구조 연구를 위한 Shigella flexneri 유래 단백질들의 클로닝, 대량발현, 정제
Cloning, Overexpression and purification of hypothetical proteins from Shigella flexneri for the structural study
- 주제(키워드) Shigella flexneri 5a M90T , structural genomics , cloning , overexpression , purification , X-ray crystallography , NMR spectroscopy
- 발행기관 아주대학교
- 지도교수 서민덕
- 발행년도 2015
- 학위수여년월 2015. 8
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000020703
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Shigella flexneri is a Gram-negative bacterium that causes shigellosis in the terminal ileum and colon. The Shigella genus is composed of four species and each species has many subspecies. Although the bacteria are closely related to human diseases, characterization and structural study are insufficient for Shigella proteins. In this study, as efforts to perform a laboratory scale structural genomics, nine proteins from S. felxneri strain 5a M90T were selected by using UniProt server and XtalPred server. The nine target proteins, including eight hypothetical proteins and one toxin protein, were cloned into the pET21a vector system. Among nine target proteins, eight proteins were overexpressed in E. coli expression system, and four proteins (SF239, SF246, SF682 and SF743) were soluble. These soluble proteins could be successfully purified by using affinity chromatography, ion-exchange chromatography (IEX) and size exclusion chromatography (SEC). In order to determine the structures of purified proteins, X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy were employed. The single crystals of SF246 were obtained and preliminary X-ray diffraction analysis revealed that the crystal diffracted to about 5.0 Å resolutions. In addition, the NMR experiments of SF239 were performed with four different conditions. As a result, we confirmed that SF239 was an intrinsically disordered protein. It is expected that this study could contribute to establish the system for structural genomics.
more목차
I. Introduction 1
1. The Shigella genus 1
1.1. The specification of Shigella 1
1.2. Treatment of shigellosis 2
1.3. The structure of type III secretion system derived
from Shigella flexneri 3
II. Materials and methods 4
1. Materials 4
1.1. Materials 4
1.2. Agents and apparatus 4
1.2.1. Agents 4
1.2.2. Apparatus 4
2. Methods 5
2.1. Target selection and cloning 5
2.2. Overexpression of SF proteins 6
2.3. Purification of target proteins 6
2.4. Initial crystal screening 7
2.5. Optimization of crystallized conditions 7
2.6. X-ray crystallography diffraction of SF246 8
2.7. Preparation of 15N-labeled SF239 for NMR 9
III. Results and discussion 11
3.1. Cloning of target proteins 11
3.2. Overexpression of SF proteins 15
3.3. Purification of SF proteins 17
3.3.1. Purification of SF239 17
3.3.2. Purification of SF246 17
3.3.3. Purification of SF682 18
3.3.4. Purification of SF743 18
3.4. Crystallization and preliminary X-ray diffraction
experiment of SF246 24
3.5. Crystallization of SF743 24
3.6. NMR measurements of SF239 27
IV. Conclusion 31
V. References 32
국문초록 35
