Luciola cruciata luciferase codon optimization for mammalian expression
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Solution Overview
Problem
Current luciferase enzymes from various organisms are not optimized for expression or production in mammalian cells, limiting their utility in medical and experimental applications.
Innovation Solution
A novel codon-optimized and stabilized luciferase gene derived from Japanese firefly Luciola cruciata, which enhances thermostability and light emission wavelength, along with improved expression levels in mammalian cells, is developed by modifying the nucleic acid sequence to remove RNAse cleavage motifs, cryptic splice sites, and palindromic sequences, and adjusting codon usage to match mammalian systems.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If native luciferase gene from Luciola cruciata is used, then the enzyme exhibits natural bioluminescence activity, but the expression levels and protein production are low in mammalian cells
Solution Approach 1:
The nucleotide sequence of the luciferase gene was modified by changing codon usage to match mammalian cell preferences, optimizing translation efficiency. Specific parameters including codon frequency, GC content, and sequence composition were adjusted to enhance expression levels in mammalian systems while preserving the amino acid sequence and bioluminescence function.
2Stability of the object's composition
If native luciferase gene is used, then the original sequence is maintained, but the mRNA stability is poor due to presence of RNAse cleavage motifs
Solution Approach 1:
Specific RNAse cleavage motifs and cryptic splice sites were identified and removed from the native luciferase gene sequence. These destabilizing elements were extracted and eliminated to prevent premature mRNA degradation and improve overall mRNA stability in mammalian cells, while maintaining the essential coding sequence.
3Temperature
If native luciferase is used, then the natural thermostability is maintained, but the enzyme shows reduced stability at elevated temperatures
Solution Approach 1:
Amino acid substitutions were introduced to enhance thermostability of the luciferase enzyme. Specific residue changes were made to strengthen structural integrity and improve thermal resistance, allowing the enzyme to maintain stability and activity at elevated temperatures commonly encountered in mammalian physiological conditions and experimental procedures.
4Illumination intensity
If native luciferase gene is used, then the natural light emission spectrum is preserved, but the light emission wavelength is not optimized for detection
Solution Approach 1:
The light emission spectrum of the luciferase was shifted to longer wavelengths through site-directed mutagenesis. Specific amino acid changes in the chromophore region were made to red-shift the emission maximum, improving tissue penetration and detection efficiency in bioluminescence imaging applications while maintaining enzyme activity.
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 modified luciferase gene exhibits increased mRNA stability and expression levels, improved thermostability, and shifted light emission, enabling more effective use in mammalian cells for bioluminescence assays and cellular analysis.
Implementation Method 1
Bioluminescence in certain organisms via the reaction of luciferin and luciferase is well known in the art
Data Source
AI summary
A codon optimized and stabilized luciferase gene based upon the sequence of the natural luciferase gene isolated from Luciola cruciata (Japanese firefly) and a novel recombinant DNA characterized by incorporating this new gene coding for a novel luciferase into a vector DNA for improved activities in mammalian cells, are disclosed. This new luciferase exhibits long-wavelength light emission, as well as improved thermostability and higher expression levels in mammalian cell systems, compared to native luciferase. Assays using this new enzyme for measuring various biological metabolic functions are described.