Yeast Surface Display Screening for Protein Interaction Networks
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Solution Overview
Problem
Existing methods for high-throughput screening of protein-protein interactions, particularly in liquid culture, are limited by the inability to efficiently characterize dynamic interactions with non-membrane permeable or toxic ligands, and lack a suitable platform for evaluating engineered protein networks.
Innovation Solution
Re-engineering yeast sexual agglutination by replacing native proteins with synthetic adhesion proteins (SAPs) expressed on the cell surface, linking protein interaction strength to mating efficiency, enabling high-throughput screening of protein-protein interactions using recombinant yeast strains with specific nucleic acid constructs and markers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If native sexual agglutination mechanism is used in yeast, then cells can mate in liquid culture, but the binding strength cannot be coupled to arbitrary protein interactions
Solution Approach 1:
The patent removes the native sexual agglutination mechanism (Aga1/Aga2/Sag1 proteins) from yeast cells through genetic knockout. This extraction eliminates the interference of endogenous binding proteins, allowing arbitrary protein-protein interactions to be studied without background noise from native agglutination systems.
Solution Approach 2:
The patent creates a universal yeast mating platform that can screen any protein-protein interaction by expressing user-defined proteins on the cell surface. The system is no longer limited to native yeast protein interactions but can universally accommodate any protein pair by fusing them to cell surface anchors, making the system multi-functional for diverse screening applications.
2Productivity
If yeast mating is performed in turbulent liquid culture, then high throughput screening is enabled, but strong binding interactions are required to maintain cell contact
Solution Approach 1:
The patent changes the binding parameters by using high-affinity protein interactions (low nM KD values) between the proteins of interest. This allows cells to maintain sustained contact in turbulent liquid culture conditions, enabling high-throughput screening while studying weak to moderate affinity interactions that would otherwise be disrupted by shear forces.
3Strength
If native agglutinin expression is induced, then cell binding strength increases, but the system cannot evaluate arbitrary protein interactions
Solution Approach 1:
The patent uses yeast surface display technology to present copies of arbitrary proteins on the cell surface in a controlled manner. By fusing target proteins to cell surface anchors, the system creates a stable, displayable copy of the protein that maintains its binding properties while enabling high-throughput screening through yeast mating.
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
Facilitates high-throughput characterization of protein interaction networks, screening of therapeutic candidates, and evaluation of engineered protein networks by quantifying mating efficiency through diploid cell formation, overcoming limitations of existing assays.
Implementation Method 1
The cell membranes of a single MATĪ± haploid cell and a single MATalpha (MATa) haploid cell must be in direct and sustained contact in order for membrane fusion to begin
Implementation Method 2
Yeast mating in a turbulent liquid culture is dependent on an initial binding step, called sexual agglutination
Data Source
AI summary
The present invention relates to methods and compositions for the high throughput screening of protein-protein interactions in yeast liquid culture. Protein fusions non-native to yeast may be expressed to replace endogenous sexual agglutination proteins and mediate library-by-library interrogation of protein interactions. The methods and compositions of the invention can be utilized for the characterization of protein interaction networks in high throughput for both binding affinity and specificity, which is crucial for understanding cellular functions, screening therapeutic candidates, and evaluating engineered protein networks.