Production and characterization of the recombinant wheat chitinase Wch1 and generation of chitin-specific antibodies [Elektronische Ressource] / vorgelegt von Siham Agdour

Siham

Sujets

• Biologie

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2007
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 2 Mo

Extrait

"Production and characterization of the recombinant wheat
chitinase Wch1 and generation of chitin-specific antibodies"

Von der Fakultät für Mathematik, Informatik und Naturwissenschaften der Rheinisch-
Westfälischen Technischen Hochschule Aachen zur Erlangung des akademischen Grades
einer Doktorin der Naturwissenschaften genehmigte Dissertation

vorgelegt von
Master of Science
Siham Agdour
Aus Algier, Algerien




Berichter: Universitätsprofessor Dr.rer.nat. R. Fischer
Universitätsprofessor Dr.rer.nat. F. Kreuzaler
Tag der mündlichen Prüfung: 31.08.2007


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verfügbar

Content
I Introduction ..............................................................................................................................1
I.1 Chitinases, chitin and chitosan..............................................................................................1
I.1.1 Chitinases......................................................................................................................1
I.1.1.1 Classification of chitinases...................................................................................1
I.1.1.2 Structure, mechanism and substrate binding of chitinases...................................3
I.1.1.2.1 Structure and mechanism .................................................................................3
I.1.1.2.2 Substrate binding cleft of chitinases.................................................................5
I.1.1.3 The roles of chitinases..........................................................................................6
I.1.1.4 Applications of chitinases ....................................................................................8
I.1.2 Chitin and chitosan:.......................................................................................................9
I.1.2.1 Extraction and crystallographic structure of chitin ..............................................9
I.1.2.2 Biotechnological applications of chitin and chitosan.........................................11
I.2 Antibody engineering..........................................................................................................12
I.2.1 Antibody structure and function..................................................................................12
I.2.2 Generation of carbohydrate-specific antibodies..........................................................13
I.3 Aim of the thesis:................................................................................................................15
II Materials and Methods........................................................................................................20
II.1 Materials...........................................................................................................................20
II.1.1 Chemicals and consumables........................................................................................20
II.1.2 Enzymes and reaction kits...........................................................................................20
II.1.3 Antibodies and substrates............................................................................................20
II.1.4 Bacterial strains...........................................................................................................21
II.1.5 Plants and animals.......................................................................................................22
II.1.6 Phage Libraries............................................................................................................22
II.1.7 DNA-Vectors...............................................................................................................22
II.1.8 Oligonucleotides..........................................................................................................23
II.1.9 Buffers, media and solutions.......................................................................................24
I Content
II.1.10 Matrices and membranes.............................................................................................24
II.1.11 Equipment...................................................................................................................24
II.2 Methods............................................................................................................................26
II.2.1 Recombinant DNA technologies.................................................................................26
II.2.1.1 Preparation of electrocompetent E. coli cells.....................................................26
II.2.1.2 Transformation of E. coli by electroporation .....................................................26
II.2.1.3 Preparation of electrocompetent A. tumefaciens cells........................................26
II.2.1.4 Transformation of A. tumefaciens by electroporation........................................27
II.2.1.5 Growth of recombinant A. tumefaciens and preparation of glycerol stocks ......27
II.2.1.6 Isolation of plasmid DNA from E. coli ..............................................................28
II.2.1.7 Agarose gel electrophoresis of DNA .................................................................28
II.2.1.8 Preparative agarose gel electrophoresis .............................................................28
II.2.1.9 PCR amplification..............................................................................................28
II.2.1.10 DNA sequencing................................................................................................29
II.2.2 Transient and stable transformation of tobacco plants................................................30
II.2.2.1 Transient assay in tobacco leaves by vacuum infiltration..................................30
II.2.2.1.1 Preparation of recombinant agrobacteria .......................................................30
II.2.2.1.2 Vacuum infiltration of intact tobacco leaves..................................................30
II.2.2.2 Recombinant Agrobacterium-mediated stable transformation of tobacco plants
31
II.2.2.3 Growth of N. tabacum cv. Petite Havana SR1...................................................32
II.2.3 Expression and purification of recombinant proteins and monoclonal antibodies .....32
II.2.3.1 Optimization of expression conditions of recombinant Wch1 in bacteria.........32
II.2.3.2 Bacterial expression and purification of Wch1-MBP ........................................33
II.2.3.3 Bacterial expression and purification of his6-tagged Wch1 chitinase ...............34
II.2.3.4 Bacterial expression and purification of Strep-tagged Wch1.............................35
II.2.3.5 Bacterial expression and purification of the chitin-binding domain ..................35
II.2.3.6 Expression and purification of Wch1 chitinase from plants ..............................36
II Content
II.2.3.7 Purification of monoclonal antibodies through Protein G matrix ......................37
II.2.4 Protein analysis............................................................................................................38
II.2.4.1 Extraction of total soluble proteins from plant leaves........................................38
II.2.4.2 Quantification of proteins...................................................................................38
II.2.4.3 SDS-PAA gel electrophoresis and Coomassie brilliant blue staining................38
II.2.4.4 Immunoblot analysis..........................................................................................39
II.2.5 Characterization of chitinase activity ..........................................................................40
II.2.5.1 Colorimetric activity assay.................................................................................40
II.2.5.2 Optimum pH and temperature............................................................................40
II.2.5.3 Degradation of glycol-chitin in an overlay gel...................................................41
II.2.5.3.1 Preparation of glycol-chitin substrate ............................................................41
II.2.5.3.2 Isoelectric focusing (IEF) gel and overlayer substrate gel.............................41
II.2.5.4 Degradation of colloidal chitin...........................................................................42
II.2.5.5 Thin layer chromatography (TLC).....................................................................42
II.2.5.6 Extended depolymerization of chitosan .............................................................43
II.2.5.7 HPLC analysis....................................................................................................43
1II.2.5.8 H-NMR spectroscopy analysis .........................................................................43
II.2.5.9 Hydrolysis of (GlcNAc) oligosaccharide by Wch1 chitinase...........................44 6
II.2.5.10 Determination of the anomeric form of the hydrolytic products .......................44
II.2.5.11 Investigation of the anti-fungal activity of Wch1 ..............................................45
II.2.5.11.1 Isolation of fungal spores ...................................................................