Periplasmic Ligand Trapping System for Receptor Screening
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Solution Overview
Problem
Current methods for studying receptor-ligand interactions in yeast cells face challenges such as the need for over-expression and purification of proteins, non-specific binding to assay beads, and limited information on functional selectivity, hindering the discovery of genetically encoded peptide and protein ligands for human transmembrane receptors.
Innovation Solution
A yeast periplasmic ligand-trapping system that displays peptide-, protein-, and nanobody-based ligands fused to a cell-wall anchoring protein in the periplasmic space, allowing for modulation of receptor function and assessment of ligand-receptor interactions through an inducible reporter system.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If proteins are displayed on the outside of the yeast cell wall for interaction studies, then protein-protein interactions can be identified, but the method requires over-expression and purification of sufficient amounts of protein target which may not be possible
Solution Approach 1:
The patent uses the yeast cell wall as an intermediary surface to display ligands, eliminating the need for extensive protein purification. The cell wall acts as a natural display platform that can present proteins and peptides in their native conformation without requiring large-scale purification steps, thus resolving the contradiction between interaction detection capability and protein quantity requirements
Solution Approach 2:
The patent creates genetic copies of ligand-encoding sequences that are integrated into the yeast genome. These genetic copies enable in situ production and display of ligands on the cell wall, eliminating the need to work with purified proteins. The genetic information serves as a stable copy that can be continuously expressed without depleting protein reserves
2Measurement precision
If purified protein target is used for pull-down assays, then bait and target binding can be identified, but non-specific binding to assay beads occurs which creates the need for several rounds of enrichment
Solution Approach 1:
The patent extracts the binding assay from the traditional purified protein pull-down format and relocates it to an in vivo yeast display system. By taking out the harmful element of bead-based non-specific binding and replacing it with cell wall-based display, the method maintains binding interaction identification while eliminating the source of non-specific binding artifacts
Solution Approach 2:
The yeast cell wall serves as a self-organizing display platform that presents ligands in a controlled manner. The cell wall's natural structure and composition provide a physiologically relevant environment that reduces non-specific binding, allowing the system to self-regulate interactions without requiring multiple enrichment rounds to remove false positives
3Measurement precision
If protein display methods are used on yeast surface, then protein-protein interactions can be studied, but little to no information regarding the functional selectivity of the bait-target interaction is obtained
Solution Approach 1:
The patent merges interaction detection with functional readout by combining ligand display on the cell wall with intracellular reporter systems. This integration allows simultaneous measurement of binding events and downstream functional responses, eliminating the information loss about functional selectivity while maintaining interaction detection capability
Solution Approach 2:
The patent adds a functional dimension to the interaction assay by coupling surface display with intracellular signaling readouts. This dimensional expansion transforms a simple binding detection assay into a multi-parameter system that simultaneously reports on binding affinity and functional selectivity, thereby recovering the lost functional information
Data Source
AI summary
Disclosed herein are engineered yeast cells, systems, and methods for screening for a ligand that modulates a receptor function and for determining functional consequences of receptor-ligand binding.