Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy.
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.
Haselhorst
Thomas
T
Institute for Glycomics, Griffith University (Gold Coast Campus), PMB 50 Gold Coast Mail Centre, Qld 9726, Australia.
Münster-Kühnel
Anja K
AK
Oschlies
Melanie
M
Tiralongo
Joe
J
Gerardy-Schahn
Rita
R
von Itzstein
Mark
M
D001681:Biological Assay / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D000216:N-Acylneuraminate Cytidylyltransferase / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D012685:Sepharose / Q000737:chemistry*
D004800:Enzymes, Immobilized; D008024:Ligands; D012685:Sepharose; D000216:N-Acylneuraminate Cytidylyltransferase