Expression, purification, and glycosylation of epidermal growth factor-like repeat 27 from mouse NOTCH1

Notch receptors have large extracellular domains containing up to 36 tandem epidermal growth factor-like (EGF) repeats, which facilitate cell signaling by binding ligands on neighboring cells. Notch receptors play major roles in a variety of developmental processes by controlling cell fate decisions. Each EGF repeat consists of about 40 amino acids with 3 conserved disulfide bonds. Many of the EGF repeats are modified by O-linked fucose glycans, and more than half have calcium-binding sites, but the sequences of the EGF repeats vary giving distinct roles to each repeat. EGF repeat 27 (EGF27) from mouse NOTCH1 is modified with O-fucose and is 1 of 7 repeats that is differentially modified by specific Fringe enzymes, which are known to regulate NOTCH1 activation and ligand binding. To better understand the role of EGF27 in NOTCH1 function and regulation, the 3-dimensional structures of EGF27 and its glycoforms are being pursued. E. coli cells were used to produce EGF27 in sufficient quantities for nuclear magnetic resonance analysis. Previous attempts to express the repeat alone and refold the repeat under a steady redox environment were unsuccessful due to low yields and extensive mixed-disulfide bond cross-linking. A new strategy using a cleavable maltose binding protein fusion tag increased the solubility and yield of EGF27. With the fusion tag, EGF27 was refolded to produce the correct disulfide bond arrangement, which was verified enzymatically with the glycosyltransferases, Protein O-fucosyltransferase 1 (POFUT1) and Lunatic Fringe (LFNG).