표제지
목차
초록 7
Abbreviation 12
I. 서론 13
II. 재료 및 실험 방법 17
[Part1] E.coli Expression test 17
1. 실험 재료 17
2. 실험 방법 19
II. 재료 및 실험 방법 24
[Part2] Mammallian cell Expression test 24
1. 실험 재료 24
2. 실험 방법 27
III. 실험 결과 51
[Part1] E.coli Expression test 51
3-1. E.coli에 Cathepsin K cloning 51
3-2. E.coli Expression test 52
3-3. Inclusion body Isolation: Purification & Refolding 54
III. 실험 결과 58
[Part2] Mammallian cell Expression test 58
3-1. Cathepsin K Cloning through Alanine point mutation 58
3-2. Alanine point mutation 한 Cathepsin K의 발현 확인 60
3-3. Alanine point mutation 한 Cathepsin K의 Signal Peptide Screening 62
3-4. 단백질 정제 결과 70
3-5. (G4S)3 linker를 이용한 단백질 발현, 정제 71
IV. 결론 및 고찰 72
V. 참고 문헌 74
ABSTRACT 76
Table 1. Signal Peptide library list 1~50 38
Table 2. Signal Peptide library list 51~100 39
Table 3. Signal Peptide library list 101~150 40
Table 4. Signal Peptide library list 151~200 41
Fig 1. pcDNA 3.4 TOPO Vector 25
Fig 2. Insertion of Cathepsin K gene in Vector 28
Fig 3. Scheme of PCR 30
Fig 4. Alanine point mutation on the Catalytic activity of Cathepsin K 31
Fig 5. Scheme of PCR for Alanine point mutation 33
Fig 6. Scheme of PCR for Alanine point mutation 34
Fig 7. Scheme of PCR for Alanine point mutation 35
Fig 8. PCR conditions for Alanine point mutation 36
Fig 9. Flexible linker insertion between target gene and His-tag 42
Fig 10. Scheme of PCR for G4S linker cloning 44
Fig 11. PCR conditions for using primers (1,3), (2,4) 45
Fig 12. OverlapPCR conditions for using primers 1,4 45
Fig 13. Cathepsin K expression results in E.coli... 53
Fig 14. Cathepsin K expression result after cell sonication 53
Fig 15. Results of purification of Cathepsin K including Urea 55
Fig 16. Results of purification of Cathepsin K by Urea refolding 57
Fig 17. PCR product for +Propeptide Cathepsin K 59
Fig 18. PCR product for Alanine point mutation... 59
Fig 19. Catalytic activity amino acid of Cathepsin K expression test result (3,5,7 day) 61
Fig 20. Signal Peptide 1~100 Screening for Cathepsin K C25A 63
Fig 21. Average error bar graph for Cathepsin K C25A Signal Peptide 1~100 Screening 64
Fig 22. Comparison of the expression of 101~150 Signal Peptide, 66
Fig 23. Average error bar graph for Cathepsin K C25A Signal Peptide... 67
Fig 24. Comparison of the expression of 151~200 68
Fig 25. Average error bar graph for Cathepsin K C25A Signal Peptide 151~200... 69
Fig 26. Ni-NTA purification results for 3.4-SP135-(+Propeptide)Cathepsin... 70
Fig 27. Expression result after purification of 3.4-SP135-... 71