title: Bacterial expression and in vitro refolding of limited fragments of the Notch receptor and its ligands.
abstract: Prokaryotic expression of limited fragments of the Notch receptor and its ligands followed by in vitro refolding has been used for the production of the significant amounts of protein required for structure determination by X-ray crystallography or nuclear magnetic resonance spectroscopy. As an illustration of the protocol for the production of these EGF-containing constructs we have focused on a limited fragment of human Notch 1 that contains three calcium-binding EGF domains, hNotch-111-13. Following characterization by the methods described here, this construct has been shown to be functionally competent in a range of assays and the structure has been solved by X-ray crystallography and NMR.
authors: Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK.
authors: Pat
authors: P
authors: Whiteman
authors: Christina
authors: C
authors: Redfield
authors: Penny A
authors: PA
authors: Handford
mesh_terms: D002118:Calcium / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D056148:Chromatography, Reverse-Phase / Q000379:methods; D003001:Cloning, Molecular / Q000379:methods; D018360:Crystallography, X-Ray; D004815:Epidermal Growth Factor / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D051880:Receptors, Notch / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection / Q000379:methods
chemical_list: D051880:Receptors, Notch; D011994:Recombinant Proteins; D004815:Epidermal Growth Factor; D002118:Calcium