Shigella flexneri 5a M90T 유래 단백질의 구조연구
Structural studies on the proteins from shigella flexneri 5a M90T
- 주제(키워드) Shigella flexneri 5a M90T , structural genomics , cloning , overexpression , purification , X-ray crystallography , structure determination
- 발행기관 아주대학교
- 지도교수 서민덕
- 발행년도 2017
- 학위수여년월 2017. 2
- 학위명 석사
- 학과 및 전공 일반대학원 분자과학기술학과
- 실제URI http://www.dcollection.net/handler/ajou/000000023983
- 본문언어 영어
- 저작권 아주대학교 논문은 저작권에 의해 보호받습니다.
초록/요약
Shigella flexneri는 대표적인 Gram-negative bacteria로, 주로 개발 도상국 및 아시아 부근에서 많이 발병하는 세균성 이질의 원인 균으로 알려져 있다. 잘 알려진 것 과는 달리 구조나 특성 연구는 활발하지 않다. 본 연구는 생물정보학 도구를 이용하여 구조연구에 적합한 S.flexneri의 단백질을 선정하여 구조와 기능에 대해 알아보고자 하였다. 선정된 9개의 단백질들은 cloning을 통하여 재조합 단백질로 합성 하였고, expression and solubility test를 통해 5개의 soluble 단백질을 얻었다. Soluble한 5개의 단백질 중 4개(SF173, SF240, SF356, SF3537)를 affinity, ion-exchange, size-exclusion chromatography를 이용해 순수하게 정제 하였다. Sel-Met SF173, SF240, SF3537이 initial screening에서 결정을 얻어 optimization을 진행 하였다. Sel-Met SF173과 SF240은 결정을 얻어 x-ray diffraction 실험을 진행 하여 1.76Å, 7.0Å의 resolution을 얻었다. SF3537은 initial 결정을 diffraction 실험 하여 약 7.0Å resolution 패턴을 얻었다. Sel-Met SF173의 pattern 결과를 이용하여 SF173의 dimer 형태의 3차원 구조를 결정 하였다. SF356은 아직 initial hit 를 잡지 못했다.
more초록/요약
Shigella is a non-motile, non-spore forming and rod-shaped Gram-negative bacterium, and causes a shigellosis. The Shigella genus consists of four species and each species has many subspecies. Among four kinds of species, Shigella flexneri is well known for causing shigellosis in Asia and developed countries. Although S. flexneri microbes are known to cause human diseases, researches about characterization and structure determination of S. flexneri are insufficient. In this study, cloning, overexpression, purification, crystallization and structure determine of the proteins from S. flexneri were performed. We selected nine proteins from S. flexneri strain 5a M90T by using various bioinformatics tools. The nine proteins were composed of seven hypothetical proteins and two characterized proteins. The genes were sub-cloned into the pET21a vector system, and overexpressed in Escherichia coli expression system. Five of nine target proteins were soluble, and four proteins of those could be purified by using affinity chromatography, ion-exchange chromatography and size exclusion chromatography. To determine the three-dimensional structure, we employed X-ray crystallography. For the crystallization screening, we could get initial hits for SF173, SF240 and SF3537. The crystallization conditions of SF173 and SF240 were further optimized. The X-ray diffraction analysis of SF240 showed the diffraction pattern to about 7.0 Å resolutions, which was not enough for the structure determination. For SF173, diffraction data were collected to 1.76 Å resolutions. We used single wavelength anomalous dispersion (SAD) for crystal structure determination of SF173. As a result, we were able to get the structure of the SF173. All these studies would contribute to establishing a system for structure determination.
more목차
Abstract……………………………………………………………………………...i
Contents…………………………………………………………………………….ii
List of figures……………………………………………………………………....v
List of tables……………………………………………………………………......vi
List of abbreviations……………………………………………………………. vii
I. Introduction……………………………………………………………………….1
1. The Shigella genus...............................................................................................1
1.1. The description of Shigella.............................................................................1
1.2. Infection mechanism of shigella.....................................................................1
1.3. Treatment of Shigellosis..................................................................................2
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 and solubility about target.....................................................5
2.3. Purification of target proteins..........................................................................6
2.4. Initial screening for crystallization.................................................................6
2.5. Optimization of crystallized conditions..........................................................7
2.6. X-ray diffraction data collection and structure determining...........................7
III. Results and discussion.........................................................................................9
3.1. Cloning of target proteins................................................................................9
3.2. Overexpression and solubility of proteins....................................................13
3.3. Purification of SF proteins............................................................................15
3.3.1. Purification of Sel-Met SF173.................................................................15
3.3.2. Purification of SF240…………………………………………………...15
3.3.3. Purification of SF356…………………………………………………...16
3.3.4. Purification of SF3537.............................................................................16
3.4. Crystallization and Structure determination of SF173..................................22
3.5. Crystallization and X-ray diffraction experiment of SF240.........................22
3.6. Crystallization of SF356...............................................................................23
3.7. Crystallization and X-ray diffraction experiment of SF3537.......................23
IV. Conclusion..........................................................................................................27
V. References............................................................................................................28
국문초록..................................................................................................................31
