Use of eRF1 mutants in unnatural amino acid incorporation
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United States of America Patent
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Aug 11, 2020
Grant Date -
Dec 14, 2017
app pub date -
Oct 21, 2015
filing date -
Oct 27, 2014
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Abstract
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.
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