Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy
- Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Qld 9726 (Australia)
- Abteilung Zellulaere Chemie, Medizinische Hochschule Hannover (MHH), Carl-Neuberg-Strasse 1, D-30625 Hannover (Germany)
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 ({alpha}/{beta}-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.
- OSTI ID:
- 20991480
- Journal Information:
- Biochemical and Biophysical Research Communications, Vol. 359, Issue 4; Other Information: DOI: 10.1016/j.bbrc.2007.05.204; PII: S0006-291X(07)01142-4; Copyright (c) 2007 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA); ISSN 0006-291X
- Country of Publication:
- United States
- Language:
- English
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