Split Intein Selection for Peptide Binder Identification
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Solution Overview
Problem
Current methods for identifying peptide binders of target proteins, such as viral receptor binding domains, are inefficient and face challenges in stability, solubility, and specificity, particularly for 'undruggable' therapeutic targets like those involved in viral infections and cancer.
Innovation Solution
The development of a split intein-based selection system using fusion proteins and an inducible promoter to identify peptide binders, which includes expressing candidate peptides with complementary split inteins that associate to form a full-length transcription factor, driving transcription and enabling the identification of peptide binders through reporter gene expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods (small molecules or antibodies) are used to identify peptide binders, then the process is simple and direct, but the efficiency is low and the candidates suffer from poor stability and solubility
Solution Approach 1:
The transcription factor is divided into two separate fragments (N-terminal and C-terminal halves), each fused to a different component (target protein or candidate peptide). These fragments cannot function alone but can reconstitute activity when both components are present and properly folded, enabling selection of stable binders through functional complementation
Solution Approach 2:
The split intein system acts as an intermediary mechanism that facilitates the selection process. The intein fragments are expressed as fusion proteins with the target and candidate peptide, and their proper folding and association serve as a mediator to enable transcription factor reconstitution and reporter gene expression, thereby identifying stable peptide binders
2Measurement precision
If traditional selection systems are used, then the system is simple to implement, but the specificity for identifying true binders is low
Solution Approach 1:
The transcription factor is segmented into two non-functional fragments that must be brought into proximity by specific binder-target interactions. This segmentation ensures that only true binders that maintain proper folding and spatial arrangement can reconstitute transcriptional activity, thereby enhancing identification specificity
Solution Approach 2:
The system incorporates feedback through reporter gene expression that is directly coupled to the reconstitution of transcription factor activity. When candidate peptides specifically bind to target proteins and maintain proper folding, the transcription factor becomes active and drives reporter gene expression, providing a measurable feedback signal that confirms specific binding
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 allows for efficient identification of peptide binders with enhanced stability and specificity, overcoming limitations of traditional drug candidates like small molecules and antibodies, and is applicable to challenging therapeutic targets including viral proteins and cancer-related proteins.
Implementation Method 1
complementary split inteins that associate to form a full-length transcription factor
Data Source
AI summary
Disclosed herein, in some embodiments, non-naturally occurring proteins (e.g., non-naturally occurring modified proteins) that may be useful in the treatment of bacterial and viral infections, including SARS-CoV-2 infection, host cells comprising the same, and methods of treating bacterial and viral infections including SARS-CoV-2 infection. Also provided herein are host cells comprising fusion proteins for split intein-based selection of peptides that bind a target protein, methods of using the same, and methods of identifying peptides that bind a target protein.