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고수압을 이용한 단백질 재접힘에 관한 연구
| DC Field | Value | Language |
|---|---|---|
| dc.contributor.advisor | 김용성 | - |
| dc.contributor.author | 이승현 | - |
| dc.date.accessioned | 2018-11-08T06:40:18Z | - |
| dc.date.available | 2018-11-08T06:40:18Z | - |
| dc.date.issued | 2006-08 | - |
| dc.identifier.other | 1672 | - |
| dc.identifier.uri | https://dspace.ajou.ac.kr/handle/2018.oak/3484 | - |
| dc.description | 학위논문(석사)--아주대학교 일반대학원 :분자과학기술학과,2006. 8 | - |
| dc.description.abstract | 고수압을 이용한 박테리아로부터 생산된 5종의 단백질 내포체 -초파리에서 유래한 세 종의 그람 음성 결합 단백질 (GNBP1, 2, and 3)과 인간에서 유래한 두 종의 인산가수분해효소 (PTPRS, DUSP7)-의 용해와 재접힘에 대한 본 연구는 redox-shuffling agent (2 mM DTT and 6 mM GSSG) 및 다양한 첨가제를 이용하여 수행되었다. 200 MPa 의 고수압에 redox-shuffling agent를 첨가한 조건에서 1 mg/ml 의 내포체는 ~42-58% 의 용해수율을 보였다. Urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), 또는 Triton X-100 (0.5 mM)을 첨가한 경우 모든 단백질의 용해수율이 증가하였다. 그러나, urea, glycerol, 비이온성 surfactant의 경우 monomeric 형태보다 oligomeric 형태가 많았으며, 반면에 arginine은 내포체 (1 mg/ml)로부터 55-78%의 재접힘 수율 중 oligomeric 형태보다 functional monomeric형태 (~70-100%)가 많았다. 이 결과는 고수압과 arginine이 결합된 조건에서 재접힘 과정 동안 부분적으로 재접힘된 중간체의 aggregation을 막고 재접힘 수율을 증가시킨다는 것을 시사한다. | - |
| dc.description.tableofcontents | Contents LIST OF TABLES ⅰ LIST OF FIGURES ⅱ ABSTRACT ⅲ Ⅰ. Introduction 1 1. Principle 1 2. Advantages of High Hydrostatic Pressure 2 3. Strategy 3 Ⅱ. Materials and Methods 4 1. Materials 4 2. Methods 6 2.1 Protein expression and purification of IBs 6 2.2 Formulation of refolding buffers and sample pressurization 7 2.3 Total protein assay and SEC 8 2.4 Functional assays of solubilized proteins 9 2.5 Atmospheric urea solubility of IBs 10 Ⅲ. Results and Discussion 11 1. Results 11 1.1 Descriptions of model proteins 11 1.2 Effects of additives on the solubilization and refolding of proteins from IBs 13 1.3 Functional assays 21 1.4 Effects of pressure and duration of pressurization 24 1.5 Relationship between atmospheric urea solubility and solubilization yield by HHP 26 2. Discussion 28 Ⅳ. Conclusions 31 Ⅴ. References 32 Ⅵ. 국문요약 38 | - |
| dc.language.iso | eng | - |
| dc.publisher | The Graduate School, Ajou University | - |
| dc.rights | 아주대학교 논문은 저작권에 의해 보호받습니다. | - |
| dc.title | 고수압을 이용한 단백질 재접힘에 관한 연구 | - |
| dc.title.alternative | Seung-Hyun Lee | - |
| dc.type | Thesis | - |
| dc.contributor.affiliation | 아주대학교 일반대학원 | - |
| dc.contributor.alternativeName | Seung-Hyun Lee | - |
| dc.contributor.department | 일반대학원 분자과학기술학과 | - |
| dc.date.awarded | 2006. 8 | - |
| dc.description.degree | Master | - |
| dc.identifier.localId | 565380 | - |
| dc.identifier.url | http://dcoll.ajou.ac.kr:9080/dcollection/jsp/common/DcLoOrgPer.jsp?sItemId=000000001672 | - |
| dc.subject.keyword | protein refolding | - |
| dc.subject.keyword | high hydrostatic pressure | - |
| dc.description.alternativeAbstract | High hydrostatic pressure (HHP)-mediated solubilization and refolding of five inclusion bodies (IBs) produced from bacteria, three Gram-negative binding proteins (GNBP1, 2 and 3) from Drosophila and two phosphatases from human, were investigated in combination of a redox-shuffling agent (2 mM DTT and 6 mM GSSG) and various additives. HHP (200 MPa) combined with the redox-shuffling agent resulted in the solubilization yields of ~42-58% from 1 mg/ml of IBs. Addition of urea (1 and 2 M), 2.5 M glycerol, L-arginine (0.5 M), Tween 20 (0.1 mM), or Triton X-100 (0.5 mM) significantly enhanced the solubilization yield for all proteins. However, urea, glycerol, non-ionic surfactants populated more soluble oligomeric species than monomeric species, whereas arginine dominantly induced functional monomeric species (~70-100%) to achieve refolding yields of approximately 55-78% from IBs (1 mg/ml). Our results suggest that the combination of HHP with arginine is most effective in enhancing refolding yield by preventing aggregation of partially folded intermediates populated during the refolding. | - |
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