Expression in E-coli and purification of recombinant fragments of wild type and mutant human prion protein

被引:34
作者
Corsaro, A
Thellung, S
Russo, C
Villa, V
Arena, S
D'Adamo, MC
Paludi, D
Principe, DR
Damonte, G
Benatti, U
Aceto, A
Tagliavini, F
Schettini, G
Florio, T [1 ]
机构
[1] Natl Inst Canc Res, Adv Biotechnol Ctr, I-16132 Genoa, Italy
[2] Univ Annunzio Chieti, Sect Pharmacol, Dept Biomed Sci, Chieti, Italy
[3] Univ Genoa, Sect Pharmacol, Dept Oncol Biol & Genet, Genoa, Italy
[4] Consorzio Mario Negri Sud, Inst Pharmacol Res Mario Negri, Chieti, Italy
[5] Univ Annunzio Chieti, Sect Biochem, Dept Biomed Sci, Chieti, Italy
[6] Univ Annunzio Chieti, Dept Sci Farmaco, Chieti, Italy
[7] Neurol Inst C Besta, Milan, Italy
[8] Univ Genoa, Dept Expt Med, Genoa, Italy
关键词
recombinant prion protein; glutathione S-transferase-fusion protein; refolding; purification;
D O I
10.1016/S0197-0186(01)00137-1
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Prion diseases are fatal neurodegenerative disorders of the CNS of men and animals, characterized by spongiform degeneration of the CNS, astrogliosis and deposition of amyloid into the brain. The conversion of a cellular glycoprotein (the prion protein, PrPC) into an altered isoform (the prion scrapie, PrPSc), which accumulates within the brain tissue by virtue of its resistance to the intracellular catabolism, is currently believed to represent the etiologic agent responsible for these diseases. Synthetic or recombinant polypeptides are commonly used to elucidate the mechanism of proteins involved in neurodegenerative diseases. Here we describe a procedure, which allows the synthesis and purification in its native folding, of the human prion protein fragment 90-231, corresponding to the protease resistant core of PrPSc. We synthesized the polypeptides 90-231 of both the wild type and the E200K mutant isoforms of PrP. Using a gluthatione S-transferase (GST) fusion protein approach, milligram amounts of polypeptides were obtained after expression in E. coli. The recovery of the purified fusion protein was monitored following the evaluation of the GST activity. The PrP fragment was released from the fusion protein immobilized on a glutathione-coupled agarose resin by direct cleavage with thrombin. The recombinant protein was identified by comassie stained acrylamide gel and by immunoblotting employing a monoclonal anti-PrP antibody. The peptide purified by gel filtration chromatography showed mainly an a-helix structure, as analysed by circular dichroism (CD) and an intact disulfide bridge. The same procedure was also successfully employed to synthesize and purify the E200 K mutant PrP fragment. (C) 2002 Elsevier Science Ltd. All rights reserved.
引用
收藏
页码:55 / 63
页数:9
相关论文
共 20 条
[1]   Recombinant protein expression in Escherichia coli [J].
Baneyx, F .
CURRENT OPINION IN BIOTECHNOLOGY, 1999, 10 (05) :411-421
[2]  
DEGIOIA L, 1994, J BIOL CHEM, V269, P7859
[3]   Intracellular calcium rise through L-type calcium channels, as molecular mechanism for prion protein fragment 106-126-induced astroglial proliferation [J].
Florio, T ;
Grimaldi, M ;
Scorziello, A ;
Salmona, M ;
Bugiani, O ;
Tagliavini, F ;
Forloni, G ;
Schettini, G .
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, 1996, 228 (02) :397-405
[4]   NEUROTOXICITY OF A PRION PROTEIN-FRAGMENT [J].
FORLONI, G ;
ANGERETTI, N ;
CHIESA, R ;
MONZANI, E ;
SALMONA, M ;
BUGIANI, O ;
TAGLIAVINI, F .
NATURE, 1993, 362 (6420) :543-546
[5]   Of mice and (mad) cows: Transgenic mice help to understand prions [J].
Gabizon, R ;
Taraboulos, A .
TRENDS IN GENETICS, 1997, 13 (07) :264-269
[6]   Multiple folding pathways for heterologously expressed human prion protein [J].
Jackson, GS ;
Hill, SF ;
Joseph, C ;
Hosszu, L ;
Power, A ;
Waltho, JP ;
Clarke, AR ;
Collinge, J .
BIOCHIMICA ET BIOPHYSICA ACTA-PROTEIN STRUCTURE AND MOLECULAR ENZYMOLOGY, 1999, 1431 (01) :1-13
[7]   The role of disulfide bridge in the folding and stability of the recombinant human prion protein [J].
Maiti, NR ;
Surewicz, WK .
JOURNAL OF BIOLOGICAL CHEMISTRY, 2001, 276 (04) :2427-2431
[8]   SUB-ACUTE SPONGIFORM ENCEPHALOPATHY (CREUTZFELDT-JAKOB DISEASE) - NATURE AND PROGRESSION OF SPONGIFORM CHANGE [J].
MASTERS, CL ;
RICHARDSON, EP .
BRAIN, 1978, 101 (JUN) :333-344
[9]   CONVERSION OF ALPHA-HELICES INTO BETA-SHEETS FEATURES IN THE FORMATION OF THE SCRAPIE PRION PROTEINS [J].
PAN, KM ;
BALDWIN, M ;
NGUYEN, J ;
GASSET, M ;
SERBAN, A ;
GROTH, D ;
MEHLHORN, I ;
HUANG, ZW ;
FLETTERICK, RJ ;
COHEN, FE ;
PRUSINER, SB .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1993, 90 (23) :10962-10966
[10]   Semipreparative chromatographic method to purify the normal cellular isoform of the prion protein in nondenatured form [J].
Pergami, P ;
Jaffe, H ;
Safar, J .
ANALYTICAL BIOCHEMISTRY, 1996, 236 (01) :63-73