Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

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Title Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy
Author Haselhorst, Thomas Erwin; Munster-Kuhnel, Anja K.; Oschlies, Melanie; Tiralongo, Giuseppe; Gerardy-Schahn, Rita; von Itzstein, Mark
Journal Name Biochemical and Biophysical Research Communications
Year Published 2007
Place of publication United States
Publisher Elsevier Inc.
Abstract We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5′-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin® Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5′-triphosphate, CTP) and a non-binding ligand (α/β-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) no specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available.
Peer Reviewed Yes
Published Yes
Publisher URI http://www.elsevier.com/wps/find/journaldescription.cws_home/622790/description#description
Alternative URI http://dx.doi.org/10.1016/j.bbrc.2007.05.204
Copyright Statement Copyright 2007 Elsevier. Please refer to the journal's website for access to the definitive, published version.
Volume 359
Page from 866
Page to 870
ISSN 0006-291X
Date Accessioned 2007-06-26
Date Available 2009-09-01T06:00:51Z
Language en_AU
Research Centre Institute for Glycomics
Faculty Faculty of Science, Environment, Engineering and Technology
Subject PRE2009-Science & Technology
URI http://hdl.handle.net/10072/18291
Publication Type Journal Articles (Refereed Article)
Publication Type Code c1

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