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Proteomic Analysis of heat, osmotic and oxidative stress responses in Thermococcus kodakarensis KOD1

두엣트 리 반 (Le, Van Duyet, 경상대학교 대학원)

원문보기

  • 발행기관 경상대학교 대학원
  • 지도교수 정강원
  • 발행년도 2006
  • 학위수여년월 2006. 8
  • 학위명 석사
  • 소속대학원 및 학과 대학원 응용생명과학부
  • 전공 생화학전공
  • 원문페이지 vi, 45
  • 본문언어 영어
초록/요약moremore
Thermococcus kodakarensis KOD1 is an anaerobic and obligated heterotrophic and grows on complex organic compounds in the presence of elemental sulfur near its optimal growth temperature, 85 oC. To analyze the response to various stresses (heat, osmotic and oxidative shock) at the protein synthesis level, we performed two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. We cloned and expressed one of the stress inducible proteins. This is a thermo-stable protein that has highly conserve domain to OsmC (osmotically inducible proteins C) and predicted redox protein. Also this protein has a hydroperoxide peroxidase activity. To examine the structural organization, we performed the size exclusion chromatography and electron microscopy analysis.
Thermococcus kodakarensis KOD1 is an anaerobic and obligated heterotrophic and grows on complex organic compounds in the presence of elemental sulfur near its optimal growth temperature, 85 oC. To analyze the response to various stresses (heat, osmotic and oxidative shock) at the protein synthesis level, we performed two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS) analysis. We cloned and expressed one of the stress inducible proteins. This is a thermo-stable protein that has highly conserve domain to OsmC (osmotically inducible proteins C) and predicted redox protein. Also this protein has a hydroperoxide peroxidase activity. To examine the structural organization, we performed the size exclusion chromatography and electron microscopy analysis.
목차moremore
I. Introduction 1
II. Materials and methods 6
II.1. Organism 6
...
I. Introduction 1
II. Materials and methods 6
II.1. Organism 6
II.2. Reagents 6
II.3. Cell culture procedure 6
II.4. Heat stress procedure 7
II.5. Oxidative stress procedure 7
II.6. Osmotic stress procedure 7
II.7. Sample preparation for 2DE 7
II.8. 2DE 7
II.9. Protein Visualization and Image Analysis 8
II.10. Protein identification 8
II.11. Isolation of genomic DNA from T. kodakarensis KOD1 10
II.12. Polymerase chain reaction (PCR) 10
II.13. Cloning and expression of OsmC gene in E. coli 11
II.14. Purification of OsmC protein 11
II.15. Assay of Peroxidase (Prx) activity 12
II.16. Size exclusion chromatography (SEC) 12
II.17. Electron microscopy 12
II.18. Image processing 12
III. Results and Discussions 15
III.1. Identification of heat shock response in T.Kodakarensis KOD1 15
III.2. Identification of oxidative shock response in T. kodakarensis KOD1 24
III.3. Identification of osmotic shock response in T. kodakarensis KOD1 31
III.4. Cloning and DNA sequencing of OsmC gene 36
III.5. Expression and purification of OsmC protein 38
III.6. Recombinant OsmC protein forms different molecular weight according to salt 41
III.7. Oligomeric structure of OsmC 41
III.8. Peroxidase activity of OsmC 45
V. References 50
Acknowledgement 54