Ramp Tag for Recombinant Protein Expression via tRNA Recycling
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Solution Overview
Problem
Current methods for recombinant protein production face challenges in achieving uniform protein expression due to rare codons in host cells, leading to inefficient translation and high production costs, particularly in eukaryotic cells with complex regulatory mechanisms.
Innovation Solution
A universal ramp tag is developed, comprising 1 to 20 codons with a ramp function that controls translation speed by recruiting and reusing tRNA, allowing for increased translation efficiency without altering the original ORF sequence or requiring costly DNA synthesis. This tag is designed to be fused with the target gene or used independently, enabling over-expression of recombinant proteins in various host cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If rare codons are present in the target gene, then translation speed is inhibited and protein expression is non-uniform, but codon optimization requires costly DNA synthesis and alteration of the original ORF sequence
Solution Approach 1:
The patent introduces a tRNA supplement plasmid as an intermediary element that mediates between the rare codons in the target gene and the host cell's translation machinery. The plasmid carries additional copies of tRNA genes corresponding to rare codons, enabling efficient translation without modifying the original target gene sequence, thus avoiding costly DNA synthesis while improving protein expression
Solution Approach 2:
The patent changes the parameter of tRNA availability in the host cell by introducing exogenous tRNA genes via a supplement plasmid. This parameter change addresses the bottleneck caused by rare codons without requiring modification of the target gene's codon sequence, thereby improving translation efficiency while maintaining the original gene structure and avoiding synthesis costs
2Productivity
If codon optimization is performed to improve translation efficiency, then protein expression increases, but the original ORF sequence is altered requiring DNA synthesis
Solution Approach 1:
The patent uses a tRNA supplement plasmid as an intermediary that resolves the conflict between maintaining the original ORF sequence and achieving efficient translation. Instead of modifying the gene, the plasmid provides the missing tRNA components, thereby improving translation efficiency while preserving gene integrity and minimizing complexity
Solution Approach 2:
The patent segments the solution into two independent parts: the original target gene remains unchanged while a separate tRNA supplement plasmid is introduced. This segmentation allows the gene to maintain its original sequence without modification while the plasmid independently provides the necessary tRNA for efficient translation of rare codons
3Quantity of substance
If strong promoter and regulatory elements are used to increase transcript amount, then expression strategy focuses on transcript increase, but there is no significant correlation between transcript amount and translated protein amount
Solution Approach 1:
The patent shifts the focus from changing transcript amount (promoter strength) to changing translation efficiency by supplementing tRNA. This parameter change addresses the reliability issue by directly enhancing the translation step, creating a more reliable correlation between genetic material and functional protein output through improved codon translation capability
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 ramp tag significantly increases protein expression levels, up to 2.5 to 7.5 times that of control groups, while maintaining protein activity and stability, and can be used with existing expression systems, reducing production costs and improving the yield of difficult-to-express proteins.
Implementation Method 1
the tag behaves (controls translation speed by recruiting of tRNA and providing re-use of tRNA) both in inside/outside of a target gene in transcription or translation steps
Data Source
AI summary
Provided is a ramp tag capable of solving instability in translation rate resulting from poor compatibility between codons in a foreign gene and a host when expressing a recombinant protein in E. coli. Unlike the conventional codon optimization or codon deoptimization method for solving the problem of rare codons, the present invention increases an expression efficiency of a target protein by merely having the ramp tag be fused with a target gene or independently expressed, without changing the original codon sequence, thereby allowing tRNA to be reused. Thus, the present invention provides a novel method for increasing recombinant protein expression which is capable of reducing costs and time in comparison to the codon optimization method that artificially synthesizes DNA sequences. Therefore, it is expected that the method of the present invention will be able to be used in production of high value-added pharmaceuticals or industrial enzymes.