Engineering of an enhanced synthetic Notch receptor by reducing ligand-independent activation

Commun Biol. 2020 Mar 13;3(1):116. doi: 10.1038/s42003-020-0848-x.

Abstract

Notch signaling is highly conserved in most animals and plays critical roles during neurogenesis as well as embryonic development. Synthetic Notch-based systems, modeled from Notch receptors, have been developed to sense and respond to a specific extracellular signal. Recent advancement of synNotch has shown promise for future use in cellular engineering to treat cancers. However, synNotch from Morsut et al. (2016) has a high level of ligand-independent activation, which limits its application. Here we show that adding an intracellular hydrophobic sequence (QHGQLWF, named as RAM7) present in native Notch, significantly reduced ligand-independent activation. Our enhanced synthetic Notch receptor (esNotch) demonstrates up to a 14.6-fold reduction in ligand-independent activation, without affecting its antigen-induced activation efficiency. Our work improves a previously reported transmembrane receptor and provides a powerful tool to develop better transmembrane signaling transduction modules for further advancement of eukaryotic synthetic biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antigens / metabolism
  • Cell Engineering / methods*
  • Cell Membrane / metabolism
  • Cloning, Molecular / methods
  • HEK293 Cells
  • Humans
  • Ligands
  • Plasmids / genetics
  • Protein Domains
  • Proteolysis
  • Receptors, Artificial / chemistry*
  • Receptors, Artificial / metabolism*
  • Receptors, Notch / chemistry*
  • Receptors, Notch / genetics
  • Receptors, Notch / metabolism*
  • Signal Transduction / genetics
  • Single-Chain Antibodies
  • Synthetic Biology / methods
  • Transfection

Substances

  • Antigens
  • Ligands
  • Receptors, Artificial
  • Receptors, Notch
  • Single-Chain Antibodies