Structural study of human antibody fragments with specificity for mucin-1 antigen [Elektronische Ressource] / vorgelegt von Rajneesh Kumar Gaur

Gaur

Sujets

• Gesundheit

Informations

• Publié par: rheinisch-westfalischen_technischen_hochschule_-rwth-_aachen
• Publié le: 01 janvier 2004
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Structural Study of Human Antibody
Fragments with Specificity
for Mucin-1 Antigen





Von der Fakultät für Mathematik, Informatik and Naturwissenschaften
der Rheinisch-Westfälischen Technischen Hochschule Aachen zur Erlangung
des akadmischen Grade eines Doktors der Naturwissenschaften
genehmigte Dissertation




vorgelegt von

Master of Technology
Rajneesh Kumar Gaur
aus
Neu-Delhi, Indien



Berichter: Universitätsprofessor Dr. rer. nat. Rainer Fischer tätsor Dr. rer. nat. Fritz Kreuzaler



Tag der mündlichen Prüfung: 27. September 2004

Diese Dissertation ist auf Internetseiten der Hochschulbibliothek online verfügbar













‘Man has never learned to simplify, only to complicate.
Often he does not say what he feels but he thinks- and
sometimes he thinks too much and, in the process, gains
much knowledge but also larger confusion and greater
grief’
From ‘Songs of Ganga’-5455 BC

















Dedicated to India
iCONTENTS

I INTRODUCTION---------------------------------------------------------------------------------1
I.1 Antibodies-------------------------------------------------------------------------------------1
I.1.1 Overview------------------------------------------------------
I.1.2 Antibody engineering----------------------------------------4
I.1.3 Therapeutic potential of antibodies ------------------------ 8
I.1.4 Recombinant small antibody fragments ------------------ 10
I.1.5 Importance of antibody fragments-----------------------------------------------------11
I.2 The human V family-------------------------------------------- 11 H
I.2.1 Variable heavy chain domain ------------------------------- 12
I.2.1.1 General structure-------------------------------------------------------------------12
I.2.1.2 Diversity of the V domain -------------------------------------------------------H
I.2.1.3 Conformations of the hypervariable loops --------- 13
I.2.1.4 V domain stability ------------------------------------ 15 H
I.2.2 Comparison between human V and camelids V H domains---------------------- 15 H H
I.2.2.1 Sequence differences----------------------------------- 15
I.2.2.2 Structural differences----------------------------------16
I.2.3 Camelization of the V domain ---------------------------- 17 H
I.3 Mucin-------------------------------------------------------------18
I.3.1 Overview-------------------------
I.3.2 Human mucin-1----------------------------------------------19
I.3.3 MUC1 and cancer ------------------------------------------------------------------------ 21
I.3.4 Antibodies against MUC1----------------------------------- 22
I.4 Research objectives-------------------------------------------------------------------------23
II MATERIALS AND METHODS--------26
II.1 Materials-----------------------------
II.1.1 Chemicals---------------------------------------------------26
II.1.2 Laboratory consumables--------------------------------------------------------------
II.1.3y equipment--------26
II.1.4 Chromatography columns--------------------------------27
II.1.5 Media, stock solutions and buffers----------------------
II.1.6 Antibodies, antisera and peptides-- 29
II.1.7 Bacterial strains------------------------------------------------------------------------29
II.1.8 Software--------------------------------------------------------------------------------
II.2 Methods-------------------------------30
II.2.1 Recombinant DNA techniques -------------------------
II.2.1.1 Analytical gel electrophoresis ----------------------- 30
II.2.1.2 Preparative gel electrophoresis --------------------------------------------------- 30
II.2.1.3 PCR amplification of DNA segments ------------------------------------------- 31
II.2.1.3.1 Native M12-V domain ------------------------------------------------------ 31 H.2 Camelized M12-V domain- 32 H
II.2.1.4 DNA digestion-----------------------------------------32
II.2.1.5 Ligation of DNA inserts -- 33
II.2.1.6 PCR based analysis of recombinant bacterial clones -------------------------- 34
II.2.1.7 Isolation of plasmid DNA from E. coli ------------------------------------------ 35
II.2.1.8 DNA sequencing and sequence analysis ---------------------------------------- 35
II.2.1.9 Preparation of E. coli competent cells ------------------------------------------- 35
II.2.1.10 Preparation of heat shock E. coli competent cells- 35
II.2.1.10.1 Transformation of heat shock E. coli competent cells ------------------ 36
iII.2.1.10.2 Efficiency of bacterial transformation------------------------------------- 36
II.2.1.10.3 Culturing and glycerol stock preparation ----- 36
II.2.2 Bacterial expression of antibody fragments---------------------------------------- 36
II.2.2.1 Small scale expression--------------------------------37
II.2.2.2 Large scale expression of scFvM12---------------------------------------------- 37
II.2.3 Purification of antibody fragments-------------------------------------------------- 38
II.2.3.1 scFvM12 purification----------------------------------38
II.2.3.1.1 Periplasmic extraction----------------------------38
II.2.3.1.2 Ni-NTA chromatography-----------------------------------------------------38
II.2.3.1.3 Cation-exchange chromatography------------------------------------------- 38
II.2.3.1.4 Anion-exchange purification-------------------------------------------------39
II.2.3.1.5 Size-exclusion chromatography --------------------------------------------- 39
II.2.3.2 Purification of the cytoplasmic M12-V fragment- 39 H
II.2.3.3 M12-V fragment inclusion bodies ---------------------------------------------- 39 H
II.2.3.3.1 Soluble M12-V fragment purification --------- 40 H
II.2.4 Protein analysis-------------------------------------------40
II.2.4.1 Concentration and dialysis --------------------------------------------------------
II.2.4.2 Protein quantification----------------------------------40
II.2.4.3 SDS-PAGE and IEF--------41
II.2.4.4 Western blotting--------------------------------------------------------------------42
II.2.4.5 Silver staining-----------------------------------------------------------------------42
II.2.4.6 Discontinuous non-denaturing electrophoresis --------------------------------- 43
II.2.5 Stability analysis--43
II.2.6 Mass spectrometry---------------------------------------
II.2.7 Flow cytometric analysis---------------------------------44
II.2.8 M12-V fragment generation ---------------------------- 45 H
II.2.9 Crystallization-------------------------------------------------------------------------46
II.2.9.1 Hanging drop method--------------------------------------------------------------
II.2.9.1.1 M12-V domain crystallization------------------- 46 H
II.2.9.1.2 scFvM12 crystallization ------------------------------------------------------ 48
II.2.9.1.3 M12-V and MUC1 complex crystallization-- 50 H
II.2.9.1.4 scFvM12 and MUC1 complex crystallization- 50
II.2.9.2 Gel crystallization------------------------------------------------------------------51
II.2.9.3 Streak seeding--------------------------------------------52
II.2.9.4 Crystal growth in meta-stable zone ------------------- 52
II.2.9.5 Macroseeding--53
II.2.10 Crystal mounting------------------------------------------53
II.2.10.1 M12-V domain alone and in complex with MUC1------------------------H
II.2.10.2 scFvM12 antibody fragment --------------------------------------------------- 53
II.2.10.3 body fragment in complex with MUC1 antigen ------------ 54
II.2.11 Data collection and data processing--------------------- 54
II.2.12 Phasing------------------------------------------------------55
II.2.12.1 Molecular replacement-----------------------------
II.2.12.1.1 M12-V domain (1.8Å) ----------------------------------------------------- 55 H1.22-V domain in complex with MUC1 peptide (1.5Å) -------------- 55 H
II.2.13 Model building and refinement------------------------------ 55
II.2.13.1 M12-V domain (1.8Å)----------------------------- 55 H
II.2.13.2 M12-V domain in complex with MUC1 peptide (1.5Å)------------------ 56 H
II.2.14 Structure validation---------------------------------------57
II.2.15 Structinterpretation-----------------------------------
III RESULTS----------------------------------58
iiIII.1 Bacterial expression of scFvM12 --------------------------------------------------------- 58
III.1.1 Small scale expression--------58
III.1.2 Large scale -
III.2 Purification of the scFvM12 ------- 60
III.2.1 Ni-NTA chromatography---------------------------------60
III.2.2 Ion-exchange chromatography-------------------------------------------------------61
III.2.3 Size-exclusion chromatography--------------------------62
III.2.4 Iso-electric focusing and mass spectrometry --------- 63
III.3 Crystallization of antibody fragments ----------------------- 65
III.3.1 Crystallization of scFvM12 ----------------------------------