METHOD OF SYNTHESIZING A SUPPRESSOR tRNA, DNA CONSTRUCT AND USE THEREOF FOR PRODUCING A NON-NATURAL AMINO ACID-INCORPORATED PROTEIN
AssigneeRiken
InventorsShigeyuki YOKOYAMA, Kensaku Sakamoto, Nobumasa Hino, Takahito Mukai, Takatsugu Kobayashi
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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.
Claims20
A DNA construct comprising a non-eukaryote-originating suppressor tRNA gene containing no internal promoter sequence that functions in a eukaryotic cell, and a eukaryote-originating promoter linked to the 5′ end of said tRNA gene.
A DNA construct comprising a non-eukaryote-originating suppressor tRNA gene containing no internal promoter sequence that functions in a eukaryotic cell, and a promoter originating from bacteriophage linked to the 5′ end of said tRNA gene.
A process for producing a non-natural amino acid-incorporated protein comprising:
- preparing a DNA construct comprising a non-eukaryote-originating suppressor tRNA gene containing no internal promoter sequence that functions in a eukaryotic cell, and a bacteriophage-originating promoter operably linked to the 5′ terminal region of said tRNA gene, wherein said suppressor tRNA is capable of binding to the non-natural amino acid in the presence of an aminoacyl-tRNA synthetase for the non-natural amino acid;
- and
- expressing, in the presence of the non-natural amino acid in a eukaryotic cell,
- a tRNA transcribed from said DNA construct,
- an aminoacyl-tRNA synthetase for the non-natural amino acid, and
- a desired protein that has a nonsense mutation or frame shift mutation at a desired position.
Dependencies are inferred from the claim text.