Title Page
Contents
Abstract 6
CHAPTER 1. INTRODUCTION 12
1.1. Biology of filamentous phage M13 13
1.1.1. Phage structure 13
1.1.2. Coat proteins used for display 16
1.1.3. Infection to host cell 19
1.2. Phage display of random peptide 19
1.2.1. Choice of phage display library 19
1.2.2. Phage display of peptides 22
1.3. Phage display principle 24
1.4. Application of phage display technology 27
1.4.1. Magnetic separation for bio-purification 28
1.4.2. Application of magnetic particles in bio-purification 28
1.5. Research objective 29
CHAPTER 2. MATERIALS AND METHOD 33
2.1. Materials 33
2.2. Methods 34
2.2.1. Biopanning towards target hIgG 34
2.2.2. Phage enzyme-linked immunosorbent assay 36
2.2.3. Biotinylation of anti- hIgG 38
2.2.4. Surface Plasmon Resonance 39
2.2.5. Immobilization of anti hIgG and peptide D1 to MP 40
2.2.6. O-phthalaldehyde assay procedure 43
2.2.7. Determination of immunobinding efficacy 43
CHAPTER 3. RESULTS AND DISCUSSION 45
3.1. Specific enrichment of positive phages 45
3.2. Identification of the positive phage clones 49
3.3. Gel electrophoresis to check the peptide mimics 49
3.4. Immunobinding of selected phage clones 51
3.5. Amino acid sequences and physicochemical parameters of synthetic peptides 55
3.6. Binding studies of the synthesized peptides 58
3.7. Immobilization of anti-hIgG by amine-coupling 61
3.8. Immobilization of biotinylated peptides on SA-MP 62
3.9. Effect of immobilization on hIgG binding efficacy 67
CHAPTER 4. CONCLUSION 71
References 73
Table 3-1. Assay for each plaque selection round (regular panning). 47
Table 3-2. Kd values of selected phages.(이미지참조) 54
Table 3-3. The amino acid sequences and properties of selected petides. 57
Table 3-4. RU values of binding analysis of 4 peptides/anti hIgG towards hIgG. 60
Table 3-5. Comparison of hIgG binding affinity to ligand-beads. 70
Fig. 1-1. Schematics of (a) expression of foreign molecules on the surface of bacteriophage M13. (b) Phage M13 vector. 14
Fig. 1-2. Schematics of filamentous phage M13 structure. 15
Fig. 1-3. The modular structure of pIII phage coat. The three domains of pIII are shown from N- to C- terminus. The N1, N2 and CT domains are each separated by glycine-rich linker (G1 and G2).... 18
Fig. 1-4. Infection of E. coli by filamentous phage. 21
Fig. 1-5. Construction of phage libraries. 23
Fig. 1-6. Biopanning procedure with a peptide library. 26
Fig. 1-7. Serum protein profile. 30
Fig. 1-8. Work flowchart diagram for this research. 32
Fig. 2-1. Schematics of two methods of biopanning. (a) Regular method and (b) modified method. 37
Fig. 2-2. Schematics of hIgG removal using anti-hIgG coated MP. 42
Fig. 3-1. Enrichment of phage-displayed peptide by biopanning. 48
Fig. 3-2. ELISA analysis of the hIgG-binding phages encoding different peptides. 50
Fig. 3-3. DNA analysis of specific clones to hIgG. IgG inserts were amplified from individual plaques of positive clones. Amplification was performed with Smart Pre-mix Potimization Kit.... 52
Fig. 3-4. Binding isotherms of the four phage clones and anti-hIgG. 53
Fig. 3-5. SPR response sensogram for hIgG interaction using different immobilized molecules. 59
Fig. 3-6. Effect of (a) pH and (b) reaction time on immobilization of anti-hIgG on COOH-MP. 64
Fig. 3-7. Scatchard plots describing the binding of anti-hIgG to COOH-MP. 65
Fig. 3-8. Effect of (a) pH and (b) reaction time on immobilization of peptide D1 on SA-MP. 66
Fig. 3-9. Scatchard plots describing the binding of peptide to SA-MP. 68