목차

General Introduction 1
Chronic myeloid leukemia 1
Cobll1, PACSIN2 and SH3BP1 5
Interaction between SH3 domain and proline-rich motif 6
Purpose of this study 8
Colorectal cancer 9
Colon cancer secreted protein-2 (CCSP-2) 11
Extracellular matrix 12
Antibody 14
Purpose of this study 15
Chapter 1 16
1.1 Introduction 16
1.2 Experimental Procedures 17
1.2.1 Protein expression and purification 17
1.2.2 Peptide 18
1.2.3 Surface plasmon resonance (SPR) 18
1.2.4 Nuclear magnetic resonance (NMR) spectroscopy 18
1.2.5 Crystallization 19
1.2.6 Multiple angle light scattering (MALS) 20
1.2.7 MALDI-TOF 20
1.3 Results 22
1.3.1 Expression and purification of PACSIN2-SH3 22
1.3.2 Preparation of three Cobll1-PRPs 25
1.3.3 NMR backbone assignments of PACSIN2-SH3 27
1.3.4 NMR titration study of PACSIN2-SH3 with the three Cobll1-PRPs 30
1.3.5 Crystallization of PACSIN2-SH3 with proline-rich peptides of Cobll1 35
1.3.6 Structure prediction and docking model of PACSIN2-SH3 with proline-rich peptides 38
1.3.7 HSQC spectroscopy spectra of PACSIN2-SH3 in the presence of Cobll1-N and SH3BP1-C 43
1.3.8 Differential binding affinity of Cobll1 and SH3BP1 for PACSIN2-SH3 48
1.3.9 Competitive binding of Cobll1 and SH3BP1 to PACSIN2-SH3 50
1.4 Discussion 53
Chapter 2 56
2-1 Introduction 56
2.2 Experimental Procedures 58
2.2.1 Construct design, expression, and purification of CCSP-2 58
2.2.2 Peptide 60
2.2.3 Construct design, expression, and purification of scFvs 60
2.2.4 Isothermal titration calorimetry (ITC) 60
2.2.5 Crystallization and data collection 61
2.2.6 Structure determination and refinement 61
2.3 Results 63
2.3.1 Expression and purification of C-terminus region of CCSP-2 63
2.3.2 Expression and purification of scFv 66
2.3.3 Binding affinities between CCSP-2 and scFv 69
2.3.4 Crystallization of apo-scFv 20 72
2.3.5 Crystallization of scFv 20:V3E2 complex 74
2.3.6 Crystallization of scFv 19:peptide 6 complex 78
2.3.7 Superimposition structure of scFv 19:peptide 6 complex and scFv 20:V3E2 complex 81
2.4 Discussion 83
Conclusion 85
References 86