Dynamics of in vitro amyloid fiber formation of yeast prion protein Sup35NM.
- Author:
Hai-yan WEI
1
;
Ying-xia LIU
;
Jian-wei WANG
;
Jian-guo QU
;
Wei-ming ZHAO
;
Xiu-ping YU
;
Tao HONG
Author Information
- Publication Type:Journal Article
- MeSH: Amyloid beta-Peptides; genetics; metabolism; ultrastructure; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; metabolism; Kinetics; Microscopy, Electron; Peptide Termination Factors; Prions; genetics; metabolism; ultrastructure; Protein Binding; Recombinant Proteins; metabolism; ultrastructure; Saccharomyces cerevisiae; genetics; metabolism; Saccharomyces cerevisiae Proteins; genetics; metabolism; ultrastructure; Thiazoles; metabolism
- From: Chinese Journal of Experimental and Clinical Virology 2006;20(1):39-42
- CountryChina
- Language:Chinese
-
Abstract:
BACKGROUNDTo investigate the dynamics of amyloid fiber formation of yeast (Saccharomyces cerevisiae) prion protein Sup35NM under the native condition to provide materials and clues for the elucidation of amyloid fiber formation.
METHODSThe Sup35NM gene was cloned and expressed in E. coli. The recombinant Sup35NM protein was purified under denaturing conditions through Nickel-Sepharose chromatography. Aliquots were removed at designated time points for transmission electron microscopy (TEM), circular dichroism (CD) spectra, protease K resistance assay, as well as thioflavin T (ThT) binding assay.
RESULTSThe Sup35NM expressed and purified under denaturing conditions. The morphological alteration of the Sup35NM in PBS (pH7.4) during the protein aggregation and amyloid fiber formation was visualized by TEM. The CD assay showed that the course of amyloid fiber formation underwent a conformational shift from alpha-helix to beta-sheet. The fibers had higher capacity of resistance to protease K digestion compared to the monomers. ThT fluorescence assay displayed a rapid growth phase before reaching a final equilibrium phase during the fiber formation, and the higher concentration of Sup35NM could greatly accelerate the fiber formation in vitro.
CONCLUSIONYeast prion protein Sup35NM forms amyloid readily under native conditions in vitro. The dynamics of Sup35NM amyloid formation may provide supporting evidences for the nucleating polymerization models of amyloid fiber formation.
