Olfactory Receptor Nanodiscs via E. coli Expression
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Producing stable nanodiscs comprising olfactory receptor proteins in E. coli is challenging due to strong hydrophobicity and differences in charge distribution, leading to difficulties in expression and productivity, especially when GPCRs are forced to be overexpressed, which can be detrimental to E. coli cells.
Innovation Solution
A method involving the production and purification of olfactory receptor proteins and membrane scaffold proteins in E. coli, followed by mixing with lipids and surfactants to form nanodiscs, optimizing the secondary structure and stability of the receptor protein, and using apolipoprotein A-I as a membrane scaffold to create a nanodisc structure that mimics the original receptor structure, stable in water and atmospheric environments.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If GPCRs are forced to be overexpressed in E. coli, then productivity is improved, but the E. coli cells die due to strong hydrophobicity and differences in charge distribution
Solution Approach 1:
The patent introduces a membrane scaffold protein (MSP) as an intermediary component that forms a protective framework around the hydrophobic GPCR. This MSP scaffold acts as a mediator between the hydrophobic receptor and the aqueous environment, preventing cell death while enabling overexpression. The MSP-GPCR complex is then reconstituted into nanodiscs with lipids, creating a stable structure that maintains receptor functionality without harming E. coli cells during production.
Solution Approach 2:
The patent changes the physical-chemical parameters of the expression system by optimizing induction conditions (IPTG concentration, temperature, time) and using modified MSP sequences with altered charge distributions. These parameter changes allow the E. coli cells to tolerate the hydrophobic stress of GPCR overexpression while maintaining high productivity.
2Stability of the object's composition
If GPCRs are expressed using animal cells and insect cells, then expression stability is improved, but productivity and cost effectiveness are lowered
Solution Approach 1:
The patent creates a simplified copy of the eukaryotic membrane environment by reconstituting GPCRs into nanodiscs composed of E. coli-expressed MSP and purified lipids. This artificial membrane copy provides the structural stability normally requiring complex animal or insect cell systems, while enabling high-yield production in the simpler prokaryotic E. coli system.
3Ease of manufacture
If GPCRs are produced in E. coli, then cost effectiveness is improved, but expression difficulty increases due to prokaryotic limitations
Solution Approach 1:
The patent segments the GPCR production process into distinct modules: (1) GPCR gene cloning into expression vectors, (2) co-expression with MSP in E. coli, (3) cell lysis and protein extraction, (4) lipid mixing and nanodisc reconstitution, and (5) purification. This segmentation transforms a complex expression problem into manageable steps, reducing overall complexity while maintaining cost effectiveness.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This method enhances the selectivity, accuracy, and reproducibility of nanodiscs in sensing cadaverine, allowing for effective detection of rotten foods and differentiation of food decomposition levels, while maintaining receptor function and improving productivity and cost-effectiveness.
Implementation Method 1
mixing the settled mixture with the membrane scaffold protein, which was produced and purified in E. coli, and stirring, thereby assembling a nanodisc
Data Source
AI summary
The present invention relates to a manufacturing method of a nanodisc comprising an olfactory receptor protein, and a nanodisc comprising an olfactory receptor protein manufactured by the same, and more specifically, a manufacturing method of a nanodisc comprising an olfactory receptor protein using E. coli, and a nanodisc comprising an olfactory receptor protein manufactured by the same.According to the present invention, nanodiscs (T13NDs) are manufactured by producing receptors used in T13NDs from E. coli, thereby being able to mimic the original receptor structure and can be stable in water and atmospheric environments, and by the same, not only selectivity, accuracy, and reproducibility can be improved, but also it was possible to selectively detect cadaverine, which is known to occur from rotten foods, through the improved performance ability.