Influence of single amino acid polymorphisms on the in vitro convertibility of goat prion protein [Elektronische Ressource] / vorgelegt von Elizabeth Ortega-Soto

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Sujets

• Biologie
• Gesundheit

Informations

• Publié par: ernst-moritz-arndt-universitat_greifswald
• Publié le: 01 janvier 2010
• Nombre de lectures: 10
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Influence of single amino acid polymorphisms on
the in vitro convertibility of goat prion protein


I n a u g u r a l d i s s e r t a t i o n

zur
Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.)
an der Mathematisch-Naturwissenschaftlichen Fakultät
der
Ernst-Moritz-Arndt-Universität Greifswald


vorgelegt von
Elizabeth Ortega-Soto

geboren am 24.04.1980
in Zacatlán Puebla, Mexiko

Insel Riems, Juli 2010

































Dekan: Prof. Dr. Klaus Fesser


1. Gutachter:
Prof. Dr. Dr. h. c. Thomas Mettenleiter

2. Gutachter:
Prof. Dr. Detlev Riesner


Tag der Promotion:
23. 09. 2010

Table of Contents


1. Objectives of the work ___________________________________________________ 1
2. Introduction ___________________________________________________________ 3
2.1 Prio n diseases ___________________________________________________________ 3
2.1.1 Prion diseases in animals ________________________________________________________ 3
2.1.1.1 Scrapie in sheep and goats __________________________________________________ 3
2.1.1.2 Bovine Spongiform Encephalopathy in cattle ___________________________________ 4
2.1.1.3 Other prion diseases in animals ______________________________________________ 5
2.1.2 Prion diseases in humans ________________________________________________________ 6
2.1.2.1 Creutzfeldt-Jakob Disease (CJD) _____________________________________________ 6
2.1.2.2 Kuru ___________________________________________________________________ 6
2.1.2.3 Gerstmann-Sträussler-Scheinker disease (GSS) and Fatal Familiar Insomnia (FFI) ______ 7
2.2 The Prion Hypothesis: Two different isoforms of one protein ____________________ 7
C2.2.1 PrP ________________________________________________________________________ 7
C2.2.1.1 Structural characteristics of PrP _____________________________________________ 7
C2.2.1.2 PrP fun c t i on ____________________________________________________________ 9
Sc2.2.2 Characteristics of PrP ________________________________________________________ 10
Sc2.2. 3 PrP as the causal agent of TSEs ________________________________________________ 11
Sc2.2.4 Replication of PrP ___________________________________________________________ 12
2.3 Genetics in TSE ________________________________________________________ 13
2.3.1 Influence of mutations of human PrP gene in prion diseases ___________________________ 14
2.3.2 Polymorphisms in sheep that modulate the susceptibility to scrapie ______________________ 14
2.3.3 PRNP polymorphism in goats ___________________________________________________ 16
Sc2.4 P r P propagation models ________________________________________________ 19
3. Materials and methods __________________________________________________ 22
3.1 M at e r i al _______________________________________________________________ 22
3.1.1 Bacteria, cells and plasmids ____________________________________________________ 22
3.1.2 Primers for PCR _____________________________________________________________ 22
3.1.3 E n z y m e s ___________________________________________________________________ 24
3.1.3.1 DNA modification enzymes _______________________________________________ 24
3.1.3.2 Protein modification enzymes ______________________________________________ 24
3.1.4 Antibodies __________________________________________________________________ 24
3.1.5 Prion strains and isolates _______________________________________________________ 25
3.1.5.1 Mouse-passaged strains ___________________________________________________ 25
3.1.5.2 Isolates ________________________________________________________________ 25
3.2 Methods _______________________________________________________________ 26
3.2.1 Molecular biological methods ___________________________________________________ 26
3.2.1.1 Insertion of point mutations by PCR _________________________________________ 26
3.2.1.2 Digestion of DNA with restriction enzymes ___________________________________ 27
3.2.1.3 Preparation of the vector for ligation _________________________________________ 27 Page I
3.2.1.4 Ligation of DNA fragments ________________________________________________ 28
3.2.1.5 Production of competent cells ______________________________________________ 28
3.2.1.6 T r a n s for m a t i on of E. coli __________________________________________________ 29
C3.2.1.7 Selection of constructs carrying the DNA of interest (PrP from goat) _______________ 29
3.2.1.8 Preparation of plasmid DNA from a bacterial culture ____________________________ 29
3.2.2 Biochemical methods _________________________________________________________ 30
C3.2.2.1 Expression of PrP in E . c ol i M15 ___________________________________________ 30
C3.2.2.2 Purification of the bacterial PrP ____________________________________________ 30
3.2.2.3 SDS polyacrylamide gel electrophoresis (SDS-PAGE) ___________________________ 30
3.2.2.4 Western blot (immunoblot) ________________________________________________ 31
3.2.2.5 Protein quantification (method by Roth) ______________________________________ 31
3.2.2.6 CD-Spectra ____________________________________________________________ 31
3.2.3 Cell culture _________________________________________________________________ 32
3.2.3.1 N2a cell culture _________________________________________________________ 32
3.2.3.2 Freezing and thawing cells ________________________________________________ 32
3.2.3.3 Transfection of N2a cells __________________________________________________ 32
3.2.4 Fluorescent activated cell sorting (FACS)__________________________________________ 33
3.2.5 Deglycosylation of glycoproteins ________________________________________________ 33
3.2.5.1 Cell homogenates________________________________________________________ 33
C3.2.5.2 Deglycosylation of PrP __________________________________________________ 33
3.2.5.3 Phospatidyl-inositol Phospholipase C release __________________________________ 34
3.2.6 Experimental part of the work ___________________________________________________ 34
C3.2.6.1 Biotinylation of recombinant PrP ___________________________________________ 34
Sc3.2.6.2 Isolation of PrP from different sources ______________________________________ 35
C Sc 3.2.6.3 Cell-fr e e c on ve r s i on of P r P into PrP with different strains ______________________ 35
3.2.6.4 Generation of plasmids MoPrP.Xho N146S and N146D __________________________ 36
C C3.2.6.5 Construction of pcDNA3.1Zeo-PrP plasmids for expression of caprine PrP in N2a cells 37
4. Results _______________________________________________________________ 39
C4.1 Prokaryotic expression of recombinant PrP ________________________________ 39
4.1.1 Cloning of PRNP haplotypes ___________________________________________________ 40
4.1.2 Expression and purification of recombinant prion proteins_____________________________ 42
C4.2 Characterization of recombinant PrP _____________________________________ 43
C4.2.1 Antigenic characterization of PrP _______________________________________________ 43
C4.2.2 CD-Spectra of PrP ___________________________________________________________ 44
Sc4.3 Characterization of PrP is o l at e s __________________________________________ 44
C Sc4.4 In vitro conversion of PrP into PrP using different strains ___________________ 47
4.4.1 Mouse-passaged strains ________________________________________________________ 47
4.4.1.1 Cell-free conversion with Me7 _____________________________________________ 47
4.4.1.2 Cell-free conversion with BSE/Bl6 __________________________________________ 56
4.4.1.3 Biotinylation of recombinant prion protein ____________________________________ 58
4.4.2 Scrapie isolates ______________________________________________________________ 63
4.4.3 BSE isolates ________________________________________________________________ 66
4.5 Dominant negative effect _________________________________________________ 69
4.6 Generation of transgenic mice carrying N146D and N146S polymorphisms _______ 70
C4.7 Expression of PrP i n N 2 a ________________________________________________ 71
4.7.1 Cloning of PRNP in pBluescript 3.1 zeo ___________________________________________ 71 Page II
C4.7.2 Expression of PrP in N2a cells _________________________________________________ 72
C4.7.3 Expression of PrP in cell membranes ____________________________________________ 73
C4.7.4 G l yc os yl a t i on of P r P in cell culture ______________________________________________ 73
5. Discussion ____________________________________________________________ 75
C5.1 Effect of caprine polymorphisms on the in vitro conversion of PrP _____________ 75
5.1.1 Cell free conversion using mouse adapted Me7 scrapie prions __________________________ 76
5.1.2 Cell-free conversion using mouse-adapted BSE prions _______________________________ 78
5.1.3 Cell-free conversion with biotinylated PrP haplotypes with scrapie and BSE isolates ________ 7