Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region

Matija Popovic, Maristella Coglievina, Corrado Guarnaccia, Giuliana Verdone, Gennaro Esposito, Alessandro Pintar, Sándor Pongor

Research output: Contribution to journalArticle

Abstract

Notch signaling plays a key role in cell differentiation and is very well conserved from Drosophila to humans. Ligands of Notch receptors are type I, membrane spanning proteins composed of a large extracellular region and a 100-150 residue cytoplasmic tail. We report here, for the first time, the expression, purification, and characterization of the intracellular region of a Notch ligand. Starting from a set of synthetic oligonucleotides, we assembled a synthetic gene optimized for Escherichia coli codon usage and encoding the cytoplasmic region of human Jagged-1 (residues 1094-1218). The protein containing a N-terminal His6-tag was over-expressed in E. coli, and purified by affinity and reversed phase chromatography. After cleavage of the His6-tag by a dipeptidyl aminopeptidase, the protein was purified to homogeneity and characterized by spectroscopic techniques. Far-UV circular dichroism, fluorescence emission spectra, fluorescence anisotropy measurements, and 1H nuclear magnetic resonance spectra, taken together, suggest that the cytoplasmic tail of human Jagged-1 behaves as an intrinsically unstructured domain in solution. This result was confirmed by the high susceptibility of the recombinant protein to proteolytic cleavage. The significance of this finding is discussed in relation to the recently proposed role of the intracellular region of Notch ligands in bi-directional signaling.

Original languageEnglish (US)
Pages (from-to)398-404
Number of pages7
JournalProtein Expression and Purification
Volume47
Issue number2
DOIs
StatePublished - Jun 2006

Keywords

  • Circular dichroism
  • Intrinsically unstructured proteins
  • Limited proteolysis
  • NMR
  • Notch signaling
  • Synthetic gene
  • Tryptophan fluorescence

ASJC Scopus subject areas

  • Biotechnology

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