Mutant Hydrolase Proteins for Stable Protein Labeling
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Solution Overview
Problem
Current methods for labeling proteins, such as using green fluorescent protein (GFP), are limited by their intrinsic properties, including a limited range of fluorescent colors and relatively low intrinsic brightness, as well as non-specific binding and poor functional expression in certain environments.
Innovation Solution
Development of mutant hydrolase sequences with improved functional expression and binding kinetics, allowing for enhanced protein labeling through stable bond formation with substrates, which can be used to tether functional groups to proteins, thereby improving fluorescence polarization and protein production.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If GFP is used for protein labeling, then fluorescence detection is enabled, but the fluorescent color range is limited and intrinsic brightness is low
Solution Approach 1:
The patent modifies the chemical structure of fluorophores by changing parameters such as conjugation extent, aromatic ring substitution, and molecular weight to achieve different fluorescent colors and brightness levels, thereby overcoming GFP's limited color range and low brightness
2Reliability
If receptor-mediated targeting is used to localize fluorophores, then high affinity binding is achieved, but functional expression is poor in reducing environments
Solution Approach 1:
The patent introduces a heterobifunctional crosslinker as an intermediary between the protein and fluorophore, allowing the protein to maintain its native environment while the crosslinker provides the necessary chemistry for stable, high-affinity bonding without requiring the protein to function in reducing environments
3Adaptability or versatility
If in vitro chemical modification is used for label attachment, then labeling flexibility is achieved, but protein purification is required first
Solution Approach 1:
The patent incorporates reactive groups into the fluorophore during the labeling process itself, eliminating the need for prior protein purification. The crosslinker is designed to react with specific amino acid residues on the native protein, enabling direct labeling in complex mixtures
4Measurement precision
If FLASH labeling method is used, then small receptor domain binds specifically, but non-specific binding to endogenous proteins occurs and fluorophore range is limited
Solution Approach 1:
The patent extracts the essential binding function from the complex FLASH receptor system and implements it through a simplified heterobifunctional crosslinker that selectively targets the protein of interest without binding to endogenous proteins, thereby eliminating non-specific binding while maintaining specificity
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
The mutant hydrolase sequences exhibit improved functional expression and binding kinetics, leading to more stable and efficient protein labeling, overcoming the limitations of existing methods by providing enhanced fluorescence and specificity.
Implementation Method 1
synthesized a tight-binding pair of molecular components... The two components bind specifically and with high affinity to one another... form a covalent bond with the substrate
Implementation Method 2
mutant hydrolase sequences that exhibit improved functional expression... mutant hydrolase proteins with improved intrinsic binding kinetics
Data Source
AI summary
The invention provides a mutant hydrolase protein with enhanced kinetics and functional expression, as well as polynucleotides encoding the mutant proteins and methods of using the polynucleotides and mutant proteins.