Protein Interaction Mapping via Mutant Library Affinity Screening
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Solution Overview
Problem
Existing methods for epitope and paratope mapping are inefficient and costly, particularly in crowded IP spaces where differentiating novel antibodies from existing ones is challenging, and there is a need for high-throughput characterization of protein-protein interactions.
Innovation Solution
A method utilizing site saturation mutagenesis and high-throughput screening to characterize the interactive surfaces of protein binding partners, identifying compensatory mutations through the AlphaSeq platform, which measures affinity values between libraries of wild-type and mutant polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If exhaustive site saturation mutagenesis and high-throughput screening are used to characterize protein-protein interactions, then measurement precision and productivity are improved, but device complexity and loss of substance increase
Solution Approach 1:
The method segments the protein-protein interaction characterization into two separate libraries: one containing mutations in the first protein and another containing mutations in the second protein. This segmentation allows systematic high-throughput screening of all mutation combinations while maintaining manageable experimental complexity.
Solution Approach 2:
The invention transitions from traditional single-protein mutagenesis to a two-dimensional screening approach by creating libraries of both proteins with mutations. This dimensional expansion enables comprehensive characterization of the interaction interface by testing all combinations of mutations across both proteins simultaneously.
2Productivity
If exhaustive site saturation mutagenesis and high-throughput screening are used to characterize protein-protein interactions, then measurement precision and productivity are improved, but loss of substance increases
Solution Approach 1:
The method creates multiple copies of protein libraries with different mutations, allowing high-throughput screening without requiring large amounts of each individual protein variant. The library approach enables parallel testing of numerous mutations using small amounts of material for each variant.
Solution Approach 2:
The screening platform is designed to universally test any combination of mutations from either library against any protein from the other library. This multi-functional capability allows the same experimental setup to characterize multiple protein-protein interactions using the same protein consumption budget.
3Measurement precision
If libraries of wild-type and mutant polypeptides are screened to identify compensatory mutations, then measurement precision is improved, but loss of time increases
Solution Approach 1:
The high-throughput screening platform continuously measures affinity values for all combinations of mutations from both libraries in a single experimental run. This continuous measurement approach eliminates the need for sequential testing of individual mutations, thereby maintaining high measurement precision while reducing total screening time.
Data Source
AI summary
Characterization of the binding dynamics at the interface between any two proteins that specifically interact plays a role in myriad biomedical applications. The methods disclosed herein provide for the high-throughput characterization of the specific interaction at the interface between two protein binding partners and the identification of functionally significant mutations of one or both protein binding partners. For example, the methods disclosed herein may be useful for epitope and paratope mapping of an antibody-antigen pair, which is useful for the discovery and development of novel therapies, vaccines, diagnostics, among other biomedical applications.