protein containing a modified amino acid that carries an azido or alkynyl group for conjugation to a second molecule such as polymer, label or drug that carries an alkynyl or azido group; translation biochemistry in eukaryotic cells; gene expression inhibitors
Inventors
Deiters, Alexander • Cropp, T. Ashton • Chin, Jason W. • Anderson, J. Christopher • Schultz, Peter G.
Assignees
US Department of Energy • Scripps Research Institute
Publication Number
US-7888063-B2
Publication Date
2011-02-15
Expiration Date
2024-04-16
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Abstract
This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.
Core Innovation
This invention provides compositions and methods for producing translational components that expand the number of genetically encoded amino acids in eukaryotic cells. The components include orthogonal tRNAs, orthogonal aminoacyl-tRNA synthetases, orthogonal pairs of tRNAs/synthetases and unnatural amino acids. Proteins and methods of producing proteins with unnatural amino acids in eukaryotic cells are also provided.
The invention provides eukaryotic cells with translation components, such as pairs of orthogonal aminoacyl-tRNA synthetases (O-RSs) and orthogonal tRNAs (O-tRNAs), that are used in eukaryotic protein biosynthetic machinery to incorporate an unnatural amino acid in a growing polypeptide chain. Compositions include eukaryotic cells (e.g., yeast, mammalian, plant, algae, fungal, insect cells) comprising an O-RS derived from a non-eukaryotic organism, which preferentially aminoacylates an orthogonal tRNA with at least one unnatural amino acid in the eukaryotic cell. The invention also discloses methods of producing orthogonal aminoacyl-tRNA synthetases that preferentially aminoacylate orthogonal tRNAs with unnatural amino acids, including positive and negative selection steps in eukaryotic cells.
The problem solved is the difficulty of genetically encoding amino acids beyond the common twenty standard amino acids in eukaryotic cells. Previous efforts in bacteria like Escherichia coli have allowed incorporation of unnatural amino acids using orthogonal tRNA/synthetase pairs, but components that function in prokaryotes often are not orthogonal or functional in eukaryotes due to differences such as RNA polymerase III transcription, nuclear export of tRNAs, and ribosome differences. The invention overcomes these limitations by providing orthogonal pairs and methods adapted for eukaryotic cells, enabling efficient, high-fidelity incorporation of unnatural amino acids into proteins in eukaryotes.
Claims Coverage
The patent includes two independent claims, each describing a method of producing a protein comprising at least one unnatural amino acid in a eukaryotic cell using orthogonal tRNA/synthetase pairs and corresponding selector codons.
Method for producing protein with unnatural amino acid using selected orthogonal aminoacyl-tRNA synthetases
A method of growing eukaryotic cells comprising a nucleic acid encoding a protein including at least one selector codon, an orthogonal tRNA (O-tRNA) that recognizes the selector codon, and an orthogonal aminoacyl tRNA synthetase (O-RS) that preferentially aminoacylates the O-tRNA with the unnatural amino acid. The O-RS comprises the amino acid sequence of any one of SEQ ID NOs: 48-53.
Method of producing protein comprising specific unnatural amino acids with specific orthogonal synthetases
A method wherein the eukaryotic cell comprises an O-RS that is either a p-propargyloxyphenylalanine O-RS or a p-azido-L-phenylalanine O-RS, aminoacylating the O-tRNA with the respective unnatural amino acid. The O-RS amino acid sequences correspond to SEQ ID NOs: 48-53 or a conservative variant at least 95% identical. The method involves incorporating the unnatural amino acid comprising a first reactive group into the protein.
Post-translational modification via [3+2] cycloaddition
After incorporating an unnatural amino acid with a first reactive group, the method includes contacting the protein with a molecule having a second reactive group; the first and second reactive groups react through a [3+2] cycloaddition to attach the molecule to the protein.
Molecules suitable for attachment via [3+2] cycloaddition
The molecule attached can be selected from dyes, polymers, polyethylene glycol derivatives, photocrosslinkers, cytotoxic compounds, affinity labels, derivatives of biotin, resin, second proteins/polypeptides, metal chelators, cofactors, fatty acids, carbohydrates or polynucleotides.
Specific example of p-propargyloxyphenylalanine incorporation and modification
Where the O-RS is the p-propargyloxyphenylalanine synthetase, the unnatural amino acid is p-propargyloxyphenylalanine, the first reactive group is an alkynyl moiety, and the second reactive group is an azido moiety.
Specific unnatural amino acid incorporation
The unnatural amino acid incorporated is p-propargyloxyphenylalanine.
Specific example of p-azido-L-phenylalanine incorporation and modification
Where the O-RS is the p-azido-L-phenylalanine synthetase, the unnatural amino acid is p-azido-L-phenylalanine, the first reactive group is an azido moiety, and the second reactive group is an alkynyl moiety.
Specific unnatural amino acid incorporation
The unnatural amino acid incorporated is p-azido-L-phenylalanine.
Use of E. coli derived orthogonal tRNA/O-RS pairs in yeast
The eukaryotic cell is a yeast cell, and the orthogonal tRNA and the orthogonal aminoacyl-tRNA synthetase are derived from E. coli.
The claims cover methods of producing proteins with unnatural amino acids in eukaryotic cells using orthogonal tRNA and aminoacyl-tRNA synthetases, focusing on specific synthetase sequences. The claims include incorporation of reactive unnatural amino acids (alkynyl or azido) and post-translational modification of proteins via [3+2] cycloaddition reactions.
Stated Advantages
The invention provides the ability to genetically encode unnatural amino acids in eukaryotic cells, enabling incorporation of amino acids beyond the twenty natural ones with high fidelity and efficiency.
It allows for efficient production of proteins containing unnatural amino acids in large useful quantities in eukaryotic cells, overcoming the limitations of prior methods such as microinjection or in vitro chemical acylation.
The use of orthogonal translation components that do not cross-react with endogenous machinery provides high selectivity and control over protein synthesis with unnatural amino acids.
The method enables site-specific post-translational modifications of proteins, e.g., via [3+2] cycloaddition reactions, allowing for versatile biochemical labeling and functionalization under physiological conditions.
Positive and negative selection schemes allow rapid and tunable isolation of aminoacyl-tRNA synthetase variants with desired substrate specificity for unnatural amino acids in eukaryotic cells.
The system is broadly applicable to a variety of eukaryotic cells and can be used to incorporate many kinds of unnatural amino acids with diverse chemical properties.
Documented Applications
Producing proteins comprising unnatural amino acids in eukaryotic cells, including therapeutic proteins, diagnostic proteins, industrial enzymes, and proteins with novel biochemical properties.
Selective post-translational modification of proteins in vitro and in vivo by conjugation to dyes, polymers, cytotoxic compounds, affinity labels, and other molecules via [3+2] cycloaddition chemistry through unnatural amino acid side chains.
Using proteins with genetically encoded unnatural amino acids for studying protein structure, function, and interactions, including photocrosslinking, spectroscopic analyses, and incorporation of heavy atoms for X-ray crystallography.
Engineering transcriptional modulator proteins (e.g., GAL4) with unnatural amino acids for selective expression and screening applications in yeast and other eukaryotic cells.
Production of glycoproteins or glycosylated proteins with unnatural amino acids and post-translational modifications only possible in eukaryotic cells.
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