대장균에서 외래 단백질 분비생산에 관한 연구
Secretory production of heterologous proteins in Escherichia coli
- 발행기관 강릉대학교·KIST 강릉분원
- 지도교수 손진기, 판철호
- 발행년도 2009
- 학위수여년월 2009. 2
- 학위명 석사
- 학과 및 전공 학연협동과정
- 원문페이지 viii, 55 p.
- 본문언어 한국어
초록/요약
The mass production of the useful protein is important and valuable in biotechnology industry. The bacreria, especially Escherichia coli, have been preferred to produce the heterologous recombinant protein. However, there are a couple of limitations to use bacterial cells as a host such as inclusion body, post-translational modification, disulfide bond, and so on. So, people have developed the alternative system such as yeast cell, insect cell, animal cell and plant cell. But, the bacterial cells are regarded as the best host for the production of the heterologous recombinant protein because the gene manipulation is easy and the production cost is cheap. Here, to overcome the limitation of E. coli expression system, we tried to express the heterologous protein as a secreted form. We adopted the signal peptide of β-agarase (ASP;23 amino acid residues) from Agarivorans albus YKW-34 which is one of marine bacteia. When we used ASP to express β-agarase, β-agarase activity was detected in the media after IPTG induction and increased. About 42% activity was secreted after 6hr IPTG induction. We also tested ASP to secret the eucaryotic protein GFP (green fluorescent protein). In this case, green fluorescence was detected in the media after IPTG induction, showing that GFP was secreted by ASP.
more목차
Ⅰ. 서 론 = 1
Ⅱ. 실험 재료 및 방법 = 7
1. 대장균 균주 및 플라스미드 = 7
2. 배지 = 7
3. 시약 및 기기 = 9
4. 형질 전환 = 10
5. 발현 플라스미드 제작 = 10
6. 재조합 단백질 발현 = 17
7. 대장균 세포 파쇄 = 17
8. Immobilized metal affinity chromatography (IMAC) = 17
9. Agarase 효소활성 측정 = 18
10. 형광광도 측정 = 18
11. 단백질 정량 = 18
12. Sodium dodecyl sulfate polyacrylamide gel = 19
Ⅲ. 연구 결과 및 고찰 = 20
1. Signal peptide ASP 분석 = 20
2. 발현 플라스미드 제작 = 24
3. Agarase 분비 발현 = 32
4. ASP의 진핵세포 유래 단백질 분비에 적용 = 43
Ⅳ. 참고문헌 = 45
