ERBB2 Variant Screening Through Saturation Mutagenesis
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Solution Overview
Problem
Current methods lack a systematic approach to identify specific residues in the ERBB2 receptor tyrosine kinase that, when mutated, result in increased autophosphorylation implicated in cancer progression, limiting effective cancer detection and treatment strategies.
Innovation Solution
A method involving saturation mutagenesis of amino acids 679-992 of ERBB2 to create a library of variants, expressed in mammalian cells, and measuring phosphorylation levels to identify variants implicated in cancer, using barcoded plasmids and high-throughput assays to generate comprehensive profiles for cancer detection and treatment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If saturation mutagenesis is performed on ERBB2 to create a comprehensive library of variants, then the ability to identify cancer-related variants is improved, but the complexity of the screening system increases
Solution Approach 1:
The ERBB2 protein sequence is segmented into multiple regions, with saturation mutagenesis applied to specific segments (e.g., residues 679-992) rather than the entire protein. This segmentation allows comprehensive variant identification while managing library complexity by focusing on cancer-relevant regions.
Solution Approach 2:
A universal screening platform is developed that can evaluate multiple ERBB2 variants simultaneously using standardized assays (phosphorylation measurements, cell-based assays). This multi-functional system handles diverse variants through a common methodology, reducing overall system complexity despite the large number of variants tested.
2Reliability
If a comprehensive library of ERBB2 variants is screened in mammalian cells, then the reliability of identifying cancer-related variants is improved, but the time and resources required for screening increase
Solution Approach 1:
In silico modeling and computational predictions are performed before experimental screening to prioritize which variants are most likely to be cancer-related. This preliminary action filters the comprehensive variant library, allowing focused experimental screening that maintains reliability while reducing time and resource requirements.
Solution Approach 2:
Cell-free systems and in silico models serve as simplified copies of the complete mammalian cell screening system. These copying approaches allow rapid preliminary evaluation of variants, with only the most promising candidates subjected to time-consuming mammalian cell assays, thereby maintaining reliability while reducing overall screening time.
3Measurement precision
If phosphorylation levels are measured to identify active ERBB2 variants, then the precision of detecting cancer-related variants is improved, but the difficulty of detection and measurement increases
Solution Approach 1:
Phosphorylation-specific antibodies serve as intermediaries that specifically bind to phosphorylated ERBB2 variants. These antibodies translate the molecular event of phosphorylation into a detectable signal through standardized assays (flow cytometry, ELISA), improving measurement precision while simplifying detection compared to direct phosphorylation measurement.
Solution Approach 2:
Traditional biochemical phosphorylation assays are replaced with cell-based phosphorylation measurements using fluorescently labeled antibodies and flow cytometry. This substitution replaces complex biochemical fractionation and detection mechanics with a more streamlined immunodetection system, maintaining precision while reducing measurement difficulty.
Data Source
AI summary
Disclosed herein are systems and methods for screening variant libraries for activity. Also disclosed herein are high throughput methods for identifying candidate variant having gain of function biological activity in an assay.