Production of recombinant human serum albumin in transgenic plants and plant cells [Elektronische Ressource] / vorgelegt von Ayse Meltem Mavituna

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Production of recombinant Human Serum
Albumin in transgenic plants and plant cells



Von der Fakultt? fr M? athematik, Informatik und Naturwissenschaften der
Rheinisch-Westflischen Technischen Hochschule Aachen
zur Erlangung des akademischen Grades einer
Doktorin der Naturwissenschaften
genehmigte Dissertation



vorgelegt von


Master of Science

A. Meltem MAVITUNA
aus
Ankara, T rkei





Berichter: Universitt? sprofessor Dr. rer. nat. Rainer Fischer
Universittsprofessor Dr. rer. nat. Fritz Kreuzaler



Tag der m?ndlichen Pr fung: 30.05.2005


Diese Dissertation ist auf den Internetseiten der Hochschulbibliothek online verf gbar.

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Annelerin en g zeline
While the work for this thesis was being completed, my mother passed away.
I will always be thankful for my mother s encouragement and unconditional love.
This thesis is dedicated with love to the memory of

M beccel Y?CELT MAVITUNA
01.12.1934-27.11.2001
She was a wonderful daughter, sister, mother and best friend to all who knew her ..
Contents
I Introduction .......................................................................................................................1
I.1 Molecular Farming ........................................................................................................2
I.1.1 Choice of the host plant..............................................................................................6
I.1.2 Plant expression cassette design .................................................................................6
I.1.3 Plant Transformation................................................................................................10
I.2 Production of therapeutic proteins in plants...............................................................11
I.2.1 Human Serum Albumin ...........................................................................................13
I.2.2 Recombinant HSA expression..................................................................................15
I.4 Aim of the Study...........................................................................................................18
II Materials and Methods.....................................................................................................22
II.1 Materials .................................................................................................................22
II.1.1 Chemicals and consumables...............................................................................22
II.1.2 Enzymes and reaction kits22
II.1.3 Media stock solutions and buffers ......................................................................23
II.1.4 Antibodies and substrates...................................................................................23
II.1.5 Plasmid Vectors.................................................................................................23
II.1.5.1 pHSA36 and pHSA20623
II.1.5.2 PCR 2.1-TOPO...........................................................................................24
II.1.5.3 pGEM-3(zf)+..............................................................................................24
II.1.5.4 pUC18........................................................................................................24
II.1.5.5 pET 21d(+)24
II.1.5.6 pET 22b(+)25
II.1.5.7 pSSH125
II.1.5.8 pAL7625
II.1.6 Oligonucleotides................................................................................................26
II.1.7 Biological Materials...........................................................................................27
II.1.7.1 Escherichia coli strains ...............................................................................27
II.1.7.2 Agrobacterium tumefaciens.........................................................................27
II.1.7.3 Plants..........................................................................................................27
II.1.7.4 Animals......................................................................................................28
II.1.8 Chromatograhpy columns matrix and membranes ..............................................28
II.1.9 Equipment ..............................................................................................................28
II.2 Molecular Biology Methods....................................................................................30
II.2.1 Transformation, selection and characterisation of recombinant bacteria ..............30
II.2.1.1 Growth and maintenance of Escherichia coli...............................................30
II.2.1.2 Growth and maintenance of Agrobacterium tumefaciens .............................30
II.2.1.3 Preparation of competent E. coli cells for heat-shock transformation ...........31
II.2.1.4 Transformation of E. coli by heat-shock ...........................................................31
II.2.1.5 Preparation of competent E. coli cells for electroporation ............................32
II.2.1.6 Transformation of E. coli by electroporation................................................32
II.2.1.7 Preparation of competent A. tumafaciens cells for electroporation................33
II.2.1.8 Transformation of Agrobacterium tumafaciens by electroporation...............33
II.2.2 Transformation of plants and suspension cultures...............................................33
II.2.2.1 Growth and maintenance of Nicotiana tabacum...........................................33
II.2.2.2 Growth and maintenance of Nicobacum cv. BY-2 cells....................34
II.2.2.3 Preparation of recombinant Agrobacterium. tumafaciens.............................34
II.2.2.4 Transformation of Nicotiana tabacum cv. BY-2 ..........................................35
II.2.2.5 Vacuum infiltration of intact plant leaves ....................................................35
II.2.2.6 Stable transformation of tobacco plants .......................................................36
II.2.2.7 Bombardment of wheat embryos .................................................................37
II.2.3 Recombinant DNA techniques ...........................................................................38
II.2.3.1 Isolation of plasmid DNA............................................................................38
I Contents
II.2.3.1.1 TELT method..............................................................................................38
II.2.3.2 Restriction enzyme digestion and precipitation of DNA...............................38
II.2.3.3 Agarose gel electrophoresis.........................................................................39
II.2.3.4 Determination of the DNA concentration.....................................................39
II.2.3.5 Ligation of DNA fragments39
II.2.3.6 PCR amplification.......................................................................................40
II.2.3.6.1 Colony check PCR......................................................................................41
II.2.3.6.2 SOE-PCR amplification..............................................................................41
II.2.3.7 DNA sequencing.........................................................................................42
II.3 Expression of recombinant proteins.......................................................................42
II.3.1 Bacterial expression...........................................................................................42
II.3.2 Expression of rHSA in the field..........................................................................43
II.4 Protein analysis...........................................................................................................43
II.4.1 Isolation of total soluble proteins from E. coli ....................................................43
II.4.2 Isolation of total soluble proteins from plant leaves ............................................44
II.4.3 Isolation of total soluble proteins from tobacco suspension cultures....................45
II.4.4 Isolation of total soluble proteins from wheat seeds45
II.4.5 Quantification of total soluble proteins ...............................................................45
II.4.6 Gel electrophoresis of proteins ...........................................................................46
II.4.6.1 One-dimensional gel electrophoresis...........................................................46
II.4.6.2 Two-dimensional gel electrophoresis46
II.4.7 Staining of protein gels ......................................................................................47
II.4.7.1 Coomassie brilliant blue staining.................................................................47
II.4.7.2 Silver Staining............................................................................................47
II.4.8 Immunoblot...................................................................................