Restriction-Free Construction of a Phage-Presented Very Short Macrocyclic Peptide Library

  • Valentin Jakob
  • Saskia Helmsing
  • Michael Hust
  • Martin EmptingEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 2070)


Phage display is a commonly used technology for the screening of large clonal libraries of proteins and peptides. The construction of peptide libraries containing very short sequences, however, poses certain problems for conventional restriction-based cloning procedures, which are rooted in the necessity to purify restricted library oligos. Herein, we present an alternative cloning method especially suitable for such very short sequences of about only 21 base pairs resulting in a 60 bp insert. The employed restriction-free hot fusion cloning strategy allows for facile library construction bypassing the need for purification of the small oligo. The library includes one well-defined disulfide bridge rendering the displayed macrocyclic peptide sequences as attractive scaffolds for novel active principles.

Key words

Library construction Hot fusion cloning Restriction-free cloning Macrocyclic oligopeptide phage display Panning 



This review contains updated and revised parts of former protocols by Zantow et al. [16] and Russo et al. [17]. We thank Rolf W. Hartmann for his continuous support.


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Copyright information

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

Authors and Affiliations

  • Valentin Jakob
    • 1
    • 2
  • Saskia Helmsing
    • 3
  • Michael Hust
    • 3
    • 4
  • Martin Empting
    • 1
    • 2
    Email author
  1. 1.Department of Drug Design and Optimization (DDOP)Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS)—Helmholtz Centre for Infection Research (HZI)SaarbrückenGermany
  2. 2.Department of PharmacySaarland UniversitySaarbrückenGermany
  3. 3.Institut für Biochemie, Biotechnologie und Bioinformatik, Abteilung BiotechnologieTechnische Universität BraunschweigBraunschweigGermany
  4. 4.YUMAB GmbHScience Campus Braunschweig SüdBraunschweigGermany

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