dimitrov

Authors
  1. Qi Zhao
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  2. Zhongyu Zhu
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  3. Dimiter S. Dimitrov
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Corresponding author

Correspondence to Dimiter S. Dimitrov .

Editor information

Editors and Affiliations

  1. MedImmune, LLC, MedImmune Way One, Gaithersburg, 20878, USA

    Vladimir Voynov

  2. , Department of Physical Biochemistry, Biogen Idec, Cambridge Center 14, Cambridge, 02142, Massachusetts, USA

    Justin A. Caravella

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Zhao, Q., Zhu, Z., Dimitrov, D.S. (2012). Yeast Display of Engineered Antibody Domains. In: Voynov, V., Caravella, J. (eds) Therapeutic Proteins. Methods in Molecular Biology, vol 899. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-921-1_5

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  • DOI: https://doi.org/10.1007/978-1-61779-921-1_5

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-920-4

  • Online ISBN: 978-1-61779-921-1

  • eBook Packages: Springer Protocols

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