Mutant eRF1 Enhances Unnatural Amino Acid Incorporation
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Solution Overview
Problem
Efficient incorporation of unnatural amino acids into proteins in eukaryotic cells is a significant challenge, limiting the potential of genetic code expansion approaches for creating site-specifically modified recombinant proteins and controlling protein function with precision.
Innovation Solution
Development of an expression system using orthogonal synthetase/tRNA pairs combined with engineered eRF1 mutants to enhance the efficiency of unnatural amino acid incorporation in eukaryotic cells, specifically increasing the yield of proteins containing unnatural amino acids by selectively enhancing the amber codon suppression without increasing read-through of other stop codons.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If orthogonal synthetase/tRNA pairs are used for unnatural amino acid incorporation in eukaryotic cells, then site-specific incorporation is achieved, but the efficiency of incorporation is low
Solution Approach 1:
The invention changes the parameters of the release factor by creating engineered variants with modified amino acid sequences. Specifically, mutations are introduced at positions 55, 129, and 130 of eRF1 to alter its codon recognition properties, enabling selective suppression of the amber stop codon while maintaining discrimination against other stop codons, thereby improving incorporation efficiency
Solution Approach 2:
The invention introduces an intermediary component - the engineered eRF1 variant - that mediates between the amber stop codon and the translation machinery. This modified release factor acts as a bridge that allows orthogonal synthetase/tRNA pairs to efficiently incorporate unnatural amino acids by competing with the engineered eRF1 for amber codon recognition, resolving the efficiency limitation
2Productivity
If eRF1 is engineered to enhance amber codon suppression, then unnatural amino acid incorporation efficiency increases, but read-through of other stop codons may increase
Solution Approach 1:
The invention applies local quality changes by introducing specific mutations at predetermined positions (55, 129, 130) of eRF1 that locally alter the protein's interaction with stop codons. These targeted mutations modify the N-terminal domain's codon recognition specificity, enhancing amber suppression while preserving discrimination against ochre and opal codons through localized structural changes
Solution Approach 2:
The invention inverts the normal function of eRF1 by engineering it to preferentially recognize the amber stop codon rather than all stop codons equally. This inverted specificity is achieved through mutations that create a unique binding interface for the amber codon, allowing the release factor to distinguish amber from other stop codons and enabling selective suppression without compromising overall stop codon fidelity
Data Source
AI summary
The invention relates to a method for incorporating an unnatural amino acid into a protein of interest in a eukaryotic cell, said method comprising the steps of: i) providing a eukaryotic cell expressing an orthogonal tRNA synthetase-t RNA pair, a nucleic acid sequence of interest encoding said protein of interest, and a mutant eRF1, said mutant eRF1 having amino acid sequence having at least 60% sequence identity to the human wild type eRF1 sequence of SEQ ID NO: 4, said nucleic acid sequence of interest comprising a codon recognised by the tRNA at the position for incorporation of an unnatural amino acid; ii) incubating the eukaryotic cell in the presence of an unnatural amino acid to be incorporated into a protein encoded by the nucleic acid sequence of interest, wherein said unnatural amino acid is a substrate for the orthogonal tRNA synthetase; and iii) incubating the eukaryotic cell to allow incorporation of said unnatural amino acid into the protein of interest via the orthogonal tRNA synthetase-t RNA pair. The invention also relates to uses, host cells, combinations and kits.