Mutant Protease Biosensors for Granzyme B Detection
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Solution Overview
Problem
Current methods for measuring cell-mediated cytotoxicity, such as the chromium release assay, rely on radioactivity and are time-intensive, necessitating the development of non-radioactive, sensitive assays for detecting cell-mediated cytotoxicity, particularly for cytotoxic lymphocytes and natural killer cells.
Innovation Solution
The use of modified circularly-permuted thermostable luciferases with linked sensor regions that interact with target molecules like granzyme B, allowing for enhanced luminescence detection in the presence or absence of these molecules, enabling the detection of granzyme B activity and cell-mediated cytotoxicity through bioluminescent assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If chromium release assay is used to measure cell-mediated cytotoxicity, then measurement capability is achieved, but radioactivity and time consumption increase
Solution Approach 1:
The patent replaces the radioactive chromium release assay with a bioluminescence-based detection system. Specifically, it uses a circularly permuted thermostable luciferase biosensor that emits light upon granzyme B cleavage, substituting radioactive measurement with a non-radioactive optical detection method that maintains high sensitivity while eliminating harmful radiation
Solution Approach 2:
The patent changes the detection parameter from radioactive signal measurement to bioluminescence signal measurement. By engineering a luciferase biosensor with a granzyme B recognition site, the system converts granzyme B activity into a measurable light signal, fundamentally changing the detection parameter from radioactivity to photons while improving safety
2Measurement precision
If chromium release assay is used to measure cell-mediated cytotoxicity, then measurement capability is achieved, but time consumption increases
Solution Approach 1:
The patent replaces the time-consuming chromium release assay with a rapid bioluminescence assay. The circularly permuted thermostable luciferase biosensor provides immediate light signal upon granzyme B cleavage, eliminating the lengthy incubation and measurement steps required by radioactive assays while maintaining detection sensitivity
Solution Approach 2:
The patent changes the temporal parameter of the assay by using a real-time bioluminescence readout. The luciferase biosensor generates an immediate light signal upon proteolytic cleavage by granzyme B, allowing for rapid assessment of cytotoxicity without the time-consuming steps inherent in chromium release assays
3Object-generated harmful factors
If circularly-permuted thermostable luciferase biosensor is used, then non-radioactive detection is achieved, but detection sensitivity must be enhanced
Solution Approach 1:
The patent applies local quality enhancement by introducing multiple point mutations at specific positions within the luciferase biosensor structure. These localized amino acid substitutions (e.g., at positions 47, 83, 107, 144, 160, 174, 188, 198, 205, 225, 233, 242, 255, 268, 308, 316, 358, 377, 403, 435, 490, 540) are designed to enhance the biosensor's responsiveness to granzyme B cleavage while maintaining its non-radioactive detection capability
Solution Approach 2:
The patent enhances detection sensitivity by modifying the biochemical parameters of the luciferase biosensor through site-directed mutagenesis. The engineered mutations optimize the biosensor's catalytic efficiency and luminescence output upon granzyme B activation, thereby improving measurement precision while maintaining the safety advantages of non-radioactive detection
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 provides a sensitive, non-radioactive method for detecting granzyme B activity and cell-mediated cytotoxicity, facilitating high-throughput screening and monitoring of cytotoxic functions in cellular processes, offering a more efficient and safer alternative to traditional assays.
Implementation Method 1
Luciferases are enzymes that catalyze the oxidation of a substrate (e.g., luciferin or coelenterazine) with the concomitant release of photons of light
Data Source
Figure 1
Figure 2A~2B
Figure 3A~3B
AI summary
A polynucleotide encoding a biosensor polypeptide comprising a modified circularly-permuted thermostable luciferase and a linker linking the C-terminal portion of the thermostable luciferase to the N-terminal portion of the thermostable luciferase. The modified circularly-permuted thermostable luciferase is modified relative to a parental circularly-permuted thermostable luciferase. The linker contains a sensor region capable of interacting with a target molecule in a cell. The modified circularly-permuted thermostable luciferase has an enhanced response after interaction of the biosensor with the target molecule relative to the parental circularly-permuted thermostable luciferase in the presence of the target molecule. Alternatively, the modified circularly-permuted thermostable luciferase has an enhanced response after interaction of the biosensor with the target molecule relative to the modified circularly-permuted thermostable luciferase in the absence of the target molecule.