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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.

Original publication

DOI

10.1007/978-1-4939-1139-4_15

Type

Journal article

Journal

Methods Mol Biol

Publication Date

2014

Volume

1187

Pages

193 - 208

Keywords

Calcium, Chromatography, Affinity, Chromatography, High Pressure Liquid, Chromatography, Reverse-Phase, Cloning, Molecular, Crystallography, X-Ray, Epidermal Growth Factor, Escherichia coli, Humans, Nuclear Magnetic Resonance, Biomolecular, Protein Conformation, Protein Folding, Protein Structure, Tertiary, Proteolysis, Receptors, Notch, Recombinant Proteins, Transfection