Yeast GPCR Evolution Through Cell Wall Permeabilization
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
The challenge of achieving high functional expression levels of transmembrane receptors, particularly GPCRs, in eukaryotic cells is hindered by the cell wall, which restricts access of ligands and requires methods like yeast display that are not suitable for insoluble transmembrane receptors.
Innovation Solution
A method involving permeabilization of yeast cell walls with an alkaline pH buffer containing lithium ions and reducing agents, followed by fluorescent labeling and sorting, allows for the selection and expansion of yeast cells expressing functional GPCRs, enabling multiple rounds of selection and amplification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If yeast display method is used to present proteins outside the cell wall, then accessibility of ligands to membrane receptors is improved, but this approach is not suitable for insoluble transmembrane receptors that must be located in the plasma membrane
Solution Approach 1:
The invention extracts and removes the cell wall barrier from yeast cells to enable ligand access to membrane receptors while maintaining the cells' eukaryotic expression capabilities. This is achieved through enzymatic digestion of cell wall components, allowing the system to be adapted for selecting insoluble transmembrane receptors that require plasma membrane localization.
Solution Approach 2:
Instead of presenting proteins outside the cell wall as fusion proteins (yeast display approach), the invention inverts the approach by keeping transmembrane receptors in their native plasma membrane location and removing the cell wall barrier to enable ligand access. This inversion makes the system suitable for insoluble receptors that cannot be expressed as soluble fusion proteins.
2Reliability
If mammalian or insect cells are used as eukaryotic hosts for directed evolution, then functional expression of transmembrane receptors is improved, but these cells are not easily transformed with libraries
Solution Approach 1:
The invention makes yeast cells universal by combining their ease of transformation with the ability to properly fold and functionalize eukaryotic transmembrane receptors. By removing the cell wall barrier, yeast acquire the dual capability of being easily transformable like prokaryotes while maintaining eukaryotic protein processing functions, making them suitable for directed evolution of membrane proteins.
Solution Approach 2:
The invention changes the physical-chemical parameters of yeast cells by removing the cell wall structure. This parameter change transforms yeast from having a rigid cell wall barrier to having a permeabilized state that allows ligand access, while maintaining the eukaryotic cellular machinery needed for proper receptor folding and function.
3Strength
If cell wall is present in yeast cells, then yeast cells maintain structural integrity, but this restricts access of administered ligands to expressed receptors in the plasma membrane
Solution Approach 1:
The invention extracts specific components of the cell wall (primarily glucans and mannans) while leaving the plasma membrane and cellular machinery intact. This selective removal maintains sufficient structural integrity for cell viability while creating permeability channels that allow ligand access to plasma membrane receptors.
Solution Approach 2:
The invention applies local quality change by selectively modifying the cell wall structure rather than completely removing it. The cell wall is partially degraded in specific regions to create access pathways for ligands, while maintaining overall structural integrity and cellular function. This localized modification enables ligand access without compromising cell strength.
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 significantly increases functional expression levels of GPCRs in yeast cells, allowing for the selection of high-expression variants and improving their stability and functionality.
Implementation Method 1
permeabilizing the cell wall of said plurality of yeast cells in a permeabilization step, wherein the permeabilization step comprises exposing the plurality of yeast cells to a chemical treatment
Implementation Method 2
wherein the chemical treatment is a buffer of alkaline pH comprising lithium ions, a reducing agent and/or a chelating agent
Implementation Method 3
contacting said plurality of viable permeabilized cells in a labelling step with a ligand capable of binding to said GPCR, wherein said ligand comprises a detectable label
Implementation Method 4
selecting a subset of said plurality of viable labelled cells as a function of detectable label present in said plurality of viable labelled cells in a selection step, yielding a selection of viable cells, wherein said detectable label is a fluorescent dye and said selection step is accomplished by fluorescent cell sorting
Data Source
Figure 1
Figure 2A~2F
Figure 3A~3B
AI summary
The invention relates to a method for selecting an expressed sequence from a library, comprising the following steps: Each of a plurality of eukaryotic cells comprising a cell wall comprises a nucleic acid sequence member of a library, which is expressed as a target membrane protein in said eukaryotic cells. The cell wall of the cells is permeabilized. The permeabilized cells are labeled with a ligand capable of binding to the target membrane protein. The ligand bears a detectable label. A subset of the labelled cells is selected as a function of detectable label present. Finally, an expressed nucleic acid sequence is isolated from said selection of cells in an isolation step.