대장균에서 생산된 G 단백질 연결 수용체의 기능적 재구성 및 고성능 바이오센서로의 응용

학위논문 (박사)-- 서울대학교 대학원 : 공과대학 화학생물공학부, 2018. 2. 장정식.this is termed a bioelectronic nose. The hOR1A2NDs responded to rose scent molecules specifically, which were monitored electrically using the underlying CNT-FET. This strategy allowed the quantitative assessment of the specific rose scents with sensitivity and selectivity. Significantly, the enhanced responses of hOR1A2NDs to a rose scent by enhancer material were successfully demonstrated like a human nose. Furthermore, the method provided a means to quantitatively evaluate rose scent components in real samples such as rose oil. As a result, the protein-based bioelectronic sensors exhibit high-performance in terms of sensitivity, selectivity, reproducibility, stability and quantitative analysis. Also, they could successfully mimic receptor agonism and antagonism, and the human olfactory response as well as detect the liquid and gaseous target molecule. This platform can be used as a practical method for the receptor-based sensing approach, which represents significant progress in nano-bio technology toward a practical biosensor.G protein-coupled receptors (GPCRs) are membrane proteins that have seven trans-membrane domains, which are significantly involved in many human diseases and 40% of all drug targets. For this reason, a platform containing stable and high-quality GPCR would be useful for the development of biosensor that can be applied to various fields, such as early diagnosis of diseases, environmental monitoring and drug screening. Significant efforts have been made to develop the GPCR-conjugated biosensor by combining the nanomaterials and biomolecules, such as protein, cell-derived nanovesicle and GPCR-expressing cells. Especially, the protein-based biosensor could have a great advantage in sensitivity, selectivity, reproducibility, reusability and quantitative analysis. For the production of recombinant proteins, the Escherichia coli (E. coli) has been widely used as a host cell because of its great advantage in productivity and convenience. However, the production of GPCR in E. coli remains a challenge because of their strong hydrophobicity, a complicated charge distribution and different membrane inserting mechanism. The functional reconstitution technique of GPCRs has been considered to be important tool for more stable and accurate receptor-based research. These techniques have been studied to acquire basic knowledge of the crystal structure, receptor-signaling and pharmacological research. Many studies have suggested that highly purified and well-reconstituted receptor could have native-like receptor structure and binding pocket. Especially, the receptor-embedded nanodiscs (NDs) have been considered to be a powerful tool for qualitative and quantitative biophysical analysis on receptor/ligand binding details and could be applied to practical biosensor, which can be used to various fields, such as assessment of food quality, disease diagnosis and drug-interaction researches. In this thesis, GPCRs were produced in E. coli with high productivity, solubilized with detergent and purified with column chromatography. Then, the functional reconstitutions of purified receptors as a detergent micelles or NDs were successfully optimized and their characteristics were monitored and analyzed. Their binding properties were also confirmed by comparing it with the responses in cell-based assay. And, they were applied to nano-electronic sensors using field-effect transistor (FET) to develop the protein-based platforms, which lead to high-performance biosensor. First, the receptor-based analysis method for studying receptor agonism and antagonism was developed. The dopamine receptor D1 (DRD1)-conjugated FET system was constructed by combining the FET and reconstituted DRD1 protein, which is a suitable substitute for conventional cell-based receptor assays. The DRD1 was purified and reconstituted to mimic native binding pockets that have highly discriminative interaction toward DRD1 agonists/antagonists. The real-time responses from the DRD1-nanohybrid FET were highly sensitive and selective for dopamine agonists/antagonists, and their maximal response levels were clearly different depending on their DRD1 affinities. And, they could successfully demonstrate the DRD1 agonism and antagonism. Second, for the more stabilization of the functional receptors, the receptor-embedded NDs were successfully optimized and assembled. A trace amine-associated receptor 13c (TAAR13c) binding to death-associated odor cadaverine (CV) was produced in E. coli, purified and reconstituted into NDs. Their characteristics were analyzed by tryptophan fluorescence assay, dynamic light scattering and scanning electron microscopy. Also, they were applied to a carbon nanotube (CNT)-based FET with floating electrodes with desired orientation. The ND-based biosensor showed high-performance in terms of sensitivity and selectivity. Moreover, they could measure CV in diverse real-food samples for the determination of food freshness. Third, the T13ND-conjugated Ni-decorated conducting polymer nanoparticle in FET was successfully constructed to develop the high-performance biosensor platform detecting the liquid and gaseous target. In this platform, the T13NDs could mimic the native-like binding pocket of natural receptor and exist with high-stability in the aqueous and dried conditions. The conducting polymer nanoparticle could enhance the sensor sensitivity and enable to the oriented immobilization of NDs on the FET. The ND-based biosensor can have a high stability and reliability as well as a high sensitivity and selectivity in aqueous condition. Furthermore, they also could detect the gaseous CV with high sensitivity, stability and reusability. Lastly, the human-like performance bioelectronic nose was successfully constructed to mimic the human sensory responses using receptor-based platform. The human olfactory receptor 1A2-embedded NDs (hOR1A2NDs) were assembled and applied to the CNT-FET with floating electrodeChapter 1. Research background and objective 1 Research background and objective 2 Chapter 2. Literature review 5 2.1 Expression and purification of G protein-coupled receptor 6 2.1.1 Expression of GPCR 6 2.1.2 Purification of GPCR 7 2.2 Functional reconstitution of G protein-coupled receptor 8 2.2.1 Detergent micelles 8 2.2.2 Lipid vesicles 11 2.2.3 Bicelles 13 2.2.4 Nanodiscs 16 2.3 G protein-coupled receptor-conjugated biosensor based on field effect transistor 19 Chapter 3. Experimental procedures 22 3.1 Materials 23 3.2 Gene cloning 23 3.3 Expression 24 3.3.1 Expression of GPCRs in E. coli 24 3.3.2 Expression of membrane scaffold protein in E. coli 25 3.3.3 Expression of GPCR in HEK-293 cell 25 3.3.4 Western blot and total protein assay 26 3.4 Purification 26 3.4.1 Purification of GPCR 26 3.4.2 Purification of membrane scaffold protein 27 3.5 Functional reconstitution 27 3.5.1 Reconstitution using detergent micelles 27 3.5.2 Reconstitution using nanodiscs 28 3.6 Characterization 28 3.6.1 Luciferase assay 28 3.6.2 Analyzes of functional receptor 29 3.7 Fabrication and immobilization 30 3.7.1 Dopamine receptor-conjugated cPPy-coated CNT-FET 30 3.7.2 Nanodisc-based CNT-FET with floating electrode 30 3.7.3 Nanodisc-conjugated Ni-decorated cPPyNP in FET 31 3.8 Electrical measurement 32 3.8.1 Preparation of tested samples 32 3.8.2 Signal measurement 33 Chapter 4. Functional reconstitution of human dopamine receptor into micelle for the analysis of receptor agonism and antagonism 35 4.1 Introduction 36 4.2 Purification and functional reconstitution of DRD1 38 4.3 Chemical immobilization of DRD1 on nanohybrids 41 4.4 Electrical properties of nanohybrids in the FET geometry 46 4.5 Sensing behaviors of DRD1-nanohybrid FET 51 4.6 Analyzing DRD1 agonism and antagonism characteristics 56 4.7 Conclusions 63 Chapter 5. Assembly of receptor-embedded nanodiscs and its application to the biosensor for the assessment of death associated-odor cadaverine 64 5.1 Introduction 65 5.2 Binding property analysis of TAAR13c using cell-based assay 66 5.3 Production, optimization and characterization of TAAR13c-embedded nanodiscs 70 5.4 Concept of oriented ND-functionalized bioelectronic nose (ONBN) for the assessment of death-associated odor cadaverine 75 5.5 Sensing performance of ONBN 78 5.6 Conclusions 84 Chapter 6. Development of nanodisc-based high-performance biosensor for the detection of liquid and gaseous cadaverine 85 6.1 Introduction 86 6.2 Fabrication of nanodisc-conjugated Ni-decorated cPPyNP in FET 87 6.3 Characteristics of nanodisc-conjugated Ni-decorated cPPyNP in FET 93 6.4 Highly stable and ultrasensitive nanodisc-based biosensor detecting the liquid and gaseous cadaverine 96 6.4.1 Detection of the liquid cadaverine 96 6.4.2 Detection of the gaseous cadaverine 100 6.5 Conclusions 102 Chapter 7. Production of human olfactory receptor-embedded nanodiscs for the development of human-like performance bioelectronic nose smelling a rose scent 103 7.1 Introduction 104 7.2 Structure of a bioelectronic nose comprised of hOR1A2-based nanodiscs and a floating electrode-based CNT-FET 106 7.3 Characterization of hOR1A2 expressed in HEK-293 cells 109 7.4 Reconstitution of hOR1A2 into nanodiscs 112 7.5 Electrical characterization of bioelectronic noses and its responses to geraniol and citronellol 115 7.6 Measuring the effect of the enhancer on the assessment of rose scent ingredients 120 7.7 Smelling rose scent ingredients in natural rose oil 125 7.8 Conclusions 129 Chapter 8. Overall discussion and further suggestions 130 Overall discussion and further suggestions 131 Bibliography 135 국문초록 151Docto