Red Fluorescent Protein Sequence Tuning for Brightness and Stability
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Solution Overview
Problem
There is a need for fluorescent proteins with improved spectral properties or increased stability, particularly in the field of red fluorescent proteins (RFPs).
Innovation Solution
Development of improved RFPs with specific amino acid sequences, such as SEQ ID NOs: 15, 17, and 19, and methods involving directed evolution and insertion of destabilizing loops to enhance stability and fluorescence, including fusion with other molecules and expression in host cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional fluorescent proteins are used, then they can be easily obtained and expressed, but they lack improved spectral properties and stability
Solution Approach 1:
The patent applies parameter changes by systematically mutating specific amino acid residues in the fluorescent protein sequence. Multiple point mutations at defined positions (such as F64L, S65T, T203Y, etc.) were introduced to optimize spectral properties and stability while maintaining expressibility in host cells
Solution Approach 2:
The patent creates composite fluorescent proteins by combining multiple beneficial mutations into a single optimized sequence. The improved RFP integrates mutations from different positions that collectively enhance stability, brightness, and spectral characteristics while preserving the fundamental protein structure and function
2Illumination intensity
If fluorescent proteins with various spectral variants are developed, then spectral properties are improved, but stability decreases
Solution Approach 1:
The patent resolves this contradiction by carefully selecting and combining specific amino acid substitutions that enhance fluorescence intensity without compromising stability. Mutations such as F64L and S65T improve brightness and folding efficiency, while other positions are optimized to maintain structural stability and resistance to denaturation
3Illumination intensity
If fluorescent proteins are optimized for brightness, then fluorescence intensity increases, but resistance to chemical denaturation decreases
Solution Approach 1:
The patent addresses this contradiction through coordinated amino acid substitutions that simultaneously enhance fluorescence and improve chemical stability. Specific mutations strengthen the protein core and surface interactions, increasing resistance to denaturants like urea and guanidine hydrochloride while maintaining or enhancing bright fluorescent properties
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 improved RFPs exhibit increased fluorescence, stability, and resistance to chemical denaturation, facilitating advanced imaging and purification applications.
Implementation Method 1
Fluorescent proteins widely used as protein tagging agents. GFP-like proteins are an expanding family of homologous, 25-30 kDa polypeptides sharing a conserved 11 beta-strand 'barrel' structure.
Data Source
AI summary
Improved Red Fluorescent Proteins (RFPs), nucleic acids encoding the improved RFPs, and methods of generating improved RFPs are described. The improved RFP can be used to form fusion molecule and can be used in applications for which fluorescent proteins are used.