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

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 2070; p. 95
• Main Authors: Jakob, Valentin, Helmsing, Saskia, Hust, Michael, Empting, Martin
• Format: Journal Article
• Language: English
• Published: United States 01.01.2020
• Subjects: Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Macrocyclic Compounds; Peptide Library; Protein Engineering; Hot fusion cloning; Panning; Library construction; Macrocyclic oligopeptide phage display; Restriction-free cloning
• ISSN: 1940-6029
• DOI: 10.1007/978-1-4939-9853-1_6

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.