Title Page
Contents
ABSTRACT 10
Ⅰ. Introduction 13
Ⅱ. Materials and Methods 18
2.1. Cloning and Fermentation 18
2.1.1. Cloning of BvPrP(23-231) M109 and I109 genes in bacterial expression vector 18
2.1.2. Fed-batch fermentation of BvPrP(23-231) M109 and I109 19
2.2. Purification 21
2.2.1. Washing and refolding of inclusion bodies containing BvPrP(23-231) M109 and I109 21
2.2.2. Immobilized-Metal Affinity Chromatography (IMAC) 22
2.2.3. Cation-exchange chromatography 23
2.2.4. Reverse-Phase chromatography 24
2.2.5. Freeze drying 25
2.3. Analysis 26
2.3.1. HPLC analysis 26
2.3.2. Validation of analytical methods 28
2.3.3. Image J Analysis 29
2.3.3. CD spectrometer 30
2.3.4. MALDI-TOF 31
Ⅲ. RESULTS 32
3.1. Cloning and Fermentation 32
3.1.1. Expression of BvPrP(23-231) M109 and I109 32
3.1.2. Fed-batch fermentation of BvPrP(23-231) M109 and I109 34
3.2. Purification 37
3.2.1. Inculsion body washing and refolding of BvPrP(23-231) M109 and I109 37
3.2.2. Immobilized-Metal Affinity Chromatography (IMAC) 40
3.2.3. Cation-exchange chromatography 47
3.2.4. Reverse-Phase chromatography 54
3.2.5. Purification table of BvPrP(23-231) M109 and I109 during recombinant protein purification 61
3.3. Analysis 64
3.3.1. HPLC analysis of purified BvPrP(23-231) M109 and I109 64
3.3.2. Validation of analytical methods 66
3.3.3. CD spectrometer 70
3.3.4. MALDI-FOF 72
Ⅳ. DISCUSSION 74
Ⅴ. CONCLUSION 80
Ⅵ. REFERENCE 81
국문 초록 87
Table 1. Composition of main media and feeding media 20
Table 2. Chromatograph conditions for BvPrPs analysis. 27
Table 3. Purification table of BvPrP(23-231) M109 62
Table 4. Purification table of BvPrP(23-231) I109 63
Table 5. Repeatability of HPLC analysis of BvPrP(23-231) M109 and BvPrP(23-231) I109. 67
Table 6. Robustness of BvPrP(23-231) M109 (A) and BvPrP(23-231) I109 (B). 69
Figure 1. Construction of the BvPrP(23-231) M109 and I109 expression vectors. 33
Figure 2. Expression patterns of according to culture and induction time of BvPrP(23-231) M109 expressing cells using fed-batch fermentation. 35
Figure 3. Expression patterns of according to culture and induction time of BvPrP(23-231) I109 expressing cells using fed-batch fermentation. 36
Figure 4. SDS-PAGE analysis of BvPrP(23-231) M109 during inclusion bodies washing. 38
Figure 5. SDS-PAGE analysis of BvPrP(23-231) I109 during inclusion bodies washing. 39
Figure 6. IMAC chromatogram of BvPrP(23-231) M109. 41
Figure 7. SDS-PAGE analysis of each fraction after IMAC of BvPrP(23-231) M109. 42
Figure 8. Analysis of BvPrP(23-231) M109 after IMAC. 43
Figure 9. IMAC chromatogram of BvPrP(23-231) I109. 44
Figure 10. SDS-PAGE analysis of each fraction after IMAC of BvPrP(23-231) I109. 45
Figure 11. Analysis of BvPrP(23-231) I109 after IMAC. 46
Figure 12. Cation-exchange chromatogram of BvPrP(23-231) M109 using HiTrap Sp column. 48
Figure 13. Area and purity of each fraction of BvPrP(23-231) M109 cation-exchange chromatography 49
Figure 14. Analysis of BvPrP(23-231) M109 after second purification. 50
Figure 15. Cation-exchange chromatogram of BvPrP(23-231) I109 using HiTrap Sp column. 51
Figure 16. Area and purity of each fraction of BvPrP(23-231) I109 cation-exchange chromatography 52
Figure 17. Analysis of BvPrP(23-231) I109 after second purification. 53
Figure 18. Reverse-phase chromatogram of BvPrP(23-231) M109 using C8 column. 55
Figure 19. Area and purity of each fraction of BvPrP(23-231) M109 reverse-phase chromatography 56
Figure 20. Analysis of BvPrP(23-231) M109 after third purification. 57
Figure 21. Reverse-phase chromatogram of BvPrP(23-231) I109 using C8 column. 58
Figure 22. Area and purity of each fraction of BvPrP(23-231) I109 reverse-phase chromatography 59
Figure 23. Analysis of BvPrP(23-231) I109 after third purification. 60
Figure 24. RP-HPLC profile of BvPrP(23-231) M109 (A) and BvPrP(23-231) I109 (B). 65
Figure 25. Linearity of BvPrP(23-231) M109 (A) and BvPrP(23-231) I109 (B). 68
Figure 26. Secondary structure analysis of BvPrP(23-231) M109 and I109 through circular dichroism CD spectrometry 71
Figure 27. MALDI-TOF PMF Analysis of BvPrP(23-231) M109 and I109 73