MaPyIRS Variant Enhances AzK Incorporation Efficiency
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Solution Overview
Problem
Current orthogonal non-canonical amino acid (ncAA) translation systems, particularly those using the wildtype MaPyIRS/MatRNACUAPyl pair, are less efficient in incorporating azido-containing lysine derivatives like AzK, limiting their application in site-specific protein modification and bioorthogonal coupling reactions.
Innovation Solution
A variant of pyrrolysyl-tRNA synthetase, MaPyIRS, with specific substitutions at positions 129 and 168 (M129L+V168C), enhances the efficiency of AzK incorporation, matching the performance of the benchmark MmPyIRS/MmtRNACUAPyl pair, and maintains orthogonality with canonical amino acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If the wildtype MaPyIRS/MatRNACUAPyl orthogonal translation system is used, then the system maintains orthogonality with canonical amino acids, but the efficiency of incorporating azido-containing lysine derivatives like AzK is reduced
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues in the MaPyIRS enzyme (positions 129 and 168) to alter its substrate binding properties. The M129L and V168C substitutions change the enzyme's parameters to enhance AzK incorporation efficiency while preserving orthogonality with canonical amino acids, directly resolving the contradiction between reliability and productivity
2Device complexity
If the wildtype MaPyIRS/MatRNACUAPyl pair is used, then the system structure is simple and well-established, but the performance in AzK incorporation is inferior to the benchmark MmPyIRS/MmtRNACUAPyl pair
Solution Approach 1:
The patent applies local quality by making targeted local modifications to the MaPyIRS enzyme structure at specific positions (129 and 168) rather than redesigning the entire system. This localized approach improves AzK incorporation efficiency while maintaining the overall simplicity and orthogonality of the translation system structure
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 MaPyIRS M129L V168C variant significantly improves AzK incorporation into proteins, achieving comparable or superior efficiency to the benchmark system, allowing for more cost-effective and efficient site-specific labeling and bioorthogonal modifications.
Implementation Method 1
a variant of pyrrolysyl-tRNA synthetase... wherein said variant has pyrrolysyl-tRNA synthetase activity... capable of catalyzing the aminoacylation of its cognate tRNA with a non-canonical amino acid
Data Source
AI summary
The present invention relates to the field of pyrrolysyl-tRNA synthetases, their variants and uses thereof. Particularly, the present invention relates to variants (mutants) of parent pyrrolysyl-tRNA synthetases, wherein said variants have pyrrolysyl-tRNA synthetase activity and exhibit altered properties relative to the corresponding parent pyrrolysyl-tRNA synthetase.