Aminoacyl transfer rna synthetases

An in vitro method of producing a protein comprising a noncanonical amino acid, the method comprising:

• (a) contacting an in vitro translation system or cell with a Tyrosyl-tRNA Synthetase (TyrRS) polypeptide, or a functional fragment thereof, having at least about 85% amino acid sequence identity to the following polypeptide sequence:
• MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALYCGFDPTADSLHLGHLVPLLCLK RFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVDKIRKQVAPFLDFDCG ENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMINKEAVKQRLNREDQGISFTEFSYN LLQGYDFACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTK FGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSG KAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVE MEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGRFTLLR RGKKNYCLICWK (SEQ ID NO:
• 1), and comprising an alteration at an amino acid position selected from the group consisting of Y37, L71, V72, Q179, D182, and Q195 and having tRNA synthetase activity,
• (b) contacting the in vitro translation system or cell of (a) with one or more noncanonical amino acids or a composition thereof and a polynucleotide encoding a protein comprising one or more noncanonical amino acids;
• and
• (c) expressing the protein comprising one or more noncanonical amino acids using the in vitro translation system or cell.

An in vitro method of producing a protein comprising a noncanonical amino acid, the method comprising:

• (a) contacting an in vitro translation system or cell with an expression vector encoding a Leucyl-tRNA Synthetase (LeuRS) polypeptide, or a functional fragment thereof, having at least about 85% amino acid sequence identity to the following polypeptide sequence:
• MQEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSMLPYPSGRLHMGHVRNYTI GDVIARYQRMLGKNVLQPIGWDAFGLPAEGAAVKNNTAPAPWTYDNIAYMKNQLKM LGFGYDWSRELATCTPEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQV IDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTVKTMQRNWIGRSEG VEITFNVNDYDNTLTVYTTRPDTFMGCTYLAVAAGHPLAQKAAENNPELAAFIDECRN TKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQ RDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADK LTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVV MDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESSWYYARYTCPQYKEGMLDSEA ANYWLPVDIYIGGIEHAIMHLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFY YVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQV MVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVA ALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQ DRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLV VVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVV G (SEQ ID NO:
• 2), and comprising an alteration at an amino acid position selected from the group consisting of M40, L41, 5496, Y499 and Y527 and having tRNA synthetase activity, and expressing the polypeptide(s) in the cell;
• (b) contacting the in vitro translation system or cell of (a) with one or more noncanonical amino acids or a composition thereof and producing a protein comprising one or more noncanonical amino acids in the cell;
• and
• (c) expressing said protein comprising one or more noncanonical amino acids using the in vitro translation system or cell.

A system for producing and selecting for a protein comprising a noncanonical amino acid, the system comprising:

• an in vitro translation system or cell containing a Leucyl-tRNA Synthetase (LeuRS) polypeptide, or a functional fragment thereof, having at least about 85% amino acid sequence identity to the following polypeptide sequence:
• MQEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSMLPYPSGRLHMGHVRNYTI GDVIARYQRMLGKNVLQPIGWDAFGLPAEGAAVKNNTAPAPWTYDNIAYMKNQLKM LGFGYDWSRELATCTPEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQV IDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTVKTMQRNWIGRSEG VEITFNVNDYDNTLTVYTTRPDTFMGCTYLAVAAGHPLAQKAAENNPELAAFIDECRN TKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQ RDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADK LTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVV MDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESSWYYARYTCPQYKEGMLDSEA ANYWLPVDIYIGGIEHAIMHLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFY YVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQV MVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVA ALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQ DRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLV VVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVV G (SEQ ID NO:
• 2), and comprising an alteration at an amino acid position selected from the group consisting of M40, L41, S496, Y499 and Y527 and having tRNA synthetase activity, wherein the in vitro translation system or cell comprises one or more noncanonical amino acids or a composition thereof, and wherein a protein comprising one or more noncanonical amino acids is produced in the in vitro translation system or cell.

A method of controlling the replication of a cell or virus, the method comprising:

• (a) (i) altering a gene encoding a polypeptide in the cell or a virus infecting the cell to encode the polypeptide altered to comprise a noncanonical amino acid and/or (ii) knocking out the gene in the cell or the virus infecting the cell, and contacting the cell with a polynucleotide sequence encoding the polypeptide altered to comprise the noncanonical amino acid, such that replication of the cell or virus is reduced or eliminated in the absence of expression of the polypeptide containing the noncanonical amino acid,
• (b) contacting the cell with an expression vector encoding a Tyrosyl-tRNA Synthetase (TyrRS) polypeptide, or a functional fragment thereof wherein the polypeptide has at least about 85% amino acid sequence identity to the following polypeptide sequence:
• MASSNLIKQLQERGLVAQVTDEEALAERLAQGPIALYCGFDPTADSLHLGHLVPLLCLK RFQQAGHKPVALVGGATGLIGDPSFKAAERKLNTEETVQEWVDKIRKQVAPFLDFDCG ENSAIAANNYDWFGNMNVLTFLRDIGKHFSVNQMINKEAVKQRLNREDQGISFTEFSYN LLQGYDFACLNKQYGVVLQIGGSDQWGNITSGIDLTRRLHQNQVFGLTVPLITKADGTK FGKTEGGAVWLDPKKTSPYKFYQFWINTADADVYRFLKFFTFMSIEEINALEEEDKNSG KAPRAQYVLAEQVTRLVHGEEGLQAAKRITECLFSGSLSALSEADFEQLAQDGVPMVE MEKGADLMQALVDSELQPSRGQARKTIASNAITINGEKQSDPEYFFKEEDRLFGRFTLLR RGKKNYCLICWK (SEQ ID NO:
• 1), and comprising an alteration at an amino acid position selected from the group consisting of Y37, L71, V72, Q179, D182, and Q195 and having tRNA synthetase activity, and expressing the polypeptide(s) in the cell;
• (c) controlling replication of the cell and/or virus by contacting the cell of (a) with the noncanonical amino acid, wherein replication of the cell and/or virus is reduced or eliminated in the absence of the noncanonical amino acid.

A method of controlling the replication of a cell or virus, the method comprising:

• (a) (i) altering a gene encoding a polypeptide in the cell or a virus infecting the cell to encode the polypeptide altered to comprise a noncanonical amino acid and/or (ii) knocking out the gene in the cell or the virus infecting the cell, and contacting the cell with a polynucleotide sequence encoding the polypeptide altered to comprise the noncanonical amino acid, such that replication of the cell or virus is reduced or eliminated in the absence of expression of the polypeptide containing the noncanonical amino acid,
• (b) contacting the cell an expression vector encoding a Leucyl-tRNA Synthetase (LeuRS) polypeptide, or a functional fragment thereof, wherein the polypeptide has at least about 85% amino acid sequence identity to the following polypeptide sequence:
• MQEQYRPEEIESKVQLHWDEKRTFEVTEDESKEKYYCLSMLPYPSGRLHMGHVRNYTI GDVIARYQRMLGKNVLQPIGWDAFGLPAEGAAVKNNTAPAPWTYDNIAYMKNQLKM LGFGYDWSRELATCTPEYYRWEQKFFTELYKKGLVYKKTSAVNWCPNDQTVLANEQV IDGCCWRCDTKVERKEIPQWFIKITAYADELLNDLDKLDHWPDTVKTMQRNWIGRSEG VEITFNVNDYDNTLTVYTTRPDTFMGCTYLAVAAGHPLAQKAAENNPELAAFIDECRN TKVAEAEMATMEKKGVDTGFKAVHPLTGEEIPVWAANFVLMEYGTGAVMAVPGHDQ RDYEFASKYGLNIKPVILAADGSEPDLSQQALTEKGVLFNSGEFNGLDHEAAFNAIADK LTAMGVGERKVNYRLRDWGVSRQRYWGAPIPMVTLEDGTVMPTPDDQLPVILPEDVV MDGITSPIKADPEWAKTTVNGMPALRETDTFDTFMESSWYYARYTCPQYKEGMLDSEA ANYWLPVDIYIGGIEHAIMHLLYFRFFHKLMRDAGMVNSDEPAKQLLCQGMVLADAFY YVGENGERNWVSPVDAIVERDEKGRIVKAKDAAGHELVYTGMSKMSKSKNNGIDPQV MVERYGADTVRLFMMFASPADMTLEWQESGVEGANRFLKRVWKLVYEHTAKGDVA ALNVDALTENQKALRRDVHKTIAKVTDDIGRRQTFNTAIAAIMELMNKLAKAPTDGEQ DRALMQEALLAVVRMLNPFTPHICFTLWQELKGEGDIDNAPWPVADEKAMVEDSTLV VVQVNGKVRAKITVPVDATEEQVRERAGQEHLVAKYLDGVTVRKVIYVPGKLLNLVV G (SEQ ID NO:
• 2), and comprising an alteration at an amino acid position selected from the group consisting of M40, L41, S496, Y499 and Y527 and having tRNA synthetase activity, and expressing the polypeptide(s) in the cell;
• (c) controlling replication of the cell and/or virus by contacting the cell of (a) with the noncanonical amino acid, wherein replication of the cell and/or virus is reduced or eliminated in the absence of the noncanonical amino acid.