Chapter title |
Mapping the Substrate Recognition Landscapes of Metalloproteases Using Comprehensive Mutagenesis
|
---|---|
Chapter number | 11 |
Book title |
Matrix Metalloproteases
|
Published in |
Methods in molecular biology, March 2017
|
DOI | 10.1007/978-1-4939-6863-3_11 |
Pubmed ID | |
Book ISBNs |
978-1-4939-6861-9, 978-1-4939-6863-3
|
Authors |
Colin A. Kretz |
Editors |
Charles A. Galea |
Abstract |
Protease specificity is controlled by exosites, which capture and orient the substrate, and the active site, which binds substrate residues near the P1-P1' scissile bond and catalyzes peptide hydrolysis. Techniques used to identify critical contact points between a protease and its substrate can be time consuming and labor-intensive. Screening tools such as phage display have been revitalized with the emergence of high-throughput sequencing technology, and can be used to interrogate protease substrate specificity. This article will outline a method for creating and screening a comprehensive mutagenesis substrate phage display library. High-throughput sequencing of uncleaved phage at various reaction time points enables k cat/K M determination for every possible single amino acid substitution at each position of the substrate, providing unprecedented resolution for the interaction between a protease and its substrate. |
Mendeley readers
Geographical breakdown
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Unknown | 10 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Bachelor | 3 | 30% |
Professor | 2 | 20% |
Student > Master | 2 | 20% |
Researcher | 1 | 10% |
Unknown | 2 | 20% |
Readers by discipline | Count | As % |
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Unknown | 3 | 30% |