Consensus GPCR Sequence Design for Membrane Expression Analysis
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Solution Overview
Problem
Existing methods struggle to efficiently express and functionally analyze G protein-coupled receptors (GPCRs) in cultured cells, particularly those with unknown ligands, due to insufficient membrane expression.
Innovation Solution
A method involving consensus design of GPCR amino acid sequences by altering specific residues based on a consensus amino acid sequence derived from orthologs, enhancing membrane expression and stability of GPCR polypeptides in cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional methods are used to express GPCRs in cultured cells, then the expression process is simple, but membrane expression is insufficient and functional analysis is difficult
Solution Approach 1:
The patent applies parameter changes by altering amino acid residues in the GPCR sequence based on consensus sequences derived from orthologs. Specifically, residues at positions 7.39, 7.40, and 7.41 are modified to enhance membrane expression and stability, transforming the GPCR from a poorly expressed protein to one with reliable membrane localization and functional activity.
Solution Approach 2:
The consensus sequence design creates a universal GPCR expression platform that can be applied across different GPCR families. By establishing a consensus amino acid sequence based on orthologous regions, the patent enables functional analysis of multiple GPCRs using a standardized approach, making the method applicable to various receptor types with unknown ligands.
2Productivity
If GPCRs with unknown ligands are expressed in cultured cells, then functional analysis can be performed, but insufficient membrane expression prevents effective ligand identification
Solution Approach 1:
The patent performs preliminary action by pre-altering the GPCR amino acid sequence before expression in cultured cells. The consensus sequence design is applied in advance to ensure that the GPCR achieves sufficient membrane expression and stability, thereby enabling subsequent functional analysis and ligand identification to proceed effectively without requiring additional optimization steps.
3Reliability
If GPCR amino acid sequences are altered based on consensus sequences, then membrane expression and stability are enhanced, but the process requires sequence alignment and residue identification
Solution Approach 1:
The patent applies local quality by making targeted alterations at specific amino acid positions (7.39, 7.40, 7.41) rather than modifying the entire sequence. By identifying and altering only the critical residues that affect membrane expression and stability, the patent maintains the overall integrity of the GPCR while improving its functional properties, thereby simplifying the modification process.
Data Source
AI summary
Provided is an approach which can express and function a G protein-coupled receptor (GPCR). The present invention provides a method for expressing a GPCR polypeptide, comprising expressing in a cell a GPCR polypeptide consisting of an amino acid sequence obtained by, in an amino acid sequence of a GPCR of interest (provided that an olfactory receptor is excluded), altering at least one amino acid residue different from that in a consensus amino acid sequence to an amino acid residue at a position corresponding thereto in the consensus amino acid sequence, wherein the consensus amino acid sequence is an amino acid sequence derived by alignment of the amino acid sequence of the GPCR of interest and amino acid sequences of GPCRs encoded by orthologs of the GPCR of interest in vertebrates.