Split Luciferase Polypeptide Kit for Low-Hindrance Interaction Detection
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Solution Overview
Problem
Existing probes for intermolecular interaction analysis, particularly those based on luciferases, face issues such as steric hindrance and improper expression of fusion proteins due to their large size, which can inhibit normal protein functioning.
Innovation Solution
A reagent kit comprising first and second polypeptides with specific amino acid sequences, optimized for luciferase activity, allowing for small molecular weight detection by luciferase activity when in close proximity, minimizing steric hindrance and enabling proper protein expression.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If a large probe is used for detecting intermolecular interactions, then the detection capability is provided, but the fusion protein may not be expressed normally in cells or steric hindrance occurs to inhibit normal functioning of the target protein
Solution Approach 1:
The luciferase probe is divided into two separate fragments (first polypeptide and second polypeptide) that can independently function. This segmentation reduces the size of each individual probe component, minimizing steric hindrance while maintaining detection capability through complementary binding of the fragments to the target protein.
Solution Approach 2:
The invention creates probes with different molecular weights and structural characteristics (first polypeptide with specific amino acid sequence deletions, second polypeptide with different sequence variations) to optimize local interaction properties. This allows one probe to bind the target protein while the other provides detection function with minimal interference.
2Reliability
If a small probe is used for detecting intermolecular interactions, then steric hindrance is minimized and normal protein function is maintained, but the detection sensitivity may be reduced
Solution Approach 1:
The invention combines two different small polypeptide probes (first and second polypeptides with different amino acid sequences) into a complementary detection system. Each polypeptide alone has limited detection capability, but together they provide enhanced detection sensitivity through cooperative binding to the target protein, while both maintain small sizes that minimize steric hindrance.
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 effective detection of intermolecular interactions with high sensitivity and minimal interference, facilitating normal protein function and robust signal detection.
Implementation Method 1
a second polypeptide including a part in any one of amino acid sequences (A) to (C), which are consistent of different sequences from a sequence of the first polypeptide, and exhibiting luciferase activity when in close proximity to the first polypeptide
Data Source
AI summary
A reagent kit comprising a first polypeptide including a part in any one of amino acid sequences (A) to (C), and a second polypeptide including a part in any one of amino acid sequences (A) to (C), which are consistent of different sequences from a sequence of the first polypeptide;(A) an amino acid sequence in SEQ ID NO: 1 with deletion of an amino acid sequence from position 1 to 69 and an amino acid sequence from position 204 to 221,(B) an amino acid sequence in SEQ ID NO: 1 with deletion of an amino acid sequence from position 1 to 69 and deletion or substitution of at least one of amino acid residues at positions 146 to 156,(C) the amino acid sequence (A) or (B) with further deletion of at least one of amino acid residues at positions 70 to 74.