Rapid purification of truncated tau proteins: model approach to purification of functionally active fragments of disordered proteins, implication for neurodegenerative diseases

被引:39
作者
Csokova, N
Skrabana, R
Liebig, HD
Mederlyova, A
Kontsek, P
Novak, M
机构
[1] Slovak Acad Sci, Inst Neuroimmunol, Bratislava 84510, Slovakia
[2] Axon Neurosci, A-1030 Vienna, Austria
关键词
Alzheimer's disease; tau protein; natively unfolded proteins; ion-exchange chromatography; gel filtration;
D O I
10.1016/j.pep.2004.01.012
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 10 mg 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. (C) 2004 Elsevier Inc. All rights reserved.
引用
收藏
页码:366 / 372
页数:7
相关论文
共 23 条
[1]   14-3-3 connects glycogen synthase kinase-3β to tau within a brain microtubule-associated tau phosphorylation complex [J].
Agarwal-Mawal, A ;
Qureshi, HY ;
Cafferty, PW ;
Yuan, ZF ;
Han, D ;
Lin, RT ;
Paudet, HK .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2003, 278 (15) :12722-12728
[2]   Tau aggregation into fibrillar polymers: taupathies [J].
Avila, J .
FEBS LETTERS, 2000, 476 (1-2) :89-92
[3]   Toward a unified scheme for the aggregation of tau into Alzheimer paired helical filaments [J].
Barghorn, S ;
Mandelkow, E .
BIOCHEMISTRY, 2002, 41 (50) :14885-14896
[4]   Tau filaments from human brain and from in vitro assembly of recombinant protein show cross-β structure [J].
Berriman, J ;
Serpell, LC ;
Oberg, KA ;
Fink, AL ;
Goedert, M ;
Crowther, RA .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2003, 100 (15) :9034-9038
[5]   The protein trinity - linking function and disorder [J].
Dunker, AK ;
Obradovic, Z .
NATURE BIOTECHNOLOGY, 2001, 19 (09) :805-806
[6]   Intrinsically disordered protein [J].
Dunker, AK ;
Lawson, JD ;
Brown, CJ ;
Williams, RM ;
Romero, P ;
Oh, JS ;
Oldfield, CJ ;
Campen, AM ;
Ratliff, CR ;
Hipps, KW ;
Ausio, J ;
Nissen, MS ;
Reeves, R ;
Kang, CH ;
Kissinger, CR ;
Bailey, RW ;
Griswold, MD ;
Chiu, M ;
Garner, EC ;
Obradovic, Z .
JOURNAL OF MOLECULAR GRAPHICS & MODELLING, 2001, 19 (01) :26-59
[7]   Phosphorylation-mimicking glutamate clusters in the proline-rich region are sufficient to simulate the functional deficiencies of hyperphosphorylated tau protein [J].
Eidenmüller, J ;
Fath, T ;
Maas, T ;
Pool, M ;
Sontag, E ;
Brandt, R .
BIOCHEMICAL JOURNAL, 2001, 357 :759-767
[8]  
Fasulo L., 1996, ALZHEIMERS RES, V2, P195
[9]   Structure of tau protein and assembly into paired helical filaments [J].
Friedhoff, P ;
von Bergen, M ;
Mandelkow, EM ;
Mandelkow, E .
BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR BASIS OF DISEASE, 2000, 1502 (01) :122-132
[10]  
Gamblin TC, 2003, P NATL ACAD SCI USA, V100, P10032, DOI 10.1073/pnas.1630428100