Abstract
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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Craik DJ, Lee M-H, Rehm FBH et al (2018) Ribosomally-synthesised cyclic peptides from plants as drug leads and pharmaceutical scaffolds. Bioorg Med Chem 26(10):2727–2737. https://doi.org/10.1016/j.bmc.2017.08.005
Bogdanowich-Knipp SJ, Chakrabarti S, Siahaan TJ et al (1999) Solution stability of linear vs. cyclic RGD peptides. J Pept Res 53(5):530–541. https://doi.org/10.1034/j.1399-3011.1999.00052.x
Diao L, Meibohm B (2013) Pharmacokinetics and pharmacokinetic-pharmacodynamic correlations of therapeutic peptides. Clin Pharmacokinet 52(10):855–868. https://doi.org/10.1007/s40262-013-0079-0
Lipinski CA (2004) Lead- and drug-like compounds: the rule-of-five revolution. Drug Discov Today Technol 1(4):337–341. https://doi.org/10.1016/j.ddtec.2004.11.007
Brown T, Brown N, Stollar EJ (2018) Most yeast SH3 domains bind peptide targets with high intrinsic specificity. PLoS One 13(2):e0193128. https://doi.org/10.1371/journal.pone.0193128
Sidhu SS, Lowman HB, Cunningham BC et al (2000) [21] Phage display for selection of novel binding peptides. In: Applications of chimeric genes and hybrid proteins—part C: protein-protein interactions and genomics, vol 328. Elsevier, Amsterdam, pp 333–IN5
Sakamoto K, Sogabe S, Kamada Y et al (2017) Discovery of high-affinity BCL6-binding peptide and its structure-activity relationship. Biochem Biophys Res Commun 482(2):310–316. https://doi.org/10.1016/j.bbrc.2016.11.060
Rentero Rebollo I, Heinis C (2013) Phage selection of bicyclic peptides. Methods 60(1):46–54. https://doi.org/10.1016/j.ymeth.2012.12.008
Diderich P, Heinis C (2014) Phage selection of bicyclic peptides binding Her2. Tetrahedron 70(42):7733–7739. https://doi.org/10.1016/j.tet.2014.05.106
Ryvkin A, Ashkenazy H, Weiss-Ottolenghi Y et al (2018) Phage display peptide libraries: deviations from randomness and correctives. Nucleic Acids Res 46(9):e52. https://doi.org/10.1093/nar/gky077
Watters JM, Telleman P, Junghans RP (1997) An optimized method for cell-based phage display panning. Immunotechnology 3(1):21–29. https://doi.org/10.1016/S1380-2933(96)00056-5
Nguyen X-H, Trinh T-L, Vu T-B-H et al (2018) Isolation of phage-display library-derived scFv antibody specific to Listeria monocytogenes by a novel immobilized method. J Appl Microbiol 124(2):591–597. https://doi.org/10.1111/jam.13648
Hust M, Meyer T, Voedisch B et al (2011) A human scFv antibody generation pipeline for proteome research. J Biotechnol 152(4):159–170. https://doi.org/10.1016/j.jbiotec.2010.09.945
Dretzen G, Bellard M, Sassone-Corsi P et al (1981) A reliable method for the recovery of DNA fragments from agarose and acrylamide gels. Anal Biochem 112(2):295–298. https://doi.org/10.1016/0003-2697(81)90296-7
Fu C, Donovan WP, Shikapwashya-Hasser O et al (2014) Hot Fusion: an efficient method to clone multiple DNA fragments as well as inverted repeats without ligase. PLoS One 9(12):e115318. https://doi.org/10.1371/journal.pone.0115318
Zantow J, Moreira GMSG, Dübel S et al (2018) ORFeome phage display. Methods Mol Biol 1701:477–495. https://doi.org/10.1007/978-1-4939-7447-4_27
Russo G, Meier D, Helmsing S et al (2018) Parallelized antibody selection in microtiter plates. Methods Mol Biol 1701:273–284. https://doi.org/10.1007/978-1-4939-7447-4_14
Frenzel A, Schirrmann T, Hust M (2016) Phage display-derived human antibodies in clinical development and therapy. MAbs 8(7):1177–1194. https://doi.org/10.1080/19420862.2016.1212149
Maurer CK, Fruth M, Empting M et al (2016) Discovery of the first small-molecule CsrA-RNA interaction inhibitors using biophysical screening technologies. Future Med Chem 8(9):931–947. https://doi.org/10.4155/fmc-2016-0033
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2020 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Jakob, V., Helmsing, S., Hust, M., Empting, M. (2020). Restriction-Free Construction of a Phage-Presented Very Short Macrocyclic Peptide Library. In: Zielonka, S., Krah, S. (eds) Genotype Phenotype Coupling. Methods in Molecular Biology, vol 2070. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9853-1_6
Download citation
DOI: https://doi.org/10.1007/978-1-4939-9853-1_6
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-4939-9852-4
Online ISBN: 978-1-4939-9853-1
eBook Packages: Springer Protocols