Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region.
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 His(6)-tag was over-expressed in E. coli, and purified by affinity and reversed phase chromatography. After cleavage of the His(6)-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 (1)H 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.
Popovic
Matija
M
International Centre for Genetic Engineering and Biotechnology (ICGEB), Protein Structure and Bioinformatics Group, AREA Science Park, Trieste, Italy.
Coglievina
Maristella
M
Guarnaccia
Corrado
C
Verdone
Giuliana
G
Esposito
Gennaro
G
Pintar
Alessandro
A
Pongor
Sándor
S
D002135:Calcium-Binding Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D002853:Chromatography, Liquid; D002942:Circular Dichroism; D029721:Drosophila Proteins; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D005813:Genes, Synthetic / Q000235:genetics; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000072100:Jagged-1 Protein; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D051880:Receptors, Notch / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000072099:Serrate-Jagged Proteins
D002135:Calcium-Binding Proteins; D029721:Drosophila Proteins; D036341:Intercellular Signaling Peptides and Proteins; C000606273:JAG1 protein, human; D000072100:Jagged-1 Protein; D008565:Membrane Proteins; D051880:Receptors, Notch; D011994:Recombinant Proteins; C000606278:Ser protein, Drosophila; D000072099:Serrate-Jagged Proteins