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Mapping Protein–Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries and Alanine Scanning
One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry suc...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
2014
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Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122165/ https://www.ncbi.nlm.nih.gov/pubmed/25616333 http://dx.doi.org/10.1007/978-1-4939-2020-4_12 |
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author | Kokoszka, Malgorzata E. Kay, Brian K. |
author_facet | Kokoszka, Malgorzata E. Kay, Brian K. |
author_sort | Kokoszka, Malgorzata E. |
collection | PubMed |
description | One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. With the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. To illustrate this approach, we use a library of bacteriophage M13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the SH3 domain of the human Lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase Cbk1. The binding properties of the selected peptide ligands are then dissected by sequence alignment, Kunkel mutagenesis, and alanine scanning. Finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery. |
format | Online Article Text |
id | pubmed-7122165 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2014 |
record_format | MEDLINE/PubMed |
spelling | pubmed-71221652020-04-06 Mapping Protein–Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries and Alanine Scanning Kokoszka, Malgorzata E. Kay, Brian K. Peptide Libraries Article One avenue for inferring the function of a protein is to learn what proteins it may bind to in the cell. Among the various methodologies, one way for doing so is to affinity select peptide ligands from a phage-displayed combinatorial peptide library and then to examine if the proteins that carry such peptide sequences interact with the target protein in the cell. With the protocols described in this chapter, a laboratory with skills in microbiology, molecular biology, and protein biochemistry can readily identify peptides in the library that bind selectively, and with micromolar affinity, to a given target protein on the time scale of 2 months. To illustrate this approach, we use a library of bacteriophage M13 particles, which display 12-mer combinatorial peptides, to affinity select different peptide ligands for two different targets, the SH3 domain of the human Lyn protein tyrosine kinase and a segment of the yeast serine/threonine protein kinase Cbk1. The binding properties of the selected peptide ligands are then dissected by sequence alignment, Kunkel mutagenesis, and alanine scanning. Finally, the peptide ligands can be used to predict cellular interacting proteins and serve as the starting point for drug discovery. 2014-10-20 /pmc/articles/PMC7122165/ /pubmed/25616333 http://dx.doi.org/10.1007/978-1-4939-2020-4_12 Text en © Springer Science+Business Media New York 2015 This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic. |
spellingShingle | Article Kokoszka, Malgorzata E. Kay, Brian K. Mapping Protein–Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries and Alanine Scanning |
title | Mapping Protein–Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries and Alanine Scanning |
title_full | Mapping Protein–Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries and Alanine Scanning |
title_fullStr | Mapping Protein–Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries and Alanine Scanning |
title_full_unstemmed | Mapping Protein–Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries and Alanine Scanning |
title_short | Mapping Protein–Protein Interactions with Phage-Displayed Combinatorial Peptide Libraries and Alanine Scanning |
title_sort | mapping protein–protein interactions with phage-displayed combinatorial peptide libraries and alanine scanning |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7122165/ https://www.ncbi.nlm.nih.gov/pubmed/25616333 http://dx.doi.org/10.1007/978-1-4939-2020-4_12 |
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