Engineered Fluorescent Proteins Maturation Speed
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Solution Overview
Problem
Wild-type A. macrodactyla green fluorescent protein (GFPxm) has a low maturation speed at temperatures relevant for mammalian cell studies, limiting its usefulness as a fluorescent marker.
Innovation Solution
Engineered fluorescent proteins with specific amino acid substitutions, such as F220L, and additional mutations like K3G, E6D, T9A, P58T, F99L, M128K, M128E, I136M, Y151H, N144S, K162E, K156M, T214A, G228C, G228S, and K238R, which enhance maturation speed and fluorescent properties, are developed.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If wild-type A. macrodactyla GFPxm is used as a fluorescent marker, then the fluorescent properties are maintained, but the maturation speed is low at temperatures relevant for mammalian cell studies
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid substitutions (F220L, K3G, E6D, T9A, P58T, F99L, M128K, M128E, I136M, Y151H, N144S, K162E, K156M, T214A, G228C, G228S, and K238R) into the GFPxm protein sequence. These molecular-level parameter changes directly increase the maturation speed of the fluorescent protein at physiological temperatures (20°C or above) while preserving its fluorescent properties, thereby resolving the contradiction between maturation speed and marker utility.
2Measurement precision
If GFPxm is modified to increase maturation speed at 37°C, then the sensitivity and detection level in mammalian cells improve, but the protein sequence complexity increases
Solution Approach 1:
The patent applies local quality by introducing specific point mutations at particular positions (e.g., F220L, K3G, E6D) within the GFPxm sequence rather than global modifications. Each substitution is strategically placed to optimize maturation speed and detection sensitivity at specific locations in the protein structure, thereby improving measurement precision while minimizing overall sequence complexity compared to comprehensive redesign approaches.
Data Source
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AI summary
The present invention provides nucleic acid molecules encoding mutant fluorescent proteins as well as proteins encoded by these nucleic acids. In addition, host-cells, stable cell lines and transgenic organisms comprising the above- referenced nucleic acid molecules are provided. The subject protein and nucleic acid compositions find use in a variety of different applications and methods, particularly for labeling of biomolecules, cells, or cell organelles.