Abstract: The present invention relates to a process for producing a protein having an unnatural amino acid introduced therein, the process including: expressing in a eukaryotic cell an aminoacyl-tRNA synthetase, a nucleic acid having a sequence containing a eukaryote-derived tRNA nucleotide sequence linked to the 5? end of a tRNA nucleotide sequence that is ligated with to an unnatural amino acid in the presence of the aminoacyl-tRNA synthetase, an unnatural amino acid, and a gene of a desired protein having a nonsense mutation at a predetermined position, to integrate the unnatural amino acid at the nonsense mutation position into the protein, thereby expressing a protein having an unnatural amino acid introduced therein.

Abstract: Method for incorporating a lysine derivative (particularly an N?-benzyloxycarbonyl-lysine (Z-Lys) derivative) having useful functional group such as heavy atom, selenium, reactive functional group, fluorescent group or crosslinker, which is suitable as a non-natural amino acid, into a desired protein in a site-specific manner. A mutant pyrrolysyl-tRNA synthetase has substitution of at least one amino acid residue selected from tyrosine residue at position 306, leucine residue at position 309 and cysteine residue at position 348 each constituting a pyrrolysine-binding site in the amino acid sequence for pyrrolysyl-tRNA synthetase of SEQ ID NO:2. The substitution of the amino acid residue is: of tyrosine residue at position 306 by glycine or alanine residue, of leucine residue at position 309 by glycine or alanine residue, and/or of a cysteine residue at position 348 by valine, serine or alanine residue.

Abstract: The present invention discloses an expression method for non-naturally-occurring amino acid-containing protein comprising: expressing in animal cells: (A) a mutant tyrosyl-tRNA synthetase that is a mutation of tyrosyl-tRNA synthetase originating in E. coli with an enhanced specificity for a non-naturally-occurring tyrosine derivative as compared with the specificity for tyrosine; (B) suppressor tRNA originating in Bacillus species, Mycoplasma species or Staphylococcus species of eubacteria and capable of binding with the tyrosine derivative in the presence of the mutant tyrosyl tRNA synthetase; and, (C) a desired protein gene that has undergone a nonsense mutation at a desired site; wherein, the tyrosine derivative is incorporated at the location of this nonsense mutation.

Abstract: The present invention discloses an expression method for non-naturally-occurring amino acid-containing protein comprising: expressing in animal cells: (A) a mutant tyrosyl-tRNA synthetase that is a mutation of tyrosyl-tRNA synthetase originating in E. coli with an enhanced specificity for a non-naturally-occurring tyrosine derivative as compared with the specificity for tyrosine; (B) suppressor tRNA originating in Bacillus species, Mycoplasma species or Staphylococcus species of eubacteria and capable of binding with the tyrosine derivative in the presence of the mutant tyrosyl tRNA synthetase; and, (C) a desired protein gene that has undergone a nonsense mutation at a desired site; wherein, the tyrosine derivative is incorporated at the location of this nonsense mutation.

Abstract: This invention provides a method for producing proteins into which non-natural amino acids have been incorporated at desired positions involving the use of either prokaryote-derived or eukaryote-type aaRS* and prokaryotic cells or eukaryote-type as host cells, the method comprising steps of: (a) introducing genes encoding prokaryote-derived aminoacyl tRNA synthetase mutants having enhanced specificity to non-natural amino acids similar to given natural amino acids (compared with specificity to the natural amino acids) and tRNA genes for non-natural amino acids capable of binding to the non-natural amino acids in the presence of the prokaryote-derived aminoacyl tRNA synthetase mutants into prokaryotic cells that express genes encoding eukaryote-type aminoacyl tRNA synthetase having specificity to the given natural amino acids and tRNA genes capable of binding to the natural amino acids in the presence of eukaryote-type aminoacyl tRNA synthetase; and (b) knocking out genes encoding aminoacyl tRNA synthetase hav

Abstract: A polypeptide according to the present invention includes: an altered polypeptide obtained by altering an ArgRS, a CysRS, a MetRS, a GlnRS, a GluRS, a LysRS, a TyrRS, or a TrpRS so that an unnatural amino acid is recognized; and an editing polypeptide derived from a PheRS, a LeuRS, an IleRS, a ValRS, an AlaRS, a ProRS, or a ThrRS, the editing polypeptide having been either inserted between a Rossman-fold N domain and a Rossman-fold C domain that exist in the altered polypeptide, or bound to an N terminal of the altered polypeptide. Thus provided are a new aaRS that exhibits high substrate specificity to an unnatural amino acid and a technique that involves the use of such an aaRS.

Abstract: A method of expressing a protein having an unnatural amino acid integrated thereinto comprising expressing (A) a mutant tyrosyl tRNA synthase that is derived from Escherichia coli-origin tyrosyl tRNA synthase and has an elevated specificity for an unnatural tyrosine derivative compared with the specificity for tyrosine, (B) a suppressor tRNA originating in an eubacterium belonging to the genus Bacillus, Mycoplasma or Staphylococcus which is capable of binding to the above tyrosine derivative in the presence of the above mutant TyrRS, and (C) a desired protein gene having a nonsense mutation at a desired site in animal cells to thereby incorporate the above tyrosine derivative into the nonsense mutation site in the above protein.

Abstract: There are provided a DNA construct comprising non-eukaryote-derived suppressor tRNA gene containing no internal promoter functioning in a eukaryotic cell, and a eukaryote-derived or bacteriophage-derived promoter linked at the 5? end of the tRNA gene, a method for synthesizing a suppressor tRNA by using the DNA construct, and a process for producing a non-natural amino acid-incorporated protein by using the same.

Abstract: Tyrosyl-tRNA synthetases having a modified amino acid sequence whereby unnatural amino acids can be more efficiently incorporated than original natural amino acids. More specifically, tyrosyl-tRNA synthetases having been modified at the amino acid(s) at the 37- and/or 195-positions. A method of modifying tyrosyl-tRNA synthetase characterized in that the position of an amino acid to be modified is determined based on the 3-D structure of a tyrosyl-AMP and of tyrosyl synthetase complex and an amino acid to which this enzyme binds.

Abstract: A method of expressing a protein having an unnatural amino acid integrated thereinto characterized by comprising expressing (A) a mutant tyrosyl tRNA synthase which is a mutant of Escherichia coli-origin tyrosyl tRNA synthase and has an elevated specificity for a unnatural tyrosine derivative compared with the specificity for tyrosine, (B) a suppressor tRNA originating in an eubacterium belonging to the genus Bacillus, Mycoplasma or Staphylococcus which is capable of binding to the above tyrosine derivative in the presence of the above mutant TyrRS, and (C) a desired protein gene having a nonsense mutation at a desired site in animal cells to thereby incorporate the above tyrosine derivative into the nonsense mutation site in the above protein.

Abstract: A novel nucleic acid (aptamer) which binds specifically to the target protein of Ras, more particularly, a novel RNA aptamer which binds specifically to Raf-1; a method for screening an RNA capable of binding specifically to the target protein of Ras which comprises selecting an RNA capable of binding to the target protein of Ras from a pool of RNAs having various base sequences; a method for regulating the signal transduction causing the proliferation or differentiation of cells by using the above-described nucleic acid; and medicinal compositions with the use of the same.

Abstract: Tyrosyl-tRNA synthetases having a modified amino acid sequence whereby unnatural amino acids can be more efficiently incorporated than original natural amino acids. More specifically, tyrosyl-tRNA synthetases having been modified at the amino acid(s) at the 37- and/or 195-positions. A method of modifying tyrosyl-tRNA synthetase characterized in that the position of an amino acid to be modified is determined based on the 3-D structure of a tyrosyl-AMP and of tyrosyl synthetase complex and an amino acid to which this enzyme binds.