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Purification and Functional Reconstitution of Human olfactory Receptor Expressed in Escherichia. coli : 대장균 발현 시스템 기반 인간 후각 수용체의 생산과 정제 및 구조 형성
DC Field | Value | Language |
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dc.contributor.advisor | 박태현 | - |
dc.contributor.author | 양희홍 | - |
dc.date.accessioned | 2017-07-17T08:44:12Z | - |
dc.date.available | 2017-07-17T08:44:12Z | - |
dc.date.issued | 2014-02 | - |
dc.identifier.other | 000000016645 | - |
dc.identifier.uri | https://hdl.handle.net/10371/127074 | - |
dc.description | 학위논문 (석사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 2. 박태현. | - |
dc.description.abstract | Olfactory receptors (ORs), belonging to G-protein coupled receptors (GPCRs), composed of seven transmembrane domains are very difficult to be overexpressed, solubilized, purified and reconstituted because of their hydrophobicity and complicated structure. These receptors bind to their specific ligands among the thousands of odorant molecules, thus its specificity is very useful for the application of bioelectronic nose. Especially, highly purified and well- reconstituted human olfactory receptor (hOR) has a strong advantage and is used to various fields, such as protein-interaction researches, drug screening, and analyzing the hOR structure. In this study, hOR2AG1 was overexpressed with N-terminus glutathione S-transferase (GST), and C-terminus 6xHis-tag as an inclusion body. The hOR2AG1 fusion protein was solubilized in buffer containing sodium dodecyl sulfate (SDS) and fusion protein was binding to Ni-NTA chromatography based on a C-terminal 6xHis-tag and GST domain was removed using proteolytic cleavage. Then hOR2AG1 was eluted and successfully reconstituted using nonionic detergents and methyl-ß-cyclodextrin as protein folding assistants. Finally the highly purified and well-reconstituted hOR was obtained. The functional activity of purified receptor was confirmed by measuring circular dichroism (CD) spectrum, recording the quenching of the intrinsic receptor fluorescence on the addition of ligand and studying the selective binding of receptor with specific ligand. This study can be applied to develop protein-based olfactory biosensor and other GPCR receptor sensing system and to analyze the native GPCR structure using solid-state NMR, X-ray crystallography, or neutron scattering. | - |
dc.description.tableofcontents | Contents
Abstract iii Contents ⅵ List of figures ⅶ List of table ⅷ 1. Introduction 1 1.1. Difficulty of G-protein coupled receptors (GPCRs) expression, purification and reconstitution in E. coli 1 1.2. Property of human olfactory receptor (hOR) and advantage of purified and reconstituted hOR 3 1.3. Summary of this work 5 2. Materials and methods 6 2.1. 6xHis gene insertion in bacterial expression vector containing hOR2AG1 gene 6 2.2. Overexpression of hOR2AG1 fusion protein in E. coli 6 2.3. Solubilization and column purification of hOR2AG1 fusion protein 8 2.4. Thrombin cleavage of GST-hOR2AG1 bound to column 9 2.5. Reconstitution of purified hOR2AG1 11 2.6. CD spectrum and tryptophan fluorescence measurements 14 3. Results and discussion 21 3.1. Expression vector design and overexpression of hOR2AG1 fusion protein 15 3.2. Solubilization, purification and reconstitution of hOR2AG1 20 3.3. Characterization and functional study of hOR2AG1 27 4. Conclusion 34 5. References 35 초록 41 List of figures Figure 1. Schematric diagram of pDEST15 vector containing thrombin cleavage site 10 Figure 2. Reconstitution strategy of purified hOR2AG1 using detergent micelles 12 Figure 3. Reconstituted hOR2AG1 in detergent micelles 13 Figure 4. Schematic representation of the plasmid pDEST15 vector containing GST, hOR2AG1 and 6xHis gene 17 Figure 5. Overexpression of human olfactory receptor, hOR2AG1 in E. coli. SDS-PAGE of the pellet of cell lysate after centrifugation 18 Figure 6. Western blot of the pellet fraction of cell lysate 19 Figure 7. SDS-PAGE of solubilization of fusion protein 23 Figure 8. SDS-PAGE and western blot of the purified hOR2AG1 fusion protein 24 Figure 9. Thrombin cleavage of GST-hOR2AG1 bound to column 25 Figure 10. Western blot of thrombin-cleaved fusion protein using anti-His-probe antibody 26 Figure 11. CD spectrum of hOR2AG1. It shows appropriately results that have α-helical features 30 Figure 12. Tryptophan fluorescence quenching of hOR2AG1 upon AB binding 32 Figure 13. Normalized tryptophan fluorescence quenching of hOR2AG1 upon AB binding 33 List of table Table. 1. Secondary structure analysis of reconstituted hOR2AG1 31 | - |
dc.format | application/pdf | - |
dc.format.extent | 936657 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | ko | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject.ddc | 660 | - |
dc.title | Purification and Functional Reconstitution of Human olfactory Receptor Expressed in Escherichia. coli | - |
dc.title.alternative | 대장균 발현 시스템 기반 인간 후각 수용체의 생산과 정제 및 구조 형성 | - |
dc.type | Thesis | - |
dc.contributor.AlternativeAuthor | Heehong Yang | - |
dc.description.degree | Master | - |
dc.citation.pages | ⅷ,43 | - |
dc.contributor.affiliation | 공과대학 화학생물공학부 | - |
dc.date.awarded | 2014-02 | - |
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