Engineered Aminoacyl-tRNA Synthetase for Aib Incorporation
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Solution Overview
Problem
Current methods fail to genetically encode 2-Aminoisobutyric acid (Aib) for recombinant expression of polypeptides, limiting the incorporation of this amino acid into polypeptides with desirable properties.
Innovation Solution
Development of an aminoacyl-tRNA synthetase (AibRS) that aminoacylates tRNA with Aib, enabling its incorporation into a growing polypeptide chain during translation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If standard translation components are used for recombinant expression, then polypeptides can be produced efficiently, but only the twenty naturally occurring amino acids can be incorporated
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of alanine-tRNA synthetase through specific mutations (P197A, T213S, V217I, N249H) to alter its substrate specificity. These parameter changes in the enzyme's active site enable it to recognize and aminoacylate tRNA with Aib instead of alanine, thus expanding the amino acid incorporation capability while maintaining recombinant expression efficiency
Solution Approach 2:
The patent achieves universality by creating an engineered AibRS that can function in the standard bacterial translation machinery while incorporating non-standard amino acids. The modified synthetase maintains compatibility with endogenous tRNAs and ribosomes, allowing the system to produce polypeptides containing both natural and unnatural amino acids using the same translation apparatus
2Ease of operation
If translation components cross-react with endogenous components, then the system can function in the host organism, but efficiency is reduced
Solution Approach 1:
The patent applies local quality by making specific localized changes to the AibRS active site through targeted mutations. These localized modifications at positions 197, 213, 217, and 249 create a specialized binding pocket that selectively recognizes Aib while maintaining interaction with endogenous tRNA and translation components, thus resolving the conflict between system functionality and incorporation efficiency
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
Enables the efficient recombinant expression of polypeptides containing Aib, such as semaglutide, by providing a means to aminoacylate tRNA with Aib, thereby overcoming the limitations of existing translation components.
Implementation Method 1
an aminoacyl-tRNA synthetase (RS) that aminoacylates a tRNA with a desired unnatural amino acid
Data Source
AI summary
The invention relates to aminoacyl-tRNA synthetases that aminoacylate tRNA with 2-Aminoisobutyric acid (Aib), thus enabling the incorporation of the Aib into a growing polypeptide chain during translation, e.g. in eubacterial host cells such as E. coli. For example, but not limited to, the invention relates to new aminoacyl-tRNA synthetases and the uses hereof, as well as a method for producing polypeptides that contain one or more Aib.