Advertisement

In Vitro Selection of Single-Domain Antibody (VHH) Using cDNA Display

  • Naoto Nemoto
  • Shigefumi Kumachi
  • Hidenao Arai
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1827)

Abstract

Single-domain antibody (e.g., Nanobody, VHH antibody) is a promising scaffold for therapeutic and diagnostic reagents. To expand the range of target molecules, in vitro selection using cell-free display technologies such as cDNA display is useful and powerful because of their huge libraries and robust stability. We provide technical details for in vitro selection of single-domain antibodies using cDNA display.

Key words

cDNA display mRNA display In vitro selection Nanobody VHH Cell-free translation Puromycin Evolutionary molecular engineering Directed evolution Protein engineering Antibody engineering 

Notes

Acknowledgments

The authors thank previous members of the Nemoto Laboratory who helped to develop this protocol, especially Dr. Yuki Mochizuki, Mr. Shinnosuke Kimura, and Mr. Takeru Suzuki. This work was supported by JSPS KAKENHI Grant Number T15H05723 and Brain and Body System Science Institute (BBSSI), Saitama University.

References

  1. 1.
    Ullman CG, Frigotto L, Cooley RN (2011) In vitro methods for peptide display and their applications. Brief Funct Genomics 10:125–134CrossRefPubMedGoogle Scholar
  2. 2.
    Yamaguchi J, Naimuddin M, Biyani M, Sasaki T, Machida M, Kubo T, Funatsu T, Husimi Y, Nemoto N (2009) cDNA display: a novel screening method for functional disulfide-rich peptides by solid-phase synthesis and stabilization of mRNA-protein fusions. Nucleic Acids Res 37:e108CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Mochizuki Y, Biyani M, Tsuji-Ueno S, Suzuki M, Nishigaki K, Husimi Y, Nemoto N (2011) One-pot preparation of mRNA/cDNA display by a novel and versatile puromycin-linker DNA. ACS Comb Sci 13:478–485CrossRefPubMedGoogle Scholar
  4. 4.
    Ueno S, Nemoto N (2012) cDNA display: rapid stabilization of mRNA display. Methods Mol Biol 805:237–250CrossRefGoogle Scholar
  5. 5.
    Mochizuki Y, Nemoto N (2012) Evolution of disulfide-rich peptide aptamers using cDNA display. Methods Mol Biol 805:113–135CrossRefGoogle Scholar
  6. 6.
    Nemoto N, Miyamoto-Sato E, Husimi Y, Yanagawa H (1997) In vitro virus: bonding of mRNA bearing puromycin at the 3′-terminal end to the C-terminal end of its encoded protein on the ribosome in vitro. FEBS Lett 414:405–408CrossRefPubMedGoogle Scholar
  7. 7.
    Roberts RW, Szostak JW (1997) RNA-peptide fusions for the in vitro selection of peptides and proteins. Proc Natl Acad Sci U S A 94:12297–12302CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ueno S, Yoshida S, Mondal A, Nishina K, Koyama M, Sakata I, Miura K, Hayashi Y, Nemoto N, Nishigaki K, Sakai T (2012) In vitro selection of a novel peptide antagonist of growth hormone secretagogue receptor using cDNA display. Proc Natl Acad Sci U S A 109:11121–11126CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Ueno S, Kimura S, Ichiki T, Nemoto N (2012) Improvement of a puromycin-linker to extend the selection target varieties in cDNA display method. J Biotechnol 162:299–302CrossRefPubMedGoogle Scholar
  10. 10.
    Mochizuki Y, Suzuki T, Fujimoto K, Nemoto N (2015) A versatile puromycin-linker using cnvK for high-throughput in vitro selection by cDNA display. J Biotechnol 212:174–180CrossRefPubMedGoogle Scholar
  11. 11.
    Moutel S, Bery N, Bernard V, Keller L, Lemesre E, de Marco A, Ligat L, Rain JC, Favre G, Olichon A, Perez F (2016) NaLi-H1: a universal synthetic library of humanized nanobodies providing highly functional antibodies and intrabodies. elife 5:e16228CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Monegal A, Ami D, Martinelli C, Huang H, Aliprandi M, Capasso P, Francavilla C, Ossolengo G, de Marco A (2009) Immunological applications of single-domain llama recombinant antibodies isolated from a naïve library. Protein Eng Des Sel 22:273–280CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Naoto Nemoto
    • 1
    • 2
  • Shigefumi Kumachi
    • 2
  • Hidenao Arai
    • 2
  1. 1.Graduate School of Science and Engineering, Saitama UniversitySaitamaJapan
  2. 2.Epsilon Molecular Engineering, Inc.SaitamaJapan

Personalised recommendations