Rapid purification of truncated tau proteins: model approach to purification of functionally active fragments of disordered proteins, implication for neurodegenerative diseases.
Truncated tau is of great interest because of its important role in neurofibrillary pathogenesis in Alzheimer's disease (AD). A major obstacle for characterization of detailed biochemical and biological properties of truncated tau species and their fragments has been the lack of reliable and quick purification methods. Uneven distribution of acidic and basic residues in tau determines that the N- and C-terminal tau fragments require entirely different purification conditions. Conventional methods take several days; they do not allow purification of the acidic N-terminal tau fragments and do not prevent aggregation during purification that makes purified truncated tau unusable in functional studies. To prevent these inherent problems, we have designed a two-step, highly efficient purification procedure yielding a fully functional, non-aggregated homogeneous population of truncated tau molecules. Various forms of tau produced in bacteria without the need for a heat pre-treatment step were subjected to anion- and cation-exchange chromatography. Conditions were developed that allowed effective separation and purification of acidic and/or basic tau species. Following the gel filtration step, up to 10mg of tau proteins with 96% purity was obtained within one working day. Purified truncated tau exhibited an unmodified immunoreactivity and allowed its functional activity analysis. Since many neurodegenerative diseases have implicated similar disordered proteins in their pathogenesis, our procedure will allow their detailed analysis and characterization.
Csokova
Natalia
N
Institute of Neuroimmunology Slovak Academy of Sciences, Dubravska cesta 9, 845 10 Bratislava, Slovakia.
Skrabana
Rostislav
R
Liebig
Hans-Dieter
HD
Mederlyova
Anna
A
Kontsek
Peter
P
Novak
Michal
M
D015153:Blotting, Western; D002850:Chromatography, Gel / Q000379:methods*; D002852:Chromatography, Ion Exchange / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D019636:Neurodegenerative Diseases / Q000503:physiopathology*; D016875:tau Proteins / Q000302:isolation & purification* / Q000502:physiology
D016875:tau Proteins