Purification and Functional Reconstitution of Human olfactory Receptor Expressed in Escherichia. coli ; 대장균 발현 시스템 기반 인간 후각 수용체의 생산과 정제 및 구조 형성

학위논문 (석사)-- 서울대학교 대학원 : 화학생물공학부, 2014. 2. 박태현. ; 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. ; 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 ...