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Volumn 283, Issue 5406, 1999, Pages 1339-1343

Prion domain initiation of amyloid formation in vitro from native Ure2p

Author keywords

[No Author keywords available]

Indexed keywords

AMYLOID; PRION PROTEIN;

EID: 0033605278     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.283.5406.1339     Document Type: Article
Times cited : (268)

References (44)
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    • 12, 106 μM ribonuclease A, 54 μM BSA, 23 μM aldolase (Bio-Rad and Pharmacia gel filtration standards) or 54 μM bovine serum albumin alone was prepared in 50 mM tris-HCl containing 0.2 M NaCl (pH 8.0). The aggregation reaction mixtures (126 μl) containing 13 μM Ure2p (17) or 0.16 volume of protein mixture or both in 50 mM tris-HCl (pH 8.0) containing 0.2 M NaCl were filter-sterilized and peptides were diluted into the protein solutions to a final concentration of 13 μM. Controls included 0.14 M guanidine and 13 μM Ure2p without the peptide. Reaction mixtures were gently rocked continuously at 8 °C for 20 hours. The aggregated protein was collected by centrifugation for 1 min at 8500g and washed with buffer. Pellets were boiled for 5 min in 10 μl of SDS-polyacrylamide gel electrophoresis (SDS-PAGE) loading buffer and 30 μl of 8 M urea and then analyzed by SDS-PAGE.
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    • After the Ni-NTA step, Ure2p aggregates were formed on dialysis against <0.15 M NaCl in 50 mM tris-HCl (pH 7.5). However, these aggregates were amorphous and had no filament structure.
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    • 6-URE2 fusion) did not take up ureidosuccinate, showing that the URE2 fusion was functional. [URE3] could be cytoduced from strain 3310 [URE3-1] (MATa kar1 arg1 [URE3]) into strain 3947 containing pKT18 showing that the fusion protein could assume the prion form. These clones, made [ure-o] by growth to single colonies on minimal medium containing 5 mM CuHCl, were used for purification of Ure2p.
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    • note
    • We thank P. McPhie for help with spectroscopy, G. Poy for synthetic peptides, L. Pannell for mass spectrometry, and H. Edskes for plasmid pH7.


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