Expression and characterisation of biopharmaceuticals in heterologous expression systems [Elektronische Ressource] / vorgelegt von Antje Pätz

Joerg_Nae

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• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2005
• Nombre de lectures: 8
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

Expression and characterisation of
biopharmaceuticals in heterologous expression systems


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




vorgelegt von





Master of Science (Biotechnology)
Antje Pätz
aus Berlin




Referent: Prof. Rainer Fischer, Biologie VII, RWTH Aachen
Co-Referent: Prof. Klaus Ritter, Medizinsche Mikrobiologie, RWTH Aachen

Tag der mündlichen Prüfung: 12.09.2005
















































Nicht der ist ein Verlierer, der hinfällt, sondern der, der liegenbleibt.
unbekannt Table of contents A
Table of contents
I. Introduction ___________________________________________________________ 1
I.1 Human therapeutic proteins ............................................................................................... 1
I.1.1 Monoclonal antibodies for HIV-therapy ......................................................................................... 1
I.1.1.1 General features of viral replication and infection __________________________________ 1
I.1.1.2 The Human Immunodeficiency Virus (HIV) ______________________________________ 2
I.1.1.3 Neutralising antibodies to HIV_________________________________________________ 5
I.1.2 Cell surface receptor as therapeutic target....................................................................................... 8
I.1.2.1 Fc γR receptors _____________________________________________________________ 8
I.1.2.2 Fc gamma receptor I (Fc γRI) 10
I.2 Expression of human therapeutic proteins in heterologous expression systems.......... 12
I.2.1 Bacterial expression ...................................................................................................................... 12
I.2.2 Mammalian expression...... 14
I.2.3 Plant expression ............................................................................................................................ 15
I.3 Goal of this thesis ............................................................................................................... 19
II. Material and methods __________________________________________________ 21
II.1 Material............................................................................................................................... 21
II.1.1 Chemicals and consumables.......................................................................................................... 21
II.1.2 Media, stock solutions and buffers................................................................................................ 21
II.1.2.1 General solutions __________________________________________________________ 21
II.1.2.2 Media for bacterial cultivation ________________________________________________ 23
II.1.2.3 Media for the cultivation of mammalian cells ____________________________________ 23
II.1.2.4 Media for the cultivation of A.tumefaciens and plant cells___________________________ 23
II.1.3 Enzymes and reaction kits....... 24
II.1.4 Antibodies and antigens ................................................................................................................ 25
II.1.4.1 Antibodies _______________________________________________________________ 25
II.1.4.2 Antigens _________________________________________________________________ 26
II.1.5 Synthetic oligonucleotides ............................................................................................................ 26
II.1.6 Recipients of recombinant DNA ...................................................................................................26
II.1.6.1 Bacterial strains ___________________________________________________________ 26
II.1.6.2 Mammalian cells 26
II.1.6.3 Agrobacteria strain _________________________________________________________ 27
II.1.6.4 Plants and plant cells _______________________________________________________ 27
II.1.7 Vectors .......................................................................................................................................... 27
II.1.7.1 pMT-M12________________________________________________________________ 27
II.1.7.2 pMS-ILAng ______________________________________________________________ 28
II.1.7.3 pTRAkc-AH 28
II.1.7.4 pTRAkc-ERH_____________________________________________________________ 29
II.1.7.5 pTRAkt-rfp_______________________________________________________________ 29
II.1.8 Equipment and applications .......................................................................................................... 30
II.1.9 Approbation for the conducted work............................................................................................. 32
II.2 Methods....... 32
II.2.1 Recombinant DNA technologies................................................................................................... 32
II.2.1.1 Isolation of plasmid-DNA from E.coli __________________________________________ 32
II.2.1.2 Polymerase-Chain-Reaction __________________________________________________ 32
II.2.1.3 PCR based analysis of recombinant bacterial clones _______________________________ 33
II.2.1.4 Total RNA extraction from whole blood ________________________________________ 33
II.2.1.5 Specific RT-PCR for cDNA synthesis 34
II.2.1.6 DNA sequencing and sequencing analysis_______________________________________ 34
II.2.1.7 Agarose gel electrophoresis 35
II.2.1.8 Quantification of nucleic acids________________________________________________ 35
II.2.1.9 Restriction digest of DNA ___________________________________________________ 36
II.2.1.10 Dephosporylation __________________________________________________________ 36
II.2.1.11 Klenow Fill-in ____________________________________________________________ 36
II.2.1.12 Ligation of DNA 36 Table of contents B
II.2.2 Bacterial expression system (E.coli) ............................................................................................. 36
II.2.2.1 Heat-shock competent E.coli _________________________________________________ 36
II.2.2.2 Heat-shock transformation of E.coli____________________________________________ 36
II.2.2.3 Culturing of E.coli and long-term storage _______________________________________ 37
II.2.2.4 Expression of rsCD64 in E.coli _______________________________________________ 37
II.2.3 Plant expression system (Nicotiana tabacum) .............................................................................. 38
II.2.3.1 Competent Agrobacterium cells 38
II.2.3.2 Transformation of Agrobacterium cells _________________________________________ 38
II.2.3.3 Cultivation of A.tumefaciens and long-term storage _______________________________ 38
II.2.3.4 Transient expression of rsCD64 38
BY-2 BY-2II.2.3.5 Expression of 2F5 and 2G12 in transgenic BY-2 cells ________________________ 39
II.2.4 Mammalian expression system (HEK 293T) ................................................................................ 40
II.2.4.1 Transfection of HEK 293T cells 40
II.2.4.2 Cultivation and long-term storage of transfected HEK 293Tcells _____________________ 41
II.2.5 Purification of recombinant proteins............................................................................................. 41
2+II.2.5.1 Purification via Ni NTA ____________________________________________________ 41
II.2.5.2 Purification via Protein-A 43
II.2.6 Large-scale protein production in 7-L and 140-L bioreactors....................................................... 43
II.2.6.1 Off-line data collection______________________________________________________ 44
II.2.6.2 Determination of substrate concentration________________________________________ 45
II.2.6.3 Determination of Phosphate __________________________________________________ 45
II.2.6.4 Downstream processing of 100-L fermentation ___________________________________ 45
II.2.7 Protein analysis ............................................................................................................................. 46
II.2.7.1 Quantification of proteins____________________________________________________ 46
II.2.7.2 SDS-PAGE and Westernblot analysis __________________________________________ 46
II.2.7.3 Dot blot analysis___________________________________________________________ 47
II.2.7.4 Enzyme-linked Immunosorbent Assay (ELISA) __________________________________ 47
II.2.7.5 Electrophoretic mobility shift assay (EMSA)_____________________________________ 48
II.2.7.6 Surface plasmon resonance (SPR) analysis ______________________________________ 48
II.2.7.7 DsRed fluorescence ________________________________________________________ 49
II.2.7.8 Mass Spectrometry _________________________________________________________ 49
III. Results ______________________________________________________________ 50
B