Cell-Free Protein Synthesis for Stop-Codon Non-Natural Amino Acid Insertion
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Solution Overview
Problem
Existing in vitro protein synthesis systems face challenges such as reversibility, instability, enzyme leakage, inactivation, and the need for additional high-cost enzymes, making them unsuitable for large-scale applications and inefficient in incorporating non-natural amino acids for protein modification.
Innovation Solution
A cell-free protein synthesis system using a cell extract, non-natural amino acids, an orthogonal aminoacyl tRNA synthetase/tRNA pair, and a template with mutated stop codons to facilitate the incorporation of non-natural amino acids into proteins, enhancing synthesis efficiency and stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional in vitro protein synthesis systems are used, then protein synthesis can be achieved, but the systems suffer from reversibility, instability, enzyme leakage, inactivation, and require additional high-cost enzymes
Solution Approach 1:
The patent extracts and utilizes endogenous aminoacyl-tRNA synthetases and tRNAs from the cell extract itself, rather than requiring additional exogenous enzymes. The cell extract naturally contains the full complement of aminoacyl-tRNA synthetases that can charge tRNAs with both natural and non-natural amino acids, eliminating the need for adding expensive external enzymes and reducing system complexity.
Solution Approach 2:
The cell extract serves itself by containing all necessary components (aminoacyl-tRNA synthetases, tRNAs, translation machinery) required for protein synthesis. The endogenous enzymes automatically perform the aminoacylation of tRNAs with non-natural amino acids without requiring external addition of specialized enzymes, making the system self-sufficient and more stable.
2Manufacturing precision
If stop codons are mutated in the template to enable non-natural amino acid incorporation, then insertion efficiency is improved, but the system requires precise mutagenesis and orthogonal translation components
Solution Approach 1:
The patent uses the natural endogenous aminoacyl-tRNA synthetase/tRNA pairs that already exist in the cell extract to recognize and incorporate non-natural amino acids at stop codon sites. This approach copies the natural translation mechanism rather than requiring entirely orthogonal synthetic systems, maintaining high insertion efficiency while reducing complexity.
Solution Approach 2:
The patent changes the genetic code parameters by mutating specific codons to stop codons (UAA, UAG, or UGA) in the template, which then serve as recognition sites for non-natural amino acid incorporation. The endogenous aminoacyl-tRNA synthetases naturally recognize these stop codons in context and facilitate non-natural amino acid insertion, achieving precise control through simple codon substitution.
3Productivity
If cell-free systems are used for protein synthesis, then rapid and efficient translation can be achieved, but the systems face challenges with ATP imbalance and enzyme stability
Solution Approach 1:
The patent prepares the cell extract in advance with all necessary translation components, including aminoacyl-tRNA synthetases and tRNAs, already present and functional. This preliminary preparation ensures that when the translation reaction is initiated, all components are immediately available and stable, avoiding ATP imbalance and enzyme instability issues during the actual protein synthesis process.
Solution Approach 2:
The patent uses a composite cell-free system that combines cell extract (containing stabilized enzymes and cofactors) with purified template and controlled addition of non-natural amino acids. This composite approach leverages the stability of the cell extract matrix while maintaining the productivity of cell-free translation, protecting enzymes from inactivation and ensuring consistent performance.
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 system achieves high efficiency in incorporating non-natural amino acids into proteins, with up to 99.81% insertion efficiency and the ability to perform click chemistry modifications, improving protein synthesis yield and functionality.
Implementation Method 1
an exogenous orthogonal aminoacyl tRNA synthetase/orthogonal tRNA pair
Implementation Method 2
The translation of mRNA into protein refers to the assembly process of activated amino acids into protein polypeptide chains on ribosomes
Data Source
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AI summary
Provided is an in-vitro cell-free protein synthesis system for inserting a non-natural amino acid. The reaction system comprises: (1) a yeast cell extracting solution; (2) a non-natural amino acid; (3) an exogenous orthogonal aminoacyl tRNA synthetase/orthogonal tRNA pair; and (4) a template containing a target protein gene sequence, wherein at least one codon encoding an amino acid in the target protein gene sequence is mutated into a stop codon. By means of the reaction system, the insertion efficiency of the non-natural amino acid and the expression quantity of the non-natural amino acid protein can be increased.