DsRed Variant Polypeptides for Reduced Aggregation and Brightness
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Solution Overview
Problem
Wild-type DsRed fluorescent protein suffers from slow maturation, tetramerization issues leading to aggregation, and cellular toxicity, which limits its effectiveness as a marker in biological research.
Innovation Solution
Development of variant polypeptides with specific amino acid substitutions such as A2D, S4T, E10P, Q188K, K121H, W58Y, and D115G to enhance bacterial expression, reduce aggregation, and improve brightness and solubility, while maintaining or shifting the emission spectra.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If wild-type DsRed is used as a fluorescent marker, then it provides bright red fluorescence and is excited by lower energy light, but it forms homotetramers that cause aggregation and cellular toxicity
Solution Approach 1:
The patent divides the wild-type DsRed sequence into multiple fragments and reassembles them with specific mutations to create monomeric variants. This segmentation breaks the tetramerization interface, preventing aggregation while preserving fluorescence properties
Solution Approach 2:
The patent introduces specific amino acid substitutions (e.g., A2D, S4T, E10P, Q188K, K121H, W58Y, D115G) that change the molecular parameters of DsRed, altering its oligomerization state from tetramer to monomer and improving its solubility and expression characteristics
2Illumination intensity
If wild-type DsRed is expressed in bacteria, then it provides red fluorescence, but it exhibits slow maturation with a half-time of approximately 12 hours at 37°C
Solution Approach 1:
The patent introduces specific amino acid substitutions that accelerate the maturation process by modifying the chromophore formation kinetics, reducing the half-time from 12 hours to a faster rate while maintaining red fluorescence
3Illumination intensity
If DsRed is used for imaging thick tissue samples, then it provides red fluorescence, but light scattering makes it difficult to image thick tissues
Solution Approach 1:
The patent develops DsRed variants with shifted emission spectra toward the far-red region, where light scattering is reduced, enabling better penetration and imaging of thick tissue samples
4Object-generated harmful factors
If monomeric DsRed variants are created to prevent aggregation, then aggregation is reduced, but they are not as bright or photostable as DsRed tetramers
Solution Approach 1:
The patent introduces multiple amino acid substitutions that simultaneously improve brightness and photostability while maintaining monomeric structure, achieving a balance between preventing aggregation and enhancing optical properties
Data Source
AI summary
Polynucleotides encoding variant polypeptides of DsRed are provided herein. The DsRed variants have increased bacterial expression, reduced aggregation, increased solubility, shifted emission spectra or increased brightness relative to a wild-type DsRed.