Recombinant expression of molluscan hemocyanin (KLH) substructures in a prokaryotic system: E. coli [Elektronische Ressource] / Valesca Boisguérin

Boisv000

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2006
• Nombre de lectures: 27
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Recombinant expression of molluscan hemocyanin
(KLH) substructures in a prokaryotic system: E. coli



D i s s e r t a t i o n
Zur Erlangung des Grades
„D o k t o r d e r N a t u r w i s s e n s c h a f t e n“



Am Fachbereich Biologie der Johannes Gutenberg-Universität
in Mainz



Valesca Boisguérin
geb. in Tunis/ Tunesien



Mainz, 2006



































Dekan:

1. Berichterstatter:

2. Berichterstatter:

Tag der mündlichen Prüfung: 14.09.2006Index I
Index
A INTRODUCTION .........................................................................1
1 Respiratory proteins............................................................................................ 1
2 Molluscan hemocyanins ...................................................................................... 2
2.1 The crystal structure of FU-g of Octopus dofleini and FU-2e of Rapana
thomasiana elucidates the relationship between structure and function................... 4
2.2 The hemocyanin of most gastropods forms didecamers and sometimes
multidecamers............................................................................................................ 5
3 The hemocyanin of the Keyhole Limpet Megathura crenulata........................ 6
4 Clinical relevance of KLH................................................................................... 7
5 Carbohydrates of KLH ....................................................................................... 9
6 Goal of the present work 10
B MATERIALS AND METHODS..................................................12
1 Animals ............................................................................................................... 12
2 Applied chemicals and equipments.................................................................. 13
3 General precautions........................................................................................... 14
4 Common microbiological methods................................................................... 14
4.1 Medium and Agar plates ............................................................................. 14
4.2 Bacterial strains and vectors....................................................................... 15
4.3 Bacterial cultures........................................................................................ 15
- Plate cultures .............................................................................................................................. 15
- Overnight cultures...................................................................................................................... 16
- Glycerine cultures........... 16
- Chemically competent bacterial cells......................................................................................... 16
4.4 Propagating the vectors .............................................................................. 16
5. Common molecular biological methods........................................................... 17
5.1 Total RNA extraction................................................................................... 17
5.2 Primer design.............................................................................................. 17
5.3 Polymerase chain reaction (PCR) and its variations.................................. 18
- Standard PCR ............................................................................................................................. 18
- att-site PCR ................................................................................................................................ 20
- Clone PCR.................................................................................................................................. 21
- Sequencing PCR......................................................................................................................... 21 Index II
- SOE-PCR (Splicing by Overlap Extension)............................................................................... 22
- Asymmetric PCR........................................................................................................................ 23
- Touchdown PCR ........................................................................................................................ 23
5.4 Reverse transcription.................................................................................. 24
5.5 RT-PCR....................................................................................................... 25
5.6 PCR clean up............................................................................................... 25
5.7 DNA gel electrophoresis ............................................................................. 26
- Solutions..................................................................................................................................... 26
- Documentation and evaluation................................................................................................... 26
5.8 DNA extraction from agarose gels.............................................................. 27
- Gel extraction Kits ..................................................................................................................... 27
- Freeze and squeeze..................................................................................................................... 28
5.9 Digestion of nucleic acids with S1 nuclease ............................................... 28
5.10 Phenol/chloroform extraction of nucleic acids ........................................... 28
5.11 Cloning........................................................................................................ 28
- Ligation ...................................................................................................................................... 29
- TOPO cloning ............................................................................................................................ 29
- Cloning without UV damage...................................................................................................... 30
- Transformation........................................................................................................................... 31
- Lethal gene selection.......... 31
5.12 Plasmid isolation......................................................................................... 31
5.13 DNA digestion using restriction endonucleases.......................................... 32
6. Routine protein biochemical methods ............................................................. 34
6.1 Determination of hemocyanin concentration and absorption spectrum..... 34
6.2 Dialysis........................................................................................................ 34
6.3 Polyacrylamide gel electrophoresis (PAGE) .............................................. 34
- SDS-PAGE................................................................................................................................. 35
- Native PAGE.............................................................................................................................. 36
- Set up of the gels ........................................................................................................................ 36
6.4 Western blot................................................................................................ 38
- Transfer of electrophoresed proteins in an SDS-PAGE ............................................................. 38
- Transfer of elsed proteins in a native PAGE 39
- Blocking ..................................................................................................................................... 40
- Detection and analysis................................................................................................................ 40
- Dot blot....................................................................................................................................... 41
6.5 Two dimensional immunoelectrophoresis................................................... 41
6.6 Carbohydrate digestion via N-glycosidase ................................................. 42
6.7 Glycan detection.......................................................................................... 43 Index III
7 Recombinant protein expression in E. coli...................................................... 43
7.1 Gateway™ Technology............................................................................... 43
- The basis of Gateway™ Technology ......................................................................................... 44
- Gateway recombination reactions .............................................................................................. 46
- Common features of the Gateway™ vectors.............................................................................. 47
- Designing attB-PCR primers.............................