Cellular Peptide Display Scaffolds for Quantitative Ligand Screening
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Solution Overview
Problem
Current combinatorial library screening methods, such as phage display, lack the ability to quantitatively and directly discriminate ligand binding parameters and are unsuitable for eukaryotic secreted proteins requiring post-translational modifications like glycosylation or extensive disulfide isomerization, which are unavailable in bacterial cells.
Innovation Solution
The development of peptide display scaffolds that present candidate peptides on the cell surface, allowing for the identification of peptide substrates or inhibitors by using transmembrane proteins and detectable moieties, enabling qualitative and quantitative characterization through fluorescence-activated cell sorting.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If phage display is used for combinatorial library screening, then polypeptide display and screening capability is provided, but quantitative and direct discrimination of ligand binding parameters is precluded
Solution Approach 1:
The patent replaces the mechanical/phage-based display system with a fluorescent protein-based system. The fluorescent protein fusion proteins are expressed in E. coli and displayed on the cell surface, allowing quantitative measurement of ligand binding parameters through fluorescence intensity. This substitution enables precise measurement while maintaining the screening capability.
Solution Approach 2:
The patent utilizes fluorescent proteins that exhibit color changes or fluorescence intensity changes upon ligand binding. This allows for quantitative and direct discrimination of ligand binding parameters through optical detection, resolving the limitation of phage display where such measurements are precluded.
2Adaptability or versatility
If bacterial cells are used for phage display, then polypeptide expression is achieved, but post-translational modifications such as glycosylation or extensive disulfide isomerization are unavailable
Solution Approach 1:
The patent uses E. coli as a universal expression system that can produce a wide variety of polypeptides including those requiring post-translational modifications. By fusing these polypeptides to fluorescent proteins and displaying them on the E. coli cell surface, the system achieves both ease of manufacture and adaptability for studying eukaryotic secreted proteins and cell surface proteins.
3Productivity
If cell surface peptide display is implemented, then efficient screening and qualitative/quantitative characterization is enabled, but system complexity increases compared to phage display
Solution Approach 1:
The patent replaces the complex phage assembly and display mechanism with a simpler fluorescent protein fusion system expressed directly in E. coli. The fluorescent proteins are fused to the polypeptides of interest and displayed on the cell surface, enabling efficient screening through flow cytometry or fluorescence microscopy without requiring phage particle assembly.
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 approach enables efficient screening and characterization of peptide interactions with enzymes, providing a scalable solution for identifying peptide ligands and inhibitors by displaying candidate peptides on the cell surface, overcoming limitations of existing methods.
Implementation Method 1
detecting the presence or absence of a D signal, wherein a decrease in the D signal in the presence of the enzyme as compared to the absence of the enzyme indicates that at least one cell of the cell library expresses a candidate peptide that is a substrate for the enzyme
Data Source
AI summary
The present invention provides compositions including peptide display scaffolds that present at least one candidate peptide and at least one detectable moiety in at least one of the N-terminal and C-terminal candidate peptide presenting domains that when expressed in a cell are accessible at a surface of the cell outermembrane. In addition, the present invention also provides kits and methods for screening a library of cells presenting the candidate peptides in peptide display scaffolds to identify a ligand for an enzyme.