Calcium Indicator Polypeptide for Insecticidal Protein Screening
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Solution Overview
Problem
Current methods for studying insecticidal protein activity and mode of action in insect cell-based assays are limited by simplicity, speed, high-throughput applicability, and invasiveness, and genetically encoded calcium indicators have not been successfully used for this purpose, especially in high-throughput screening.
Innovation Solution
A polypeptide with calcium ion indicator activity, represented by SEQ ID NO. 1 or sequences with at least 80% identity, is used to identify transmembrane pore formation capability in cellular assays, providing a genetically encoded calcium ion indicator that measures calcium influx through fluorescent signals, overcoming the limitations of existing methods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional in vitro methods (osmotic swelling assays, two-electrode voltage clamp, cytotoxicity detection) are used to study insecticidal protein activity, then measurement capability is achieved, but device complexity and ease of operation deteriorate due to invasiveness and procedural complexity
Solution Approach 1:
The patent replaces complex mechanical and electrochemical measurement systems (two-electrode voltage clamp, osmotic swelling assays) with a fluorescent optical system. The calcium indicator protein provides real-time calcium influx detection through fluorescence intensity changes, eliminating the need for invasive electrical or mechanical measurements while simplifying the assay procedure.
Solution Approach 2:
The calcium indicator protein acts as an intermediary that translates transmembrane pore formation events into measurable fluorescent signals. Instead of directly measuring pore formation or insecticidal protein activity, the system uses calcium ions as intermediaries that flow through formed pores and bind to the indicator protein, producing a readable fluorescent output that simplifies detection.
2Measurement precision
If genetically encoded calcium indicators are used to measure intracellular calcium levels, then measurement capability is provided, but high-throughput applicability deteriorates due to lack of successful implementation in insect cell-based assays
Solution Approach 1:
The calcium indicator protein is genetically encoded and expressed within the insect cells themselves, making the cells self-indicating. This eliminates the need for external indicator addition or complex sample preparation steps, enabling direct high-throughput screening of multiple insect cell samples simultaneously using standard fluorescent plate readers.
3Measurement precision
If invasive methods are used to detect insecticidal protein activity, then activity identification is achieved, but loss of time and ease of operation worsen due to repeated measurement limitations
Solution Approach 1:
The patent replaces invasive mechanical and electrical detection methods with non-invasive fluorescent optical detection. This allows repeated measurements of the same insect cell samples over time without causing damage or altering cell physiology, enabling kinetic studies and longitudinal monitoring of transmembrane pore formation dynamics.
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 easy-to-use, fast, and high-throughput identification of transmembrane pore formation capability, specifically characterizing insecticidal proteins by measuring calcium influx, which is non-invasive and suitable for repeated measurements, enhancing the understanding of insecticidal protein activity.
Implementation Method 1
a calcium indicator protein is known in which a partial sequence of calmodulin and a partial sequence of myosin light chain kinase are linked to a fluorescent protein. This calcium indicator protein utilizes the phenomenon in which binding of calcium to the partial sequence of calmodulin causes a change in the conformation of the protein, which causes a change in the intensity of fluorescence emitted by the fluorescent protein
Data Source
AI summary
Provided is the use of a polypeptide with calcium indicator activity which comprises an amino acid sequence represented by SEQ ID NO. 1, or an amino acid sequence which has at least 80% sequence identity thereto, for identifying transmembrane pore formation capability of a target polypeptide, especially an insecticidal protein, in a cellular assay.