Fusion Protein Vesicle Tagging for High-Throughput Secretion Screening
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Solution Overview
Problem
Current methods for measuring insulin secretion are expensive, time-consuming, and not suitable for high-throughput genome-wide chemical and genetic screening, limiting the investigation of genetic pathways regulating insulin secretion and hindering the development of diabetes treatments.
Innovation Solution
A non-naturally occurring nucleic acid construct encoding a fusion protein that localizes to secretory vesicles, incorporating a protein tag which binds to a cell-impermeable marker, allowing for quantitative measurement of secretion levels and sorting of cells based on secretion levels, enabling cost-effective high-throughput screening.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional immunoassays (ELISA, radioimmunoassay) are used to measure secreted proteins, then measurement precision is improved, but productivity deteriorates due to expense and time consumption
Solution Approach 1:
The patent replaces traditional mechanical/chemical immunoassay systems with a fluorescent optical detection system. Cells express fusion proteins containing vesicular domains tagged with fluorescent proteins, allowing direct visualization and quantification of secretion events through fluorescence microscopy or flow cytometry, eliminating the need for expensive immunoassay reagents and procedures
Solution Approach 2:
The invention utilizes fluorescent protein tags that emit specific colors/wavelengths when excited, enabling detection and quantification of secretion. The fluorescent signal intensity correlates with secretion amount, providing a quantitative measure that replaces traditional immunoassay readouts while enabling high-throughput screening
2Measurement precision
If traditional immunoassays are used for measuring secretion, then measurement precision is improved, but loss of time worsens due to complicated handling requirements
Solution Approach 1:
The patent replaces time-consuming immunoassay procedures with a real-time fluorescent imaging system. The fusion proteins continuously report secretion events as they occur, allowing multiple time points to be captured without repeating assay steps, thereby dramatically reducing total measurement time while maintaining precision
Solution Approach 2:
The fluorescent fusion proteins provide continuous monitoring of secretion activity throughout the experiment duration. Unlike discrete immunoassay steps, the fluorescent signal persists and can be measured at multiple time points without interrupting the secretion process, enabling dynamic tracking of secretion kinetics
3Measurement precision
If traditional immunoassays are used, then measurement precision is improved, but device complexity worsens due to restriction to 96-well format
Solution Approach 1:
The fluorescent fusion protein system is universally applicable across multiple plate formats (96-well, 384-well, high-content imaging plates) and cell types. The same basic approach of tagging vesicular proteins with fluorescent markers can be used in various screening formats without requiring format-specific reagents or procedures, thereby reducing overall system complexity
4Measurement precision
If traditional immunoassays are used for screening, then measurement precision is improved, but productivity deteriorates due to inability to perform pooled screens
Solution Approach 1:
The invention enables pooled screening by using fluorescent fusion proteins that report secretion in individual cells within pooled populations. Each cell expresses the fusion protein and can be individually resolved by fluorescence-activated cell sorting (FACS) or high-content imaging, allowing simultaneous assessment of many cells in parallel while maintaining single-cell measurement precision
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
Enables rapid and efficient measurement of secretion in single cells, facilitating high-throughput screening for modulators of secretion and improving the understanding of genetic pathways involved in insulin secretion, potentially leading to advancements in diabetes treatment.
Implementation Method 1
The cell impermeable marker may be a fluorescent marker
Data Source
AI summary
The present invention provides methods and compositions based on a non-naturally occurring nucleic acid construct encoding a fusion protein for quantitating levels of secretion in a single cell which may comprise a protein sequence which may comprise a cytoplasmic domain, a transmembrane domain and a vesicular domain, wherein the vesicular domain may comprise a protein tag sequence, wherein upon expression of the fusion protein by a cell, the fusion protein localizes to the membrane of a secretory vesicle such that the protein tag localizes to the lumen of the secretory vesicle, and wherein the protein tag binds to a cell-impermeable marker; whereby upon secretion of the contents of the secretory vesicle, the protein tag is exposed to the cell-impermeable marker, the fusion protein is recycled back into the cell, and the single cell becomes labeled with the marker relative to the amount of secretion.