Codon-Optimized Oplophorus Luciferase Gene for Soluble Expression
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Solution Overview
Problem
Current luciferase technologies face challenges in precise molecular design and substrate optimization due to significant homology in primary structures among marine bioluminescent organisms, making it difficult to predict optimized luciferins for specific luciferases, and existing methods struggle to express the mutated catalytic domain of Oplophorus luciferase efficiently in both Escherichia coli and cultured animal cells.
Innovation Solution
A codon-optimized gene for the mutated catalytic domain of Oplophorus luciferase is developed, allowing efficient expression in both Escherichia coli and cultured animal cells, and coelenterazine analogues with high activity are identified and optimized for the mutated 19 kDa protein, enhancing luminescent activity beyond native levels.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the native 19 kDa domain gene is expressed in Escherichia coli, then the protein is produced, but it forms inclusion bodies over 95% and is not expressed as soluble form
Solution Approach 1:
The patent applies codon optimization to change the nucleotide sequence parameters of the 19 kDa domain gene while maintaining the same amino acid sequence. This parameter change at the DNA level adapts the gene to Escherichia coli's codon usage preferences, enabling proper translation and soluble protein expression without forming inclusion bodies
Solution Approach 2:
The patent replaces the native signal peptide sequence with the Gaussia luciferase signal peptide sequence. This substitution changes the secretion mechanism, allowing the 19 kDa domain protein to be secreted from cultured animal cells effectively, overcoming the limitation of the native signal peptide
2Reliability
If conventional random mutagenesis is applied to produce mutated 19 kDa domain gene, then luminescence activity is enhanced, but the essential amino acid residues for luminescence function are not identified and function of mutated residues remains unclear
Solution Approach 1:
The patent performs site-directed mutagenesis based on preliminary structural and functional analysis of the catalytic domain. By targeting specific residues predicted to be important for luminescence function before experimentation, the patent achieves enhanced activity while maintaining clarity about which residues are functionally critical
Solution Approach 2:
The patent uses a systematic approach where luminescence activity measurements provide feedback to identify which mutated residues contribute to enhanced function. This feedback mechanism allows identification of essential amino acid residues while achieving improved luminescence activity
3Ease of operation
If Oplophorus luciferase is used as secreted luciferase, then it can be secreted from cells, but it is difficult to find coelenterazine analogues showing at least 5-fold higher luminescence activity than coelenterazine
Solution Approach 1:
The patent changes the substrate binding pocket parameters through site-directed mutagenesis of specific amino acid residues in the 19 kDa domain. These parameter changes in the protein structure enable accommodation and optimization of coelenterazine analogues, achieving at least 5-fold higher luminescence activity with optimized substrates like bis-coelenterazine
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 codon-optimized gene enables efficient protein expression and a light-emitting method using optimized coelenterazine analogues, achieving higher luminescent activity than native proteins, with bis-coelenterazine showing at least 10-fold higher relative maximum intensity and continuous emission without decay.
Implementation Method 1
A polynucleotide encoding a mutated catalytic 19 kDa domain of codon-optimized Oplophorus luciferase
Implementation Method 2
When a gene encoding the 19 kDa domain protein is expressed in Escherichia coli, it is expressed as inclusion bodies over 95%
Implementation Method 3
The simplest light-emitting system among luciferases is performed by the luminescence reaction only with a luciferin and molecular oxygen
Implementation Method 4
the catalytic domain responsible for the luminescent oxidation of coelenterazine
Implementation Method 5
Molecular oxygen attaches to coelenterazine and the resulting peroxide produces the dioxetanone. Subsequently, decarboxylation proceeds to form the coelenteramide anion at the excited state
Implementation Method 6
when the anion relaxes to the ground state, it is considered to produce light emission of blue (λmax=460-490 nm)
Data Source
AI summary
There has been a demand for a codon-optimized gene for the mutated catalytic domain of Oplophorus luciferase, which is capable of efficiently expressing a protein both in a cultured animal cell and Escherichia coli. There has also been a demand for a substrate coelenterazine analog showing a higher activity than that of native 19 kDa protein. The invention provides a polynucleotide comprising a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2. According to the invention, bis-coelenterazine is used as a substrate coelenterazine analog suitable for the photoprotein encoded by the polynucleotide comprising the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 2.