Process development of GPCR insertion into bare nanodisc for the efficient mass production of GPCR-embedded nanodisc = GPCR 내재 나노디스크의 효율적인 대량 생산을 위한 맨 나노디스크로의 GPCR 삽입 공정 개발

다국어 초록 (Multilingual Abstract)

G protein-coupled receptors (GPCRs) are involved in diverse physiological and biochemical processes and represent important targets for pharmaceutical and industrial applications. Nanodiscs (NDs) provide a stable lipid bilayer environment suitable for structural and functional studies of membrane proteins such as GPCRs, as well as for biosensor and drug detection platforms.
In this study, GPCRs expressed in E. coli were purified and reconstituted into NDs using two different assembly approaches. Instead of the conventional direct assembly method, in which each component is prepared separately and mixed simultaneously, we designed and evaluated an alternative strategy in which purified GPCRs were inserted into preassembled bare NDs. The bare NDs were further characterized for long-term storage stability at different temperatures and for process stability, considering their potential use in future biosensor applications. This bare ND–based process offers advantages in reproducibility, storage stability, and troubleshooting, making it a useful strategy for large-scale production of GPCR-embedded NDs.
Furthermore, to minimize aggregation and enhance GPCR incorporation efficiency into NDs, salt concentrations were optimized to improve protein solubility during expression and purification. This study provides a promising foundation for industrial applications by establishing a scalable platform for producing NDs with functional GPCRs, based on preassembled bare NDs and optimized salt conditions, using three representative GPCRs as model systems.

국문 초록 (Abstract)

G 단백질 결합 수용체(G protein-coupled receptor, GPCR)는 다양한 생리적 및 화학적 과정에 관여하며, 의약 및 산업적 응용을 위한 중요한 표적 단백질로 알려져 있다. 나노디스크는 GPCR과 같은 막단백질의 구조적, 기능적 연구 뿐만 아니라 바이오센서 및 약물 검출 플랫폼에도 적합한 안정적인 인지질 이중층 환경을 제공한다.
본 연구에서는 E. coli에서 발현된 GPCR을 정제한 후, 두 가지 서로 다른 조립 방법을 이용하여 나노디스크에 재구성하였다. 각 성분을 개별적으로 준비한 후 동시에 혼합하는 기존의 직접 조립(direct assembly) 방식 대신, 정제된 GPCR을 미리 조립된 맨 나노디스크에 삽입하는 대체 전략을 고안하고 평가하였다. 또한, 맨 나노디스크의 장기 저장 안정성과 공정 안정성을 다양한 온도 조건에서 분석하였으며, 향후 바이오센서 응용을 고려하여 그 특성을 평가하였다. 이러한 맨 나노디스크 기반 공정은 재현성, 저장 안정성, 문제 해결 용이성 측면에서 이점을 제공하며, GPCR이 삽입된 나노디스크의 대량생산에 유용한 전략으로 제시된다.
더 나아가, 단백질 발현 및 정제 과정에서의 용해도를 향상시키기 위해 염 농도를 최적화하여 응집을 최소화하고 GPCR의 나노디스크 삽입 효율을 높였다. 본 연구는 세 가지 대표적인 GPCR을 모델 시스템으로 활용하여, 미리 조립된 맨 나노디스크와 최적화된 염 조건을 기반으로 기능성 GPCR 나노디스크를 대량생산할 수 있는 확장 가능한 플랫폼을 구축함으로써, 향후 산업적 응용의 기반을 마련하였다.

목차 (Table of Contents)

• 1. Introduction 1
• 1.1. Importance of GPCR-embedded nanodisc 1
• 1.2. Need for GPCR-embedded nanodisc 3
• 2. Materials and methods 5
• 2.1. Gene cloning 5
• 2.2. Production of recombinant protein 7
• 2.2.1. Expression and purification of MSP1E3D1-c-Myc 7
• 2.2.2. Expression and purification of GPCR 9
• 2.3. Preparation of lipid 10
• 2.4. Production of bare nanodisc 11
• 2.5. Reconstitution of GPCR-embedded nanodisc 12
• 2.5.1. Direct assembly method 12
• 2.5.2. Insertion into bare ND method method 14
• 2.6. Characterization 16
• 2.6.1. SDS-PAGE and western blot analysis 16
• 2.6.2. Dynamic light scattering 17
• 2.6.3. Size-exclusion chromatography 17
• 2.6.4. Transmission electron microscopy 18
• 2.7. Optimization of salt concentration on purified GPCRs 19
• 2.8. Tryptophan fluorescence quenching assay 20
• 2.9. Structural modeling and visualization 21
• 3. Results and discussion 22
• 3.1. Expression and purification of MSP1E3D1-c-Myc 22
• 3.2. Production of bare nanodisc 24
• 3.2.1. Characterization of bare nanodisc 24
• 3.2.2. Long-term storage stability test 26
• 3.2.3. Process stability test 29
• 3.3. Expression and purification of μOR and AT-μOR 32
• 3.4. Reconstitution of AT-μOR ND 34
• 3.4.1. Comparison of ND synthesis methods 34
• 3.4.2. Characterization of AT-μOR ND 36
• 3.4.3. Tryptophan fluorescence quenching assay 38
• 3.5. Expression and purification of hOR51E1 40
• 3.6. Reconstitution of hOR51E1 ND 42
• 3.6.1. Comparison of ND synthesis methods 42
• 3.6.2. Characterization of hOR51E1 ND 44
• 3.6.3. Tryptophan fluorescence quenching assay 46
• 3.7. Expression and purification of 5-HT7R 48
• 3.7.1. Characterization of 5-HT7R 48
• 3.7.2. Comparative DLS analysis of purified GPCRs 50
• 3.7.3. Parameter-dependent aggregation tendency of 5-HT7R 52
• 3.8. Optimization of salt concentration for high solubility 57
• 3.8.1. Assessment of solubility in AT-μOR and 5-HT7R 57
• 3.8.2. SEC analysis of AT-μOR and 5-HT7R at different NaCl concentrations 59
• 3.9. Reconstitution of 5-HT7R ND 61
• 3.9.1. Comparison of ND synthesis methods 61
• 3.9.2. Characterization of 5-HT7R ND 63
• 3.9.3. Tryptophan fluorescence quenching assay 65
• 4. Conclusion 67
• Bibliography 69
• 국 문 초 록 71