Notch1 proteins function as transmembrane receptors that receive intracellular signals and determine the cell specification by binding to the membrane bound ligands. The Notch1 protein consists of 36 epidermal growth factor-like (EGF-like) repeats. EGF-like repeats are small protein domains made up of about 40 amino acids that are held together by three disulfide bonds and are known to be located in tandem and in long chains. The main focus of this study is the Abruptexregion (EGF24-29), which regulates Notch1 signaling and ligand binding by interacting with EGF11-12, the minimum ligand binding domain. EGF25 was fused with the maltose binding protein (MBP), a fusion protein expressed in the cytoplasm with high solubility and expression yield, separated with a human rhinovirus (HRV) 3C protease recognition sequence. Using C3030 Shuffle lysYcells, the MBP-EGF25 fusion was successfully expressed and purified based on SDS-PAGE results. Cleavage of EGF25 from MBP with HRV 3C protease was optimized, and EGF25 and MBP were separated using size exclusion chromatography. Purification of EGF25 isomers was achieved using reverse-phase HPLC with MALDI-MS analysis, resulting in the isolation of three isoforms.

Expression, purification, and separation of Notch1 EGF25

2019 Spring National Meeting

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