Expression and purification of a recombinant amyloidogenic peptide from transthyretin for solid-state NMR spectroscopy.
We describe the expression and purification of a model amyloidogenic peptide comprising residues 105-115 of human transthyretin (TTR105-115). Recombinant TTR105-115, which does not contain any non-native residues, was prepared as part of a fusion protein construct with a highly soluble B1 immunoglobulin binding domain of protein G (GB1), with typical yields of approximately 4 mg/L of uniformly (13)C,(15)N-enriched HPLC-purified peptide per liter of minimal media culture. Amyloid fibrils formed by recombinant TTR105-115 were characterized by transmission electron microscopy and solid-state NMR spectroscopy, and found to be comparable to synthetic TTR105-115 fibrils. These results establish recombinant TTR105-115 as a valuable model system for the development of new solid-state NMR techniques for the atomic-level characterization of amyloid architecture.
Department of Chemistry, The Ohio State University, Columbus, OH 43210, USA.
Philippe S
PS
Nadaud
Mohosin
M
Sarkar
Bo
B
Wu
Cait E
CE
MacPhee
Thomas J
TJ
Magliery
Christopher P
CP
Jaroniec
D000595:Amino Acid Sequence; D000682:Amyloid / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D015870:Gene Expression; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D011228:Prealbumin / Q000737:chemistry* / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D000682:Amyloid; D010446:Peptide Fragments; D011228:Prealbumin; D011994:Recombinant Proteins