Non-standard Amino Acid Incorporation in Recoded Hosts
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Solution Overview
Problem
Current methods for multi-site incorporation of non-standard amino acids into proteins face inefficiencies due to competition with essential translation machinery and reduced enzyme activity of evolved aminoacyl tRNA synthetases, leading to low protein yields and limited chemical diversity.
Innovation Solution
Development of improved genomically recoded organisms and variant aminoacyl tRNA synthetases with enhanced specificity and activity, integrated into the host genome, to enable efficient multi-site incorporation of non-standard amino acids into polypeptides with higher yields and purity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If conventional orthogonal translation systems are used for multi-site nsAA incorporation, then site-specific nsAA incorporation is achieved, but protein yields are low due to competition with essential translation machinery and reduced enzyme activity
Solution Approach 1:
The invention extracts and removes the competing release factor 1 (RF1) from the translation system by creating RF1-deficient host cells. This eliminates the competition for UAG stop codons between the orthogonal suppressor tRNA and the native release factor, thereby resolving the technical contradiction by removing the harmful competitive interaction while preserving the desired site-specific nsAA incorporation capability and significantly improving protein yields
Solution Approach 2:
The invention changes the genetic code parameters by reassigning the UAG stop codon from its native termination function to a sense codon function for nsAA incorporation. This parameter change in the translation system allows the orthogonal tRNA to efficiently recognize and incorporate nsAAs at UAG codons without competition from RF1, thereby improving both manufacturing precision and productivity
2Adaptability or versatility
If complete amino acid replacement is used to introduce nsAA, then chemical diversity is limited since nsAA must be a close analog, but this approach has been extensively utilized to tag and study proteomes
Solution Approach 1:
The invention creates a universal orthogonal translation system that can incorporate diverse non-standard amino acids including photocaged amino acids, bioorthogonal reactive groups, and spectroscopic labels. This multi-functional system allows the same UAG codon assignment to work with various chemically diverse nsAAs, thereby improving chemical diversity while maintaining ease of manufacture through a unified approach
Solution Approach 2:
The invention introduces an orthogonal aminoacyl-tRNA synthetase/tRNA pair as an intermediary system that mediates the incorporation of chemically diverse nsAAs. This intermediary orthogonal system bypasses the need for complete amino acid replacement and allows diverse nsAAs to be incorporated through a common UAG codon assignment, resolving the contradiction between chemical diversity and ease of manufacture
3Manufacturing precision
If evolved aminoacyl tRNA synthetases are used for nsAA incorporation, then site-specific incorporation is achieved, but enzyme activity is reduced leading to low protein yields
Solution Approach 1:
The invention extracts and removes the competing RF1 release factor from the system, creating RF1-deficient host cells. This elimination of competition allows the evolved aminoacyl-tRNA synthetase to function more efficiently at UAG codons, thereby improving protein yields while maintaining site-specific incorporation precision
Solution Approach 2:
The invention performs preliminary evolution and optimization of the aminoacyl-tRNA synthetase enzyme to improve its activity and specificity for the non-standard amino acid substrate. This preliminary optimization action addresses the reduced enzyme activity issue before the actual protein production, thereby improving both manufacturing precision and productivity
Data Source
AI summary
Compositions, systems, and methods for preparation of polypeptides having multiple iterations of non-standard amino acids are provided. The compositions and method can be used to produce recombinant proteins at a greater yield than the same or similar polypeptides made using conventional compositions, systems, and methods. Accordingly, in some embodiments, the polypeptides are ones that could not be made using conventional methods and reagents, or could not be made a sufficient yield or purity to serve a practical purpose using conventional methods and reagents. Polypeptides made using the disclosed compositions, systems, and methods are also provided.