Functional reconstitution of G protein-coupled receptor produced in Escherichia coli and its applications to the high-performance biosensor

Abstract

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 electrode