목차

1. 서 론 .......................................................................................................... 1
1.1. 혈관내피세포 성장인자의 개요 .........................................................1
1.2. 혈관내피세포 성장인자의 정제에 관한 문헌조사 ........................3
1.3. Ubiquitin 융합단백질에 대한 개요 ......................................................4
1.4. 대장균 봉입체의 가용화 및 refolding관련 정제공정에 대한 개요.......8
1.5. 연구목적.....................................................................................................12
2. 재료 및 방법 ................................................................................................14
2.1. 재조합 발현벡터의 cloning ..............................................................14
2.2. 20L-scale 회분식 배양 및 발현 ......................................................16
2.3. rhVEGF165 분리정제 ..........................................................................18
2.3.1. Inclusion body 회수 ...............................................................18
2.3.2. rhVEGF165 monomer 정제 .....................................................18
2.3.3. Refolding 및 rhVEGF165 dimer정제 .....................................20
2.4. 특성분석 .............................................................................................24
2.4.1. SDS-PAGE 전기영동 .............................................................24
2.4.2. Lowry법 ....................................................................................25
2.4.3. RP-HPLC ..................................................................................26
2.4.4. SEC-HPLC ...............................................................................27
2.4.5. Western-blot .............................................................................28
2.4.6. Biological activity assay .........................................................28
3. 결과 및 토의 ..............................................................................................31
3.1. rhVEGF165 cloning ...............................................................................31
3.1.1. rhVEGF165 발현벡터 제조 및 DNA sequence 확인 ............31
3.1.2. 재조합 대장균에서 rhVEGF165 발현 위치확인 ...................33
3.2. rhVEGF165의 배양 및 발현 확인(20L-scale) ..................................35
3.3. 분리정제 공정 별 SDS-PAGE 순도 및 fraction확인 ...................37
3.3.1. Inclusion body 회수 및 세척 ................................................37
3.3.2. 가용화 조건실험 .....................................................................40
3.3.3. 1st His-tag affinity chromatography ......................................46
3.3.4. UBP-1에의한 fusion-tag절단 결과 .......................................50
3.3.5. 2nd His-tag affinity chromatography이용한 fusion tag 제거 및 정제 결과 ............................................................................52
3.3.6. Dimerization and refolding .....................................................54
3.3.7. DEAE anion-exchange chromatography .................................56
3.3.8. Gel filtration .............................................................................59
3.3.9. 공정분석 ..................................................................................61
3.4. 특성분석 ..............................................................................................64
3.4.1. 아미노산 서열분석.....................................................................64
3.4.2. SDS-PAGE 전기영동 분석 ....................................................66
3.4.3. HPLC 분석 ...............................................................................68
3.4.5. Western-blotting ........................................................................70
3.4.6. Biological activity assay ..........................................................72
4. 결론 .....................................................................................................................74
5. 참고문헌 ..........................................................................................................76
Abstract .....................................................................................................................80