Polypeptide having activity of aminoacyl-tRNA synthetase and use thereof

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.

1 . A polypeptide having aminoacyl-tRNA synthetase activity, the polypeptide comprising:
 an altered polypeptide obtained by altering an arginyl-tRNA synthetase, a cysteinyl-tRNA synthetase, a methionyl-tRNA synthetase, a glutaminyl-tRNA synthetase, a glutamyl-tRNA synthetase, a lysyl-tRNA synthetase, a tyrosyl-tRNA synthetase, or a tryptophanyl-tRNA synthetase so that an unnatural amino acid is recognized; and   an editing polypeptide containing an editing reaction active site derived from a phenylalanyl-tRNA synthetase, a leucyl-tRNA synthetase, an isoleucyl-tRNA synthetase, a valyl-tRNA synthetase, an alanyl-tRNA synthetase, a prolyl-tRNA synthetase, or a threonyl-tRNA synthetase, 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.   
     
     
         2 . The polypeptide as set forth in  claim 1 , wherein the editing polypeptide has been inserted into a CP1 domain that lies between the Rossman-fold N domain and the Rossman-fold C domain. 
     
     
         3 . The polypeptide as set forth in  claim 1 , wherein a linker polypeptide that connects the editing polypeptide with the altered polypeptide has been further inserted. 
     
     
         4 . The polypeptide as set forth in  claim 1 , wherein the altered polypeptide is a tyrosyl-tRNA synthetase altered so as to recognize an unnatural amino acid. 
     
     
         5 . The polypeptide as set forth in  claim 1 , wherein the tyrosyl-tRNA synthetase is derived from eukaryotic organisms or eubacteria. 
     
     
         6 . The polypeptide as set forth in  claim 5 , wherein the eubacteria are  Escherichia coli.    
     
     
         7 . The polypeptide as set forth in  claim 1 , wherein the tyrosyl-tRNA synthetase is a polypeptide altered so as to recognize a tyrosine derivative. 
     
     
         8 . The polypeptide as set forth in  claim 7 , wherein the tyrosine derivative is 3-iodotyrosine. 
     
     
         9 . The polypeptide as set forth in  claim 1 , wherein the altered polypeptide is a polypeptide as set forth in either of (a) and (b):
 (a) a polypeptide consisting of an amino-acid sequence represented by SEQ ID NO: 1; and   (b) a polypeptide (i) consisting of an amino-acid sequence, represented by SEQ ID NO: 1 with a deletion, insertion, substitution, or addition of one or several amino acids and (ii) having activity to bind 3-iodotyrosine to tRNA.   
     
     
         10 . The polypeptide as set forth in  claim 1 , wherein the editing polypeptide is derived from a phenylalanyl-tRNA synthetase. 
     
     
         11 . The polypeptide as set forth in  claim 1 , wherein the phenylalanyl-tRNA synthetase is derived from eukaryotic organisms or archaebacteria. 
     
     
         12 . The polypeptide as set forth in  claim 11 , wherein the archaebacteria belong to the genus  Pyrococcus.    
     
     
         13 . The polypeptide as set forth in  claim 11 , wherein the archaebacteria are  Pyrococcus horikoshii.    
     
     
         14 . The polypeptide as set forth in  claim 1 , wherein the editing polypeptide is a polypeptide as set forth in either of (c) and (d):
 (c) a polypeptide consisting of an amino-acid sequence represented by SEQ ID NO: 2; and   (d) a polypeptide (i) consisting of an amino-acid sequence, represented by SEQ ID NO: 2 with a deletion, insertion, substitution, or addition of one or several amino acids and (ii) having activity to degrade binding of tyrosine to tRNA or activity to degrade tyrosyl adenylate intermediate into tyrosine and an inorganic phosphoric acid.   
     
     
         15 . The polypeptide as set forth in  claim 1 , wherein the polypeptide is a polypeptide as set forth in either of (e) and (f):
 (e) a polypeptide consisting of an amino-acid sequence represented by SEQ ID NO: 3; and   (f) a polypeptide (i) consisting of an amino-acid sequence, represented by SEQ ID NO: 3 with a deletion, insertion, substitution, or addition of one or several amino acids and (ii) having activity to degrade binding of tyrosine to tRNA or activity to degrade tyrosyl adenylate intermediate into tyrosine and an inorganic phosphoric acid and activity to bind an unnatural amino acid to tRNA.   
     
     
         16 . The polypeptide as set forth in  claim 1 , wherein:
 the altered polypeptide is a tyrosyl-tRNA synthetase altered so as to recognize an unnatural amino acid and the editing polypeptide contains an editing reaction active site derived from a phenylalanyl-tRNA synthetase; or   the altered polypeptide is a methionyl-tRNA synthetase altered so as to recognize an unnatural amino acid and the editing polypeptide contains an editing reaction active site derived from a leucyl-tRNA synthetase.   
     
     
         17 . A polynucleotide coding for a polypeptide having aminoacyl-tRNA synthetase activity as set froth in  claim 1 . 
     
     
         18 . A method for producing a polypeptide having aminoacyl-tRNA synthetase activity, the method comprising:
 a preparing step of preparing a polynucleotide in which a polynucleotide coding for an editing polypeptide containing an editing reaction active site derived from a phenylalanyl-tRNA synthetase, a leucyl-tRNA synthetase, an isoleucyl-tRNA synthetase, a valyl-tRNA synthetase, an alanyl-tRNA synthetase, a prolyl-tRNA synthetase, or a threonyl-tRNA synthetase has been introduced into a polynucleotide coding for an altered polypeptide obtained by altering an arginyl-tRNA synthetase, a cysteinyl-tRNA synthetase, a methionyl-tRNA synthetase, a glutaminyl-tRNA synthetase, a glutamyl-tRNA synthetase, a lysyl-tRNA synthetase, a tyrosyl-tRNA synthetase, or a tryptophanyl-tRNA synthetase so that an unnatural amino acid is recognized; and   an expressing step of expressing a polypeptide coded for by the polynucleotide obtained in the preparing step,   the preparing step includes preparing either a polynucleotide in which the editing polypeptide has been introduced so as to be positioned between a Rossman-fold N domain and a Rossman-fold C domain that exist in the altered polypeptide, or a polynucleotide in which the editing polypeptide has been introduced so as to be bound to an N terminal of the altered polypeptide.