Chapter title |
Affinity Purification Method for the Identification of Nonribosomal Peptide Biosynthetic Enzymes Using a Synthetic Probe for Adenylation Domains
|
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Chapter number | 4 |
Book title |
Nonribosomal Peptide and Polyketide Biosynthesis
|
Published in |
Methods in molecular biology, January 2016
|
DOI | 10.1007/978-1-4939-3375-4_4 |
Pubmed ID | |
Book ISBNs |
978-1-4939-3373-0, 978-1-4939-3375-4
|
Authors |
Fumihiro Ishikawa, Hideaki Kakeya |
Abstract |
A series of inhibitors have been designed based on 5'-O-sulfamoyl adenosine (AMS) that display tight binding characteristics towards the inhibition of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs). We recently developed an affinity probe for A domains that could be used to facilitate the specific isolation and identification of NRPS modules. Our synthetic probe, which is a biotinylated variant of L-Phe-AMS (L-Phe-AMS-biotin), selectively targets the A domains in NRPS modules that recognize and convert L-Phe to an aminoacyl adenylate in whole proteomes. In this chapter, we describe the design and synthesis of L-Phe-AMS-biotin and provide a summary of our work towards the development of a series of protocols for the specific enrichment of NRPS modules using this probe. |
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