Biochemical and biophysical characterization of extrinsic and intrinsic modulators of the antigen transporter TAP [Elektronische Ressource] / von Gabriele Plewnia

Gabriele Plewnia

Sujets

• Biologie

Informations

• Publié par: goethe_universitat_frankfurt_am_main
• Publié le: 01 janvier 2009
• Nombre de lectures: 28
• Langue: Deutsch
• Poids de l'ouvrage: 8 Mo

Extrait

Biochemical and Biophysical Characterization of Extrinsic
and Intrinsic Modulators of the Antigen Transporter TAP


Dissertation
zur Erlangung des Doktorgrades
der Naturwissenschaften


vorgelegt beim Fachbereich
Biochemie, Chemie und Pharmazie
der Goethe-Universität in Frankfurt am Main



von
Gabriele Plewnia
aus Reutlingen

Frankfurt am Main, 2008





















Vom Fachbereich Biochemie, Chemie und Pharmazie der Goethe-Universität
als Dissertation angenommen.

Dekan: Prof. Dr. Harald Schwalbe
1. Gutachter: Prof. Dr. Robert Tampé
2. Prof. Dr. Bernd Ludwig























In der Wissenschaft gleichen wir alle
nur den Kindern, die am Rande des
Wissens hier und da einen Kiesel
aufheben, während sich der weite
Ozean des Unbekannten vor
unseren Augen erstreckt.
Isaac Newton
(1643-1727)


























Index


INDEX
1. Deutsche Zusammenfassung............................................................................. 1
2. Summary .............................................................................................................. 3
3. Introduction.......................................................................................................... 5
3.1. The immune system and the quest against invaders..................................... 5
3.2. MHC class I antigen processing and presentation pathway ........................ 10
3.2.1. The major histocompatibility complex (MHC)............................................ 10
3.2.1.1. Genomic organization ........................................................................... 10
3.2.1.2. Structural organization .......................................................................... 11
3.2.1.3. Peptide-binding cleft.............................................................................. 12
3.2.2. The antigen transporter TAP..................................................................... 16
3.2.3. Tapasin ..................................................................................................... 18
3.2.4. Auxiliary factors......................................................................................... 23
3.2.4.1. ERp57 ................................................................................................... 23
3.2.4.2. Calnexin and calreticulin ....................................................................... 24
3.2.4.3. The immunoglobulin binding protein (BiP) ............................................ 27
3.2.5. Peptide translocation to the ER................................................................. 27
3.2.6 Assembly of the peptide loading complex................................................. 30
3.3. Objectives .................................................................................................... 33
4. Material ............................................................................................................... 36
4.1. Microorganisms............................................................................................ 36
4.2. Plasmids ...................................................................................................... 36
4.3. Eukaryotic cell lines ..................................................................................... 37
4.4. Antibodies 37
4.5. Oligonucleotides .......................................................................................... 38
4.6. Peptides ....................................................................................................... 39
4.7. Chemicals .................................................................................................... 40
4.8. Enzymes 41
4.9. Equipment 42
4.10. Chromatography material ............................................................................ 43
4.11. Supplementary material ............................................................................... 44
5. Methods .............................................................................................................. 45
5.1. Molecular biology ......................................................................................... 45
5.1.1. Mini preparation of plasmid DNA .............................................................. 45
VII Index


5.1.2. Restriction enzyme digestion .................................................................... 45
5.1.3. Ligation...................................................................................................... 46
5.1.4. Nucleic acid electrophoresis and DNA recovery ....................................... 46
5.1.5. Photometric concentration determination of nucleic acids ........................ 47
5.1.6. Amplification of V and V genes of mAb 148.3 ........................................ 47 H L
5.1.6.1. Poly-mRNA isolation ............................................................................. 47
5.1.6.2. cDNA synthesis..................................................................................... 48
5.1.6.3. Polymerase chain reaction (PCR)......................................................... 48
5.1.6.4. Colony PCR .......................................................................................... 51
5.1.6.5. Bacmid PCR 51
5.1.7. Construction of expression plasmids ........................................................ 52
5.1.7.1. Bacterial expression plasmids for recombinant antibodies ................... 52
5.1.7.2. Insect cell expression plasmids for recombinant antibodies ................. 52
5.1.7.3. ll expression plasmids for HLA-B4402 and HLA-B4405 ......... 53
5.2. Microbiology................................................................................................. 53
5.2.1. Preparation of competent E. coli cells....................................................... 53
5.2.2. Transformation of competent E. coli cells ................................................. 54
5.2.3. Transformation of competent DH10 Bac cells........................................... 54
5.3. Cell culture ................................................................................................... 55
5.3.1. Expression of recombinant antibodies in E. coli........................................ 55
5.3.2. Monolayer culture of Sf9 insect cells......................................................... 55
5.3.3. uTn5 insect cells........................................................ 56
5.3.4. Shaker culture of Sf9 and Tn5 insect cells................................................ 56
5.3.5. Transfection of Sf9 insect cells ................................................................. 56
5.3.6. Amplification of virus ................................................................................. 56
5.3.7. Infection of Sf9 and Tn5 cells for protein production................................. 57
5.3.8. Raji cells.................................................................................................... 57
5.4. Biochemical methods................................................................................... 57
5.4.1. Preparation of microsomes ....................................................................... 57
5.4.2. Preparation of membranes........................................................................ 58
5.4.3. SDS-polyacrylamide-gelelectrophoresis ................................................... 59
5.4.4. Tricine-SDS-polyacrylamide-gelelectrophoresis ....................................... 60
5.4.5. Coomassie and silver staining .................................................................. 60
5.4.6. Immunoblotting.......................................................................................... 61
5.4.7. Stripping of immunoblots........................................................................... 63
5.4.8. Protein G affinity chromatography............................................................. 63
5.4.9. Epitope coupled affinity chromatography .................................................. 63
5.4.10. Streptavidin affinity chromatography......................................................... 64
VIII Index


5.4.10.1. Refolding of core streptavidin................................................................ 64
5.4.10.2. Preparation of streptavidin sepharose .................................................. 65
5.4.10.3. Immobilized streptavidin affinity chromatography ................................. 66
5.4.11. Immobilized metal chelate affinity chromatography .................................. 67
5.4.12. Immobilized antibody fragment affinity chromatography........................... 68
5.4.13. Size exclusion chromatography 69
5.4.14. Generation of proteolytic Fab fragments................................................... 69