Expression and purification of a recombinant peptide from the Alzheimer's beta-amyloid protein for solid-state NMR.
Fibrillar protein aggregates contribute to the pathology of a number of disease states. To facilitate structural studies of these amyloid fibrils by solid-state NMR, efficient methods for the production of milligram quantities of isotopically labeled peptide are necessary. Bacterial expression of recombinant amyloid proteins and peptides allows uniform isotopic labeling, as well as other patterns of isotope incorporation. However, large-scale production of recombinant amyloidogenic peptides has proven particularly difficult, due to their inherent propensity for aggregation and the associated toxicity of fibrillar material. Yields of recombinant protein are further reduced by the small molecular weights of short amyloidogenic fragments. Here, we report high-yield expression and purification of a peptide comprising residues 11-26 of the Alzheimer's beta-amyloid protein (Abeta(11-26)), with homoserine lactone replacing serine at residue 26. Expression in inclusion bodies as a ketosteroid isomerase fusion protein and subsequent purification under denaturing conditions allows production of milligram quantities of uniformly labeled (13)C- and (15)N-labeled peptide, which forms amyloid fibrils suitable for solid-state NMR spectroscopy. Initial structural data obtained by atomic force microscopy, electron microscopy, and solid-state NMR measurements of Abeta(11-26) fibrils are also presented.
Sharpe
Simon
S
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892-0520, USA. SimonS@intra.niddk.nih.gov
Yau
Wai-Ming
WM
Tycko
Robert
R
D000595:Amino Acid Sequence; D000682:Amyloid / Q000096:biosynthesis / Q000648:ultrastructure; D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003488:Cyanogen Bromide / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D006639:Histidine / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D010446:Peptide Fragments / Q000235:genetics / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013251:Steroid Isomerases / Q000235:genetics
D000682:Amyloid; D016229:Amyloid beta-Peptides; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C033223:polyhistidine; D006639:Histidine; D013251:Steroid Isomerases; D003488:Cyanogen Bromide