Riken Corporation

Takahito Mukai

Shigeyuki Yokoyama

Kensaku Sakamoto

Takatsugu Kobayashi

Nobumasa Hino

Akiko Matsumoto

Tatsuo Yanagisawa

Kenji Oki

The present invention provides a novel method of producing a recombinant bacterium for production of a non-natural protein, including: (1) expressing tRNA in a bacterium, which tRNA recognizes UAG codon; (2) expressing an aminoacyl-tRNA synthetase in the bacterium, which aminoacyl-tRNA synthetase acylates the tRNA with a non-natural amino acid or an &#x3b1;-hydroxy acid; (3) (i) introducing a DNA construct into the bacterium, which DNA construct is for expressing, in the absence of a release factor for terminating translation at UAG codon, a function of at least one gene selected from the group consisting of genes each of which loses its function when a gene that codes for the release factor is defective and/or introducing an alteration into said at least one gene in a chromosome of the bacterium, which alteration is for expressing the function of said at least one gene in the absence of the release factor; and (4) causing the gene that codes for the release factor in the bacterium to be defective.

Method for constructing recombinant bacterium for producing non-native protein, and utilization of same

There are provided a DNA construct comprising a suppressor tRNA gene of a non-eukaryote containing no internal promoter functioning in a eukaryotic cell, and a eukaryotic or bacteriophage 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 protein incorporating a non-natural amino acid by using the same.

Method of synthesizing a suppressor tRNA, DNA construct and use thereof for producing a non-natural amino acid-incorporated protein

A non-natural protein having at least one ester bond in its polypeptide main chain is synthesized by using an in vivo translation system in a ribosome. The following components (a) to (c) are expressed in a cell or an cell extraction solution in the presence of an &#x3b1;-hydroxy acid: (a) an aminoacyl-tRNA synthetase which can activate the &#x3b1;-hydroxy acid; (b) suppressor tRNA which can bind to the &#x3b1;-hydroxy acid in the presence of the aminoacyl-tRNA synthetase; and (c) a gene encoding a desired protein having a nonsense mutation or a frame-shift mutation at a desired site.

Process for production of non-natural protein having ester bond therein

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.

Polypeptide having activity of aminoacyl-tRNA synthetase and use thereof

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.

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Yokohama, Japan

Takahito Mukai