# US 8,785,152 B2 · Process for production of non-natural protein having ester bond therein

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- Number: US 8,785,152 B2
- Document ID: US8785152B2
- Status: Granted
- Kind: B2
- Application number: 12/744,322
- Filed: 2008-11-21
- Published: 2011-06-09
- Granted: 2014-07-22
- Earliest claimed: 2007-11-22
- Assignees: Riken
- Applicants: Shigeyuki Yokoyama; Kensaku Sakamoto; Tatsuo Yanagisawa; Takahito Mukai; Takatsugu Kobayashi
- Inventors: Shigeyuki Yokoyama; Kensaku Sakamoto; Tatsuo Yanagisawa; Takahito Mukai; Takatsugu Kobayashi
- Web page: https://patentgrep.com/patent/US8785152B2

## CPC

- C12N 15/67: General methods for enhancing the expression
- C12N 9/93: {Ligases (6)}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

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 α-hydroxy acid: (a) an aminoacyl-tRNA synthetase which can activate the α-hydroxy acid; (b) suppressor tRNA which can bind to the α-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.

## Claims

### Claim 1

A method of producing a cell or a cell extract having activity of incorporating an alpha-hydroxy acid into a polypeptide to provide a polypeptide having an ester bond in the main chain, comprising expressing
(a1) a pyrrolysyl-tRNA synthetase derived from an archaebacterium that is capable of activating an α-hydroxy acid that is N-substituted 6-amino-2-hydroxyhexanoic acid; or
(a2) a pyrrolysyl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2 and having at least one amino acid substitution of:
from tyrosine to alanine at position 306,
from leucine to alanine at position 309,
from cysteine to valine at position 348, and
from tyrosine to phenylalanine at position 384,
and that is capable of activating an α-hydroxy acid that is N-substituted 6-amino-2-hydroxyhexanoic acid; or
(a3) pyrrolysyl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2 and having the five amino acid substitutions from alanine to phenylalanine at position 302, tyrosine to alanine at position 306, from asparagine to serine at position 346, from cysteine to isoleucine at position 348, and from tyrosine to phenylalanine at position 384, and that is capable of activating an α-hydroxy acid that is an optionally substituted 3-phenyllactic acid;
(b) a suppressor tRNA capable of binding to the α-hydroxy acid in the presence of said pyrrolysyl-tRNA, synthetase; and
(c) a gene encoding a desired protein that has a nonsense mutation or frameshift mutation at a desired site;
in a cell or cell extract solution in the presence of said α-hydroxy acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein a substituent at the amino group of said N-substituted 6-amino-2-hydroxyhexanoic acid is t-butoxycarbonyl group, or an optionally substituted phenyl group, benzyl group or benzyloxycarbonyl group.

### Claim 3 (depends on 1)

The method of claim 1, comprising expressing said (a1), (a2) or (a3), (b) and (c) in Escherichia coli cells.

### Claim 4 (depends on 1)

The method of claim 1, comprising expressing said (a1), (a2) or (a3), (b) and (c) in mammalian cells.

### Claim 5

A method for making an unnatural polypeptide that has at least one ester bond in a polypeptide main chain comprising culturing a cell expressing:
(a1) a pyrrolysyl-tRNA synthetase derived from an archaebacterium that is capable of activating an α-hydroxy acid that is N-substituted 6-amino-2-hydroxyhexanoic acid; or
(a2) a pyrrolysyl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2 and having at least one amino acid substitution of:
from tyrosine to alanine at position 306,
from leucine to alanine at position 309,
from cysteine to valine at position 348, and
from tyrosine to phenylalanine at position 384,
and that is capable of activating an α-hydroxy acid that is N-substituted 6-amino-2-hydroxyhexanoic acid; or
(a3) pyrrolysyl-tRNA synthetase having the amino acid sequence of SEQ ID NO: 2 and having the five amino acid substitutions from alanine to phenylalanine at position 302, tyrosine to alanine at position 306, from asparagine to serine at position 346, from cysteine to isoleucine at position 348, and from tyrosine to phenylalanine at position 384, and that is capable of activating an α-hydroxy acid that is an optionally substituted 3-phenyllactic acid;
(b) a suppressor tRNA capable of binding to the α-hydroxy acid in the presence of said aminoacyl-tRNA synthetase; and
(c) a gene encoding a desired protein that has a nonsense mutation or frameshift mutation at a desired site;
in the presence of said α-hydroxy acid.

### Claim 6 (depends on 1)

The method of claim 1, in which the pyrrolysyl-tRNA synthetase is a mutant enzyme selected from the group consisting of substitution Y384F, substitution Y306A, the double substitution Y306A/Y384F and the double substitution L309A/C348V, in the amino acid sequence of SEQ ID NO:2.

### Claim 7 (depends on 5)

The method of claim 5, in which the pyrrolysyl-tRNA synthetase is a mutant enzyme selected from the group consisting of substitution Y384F, substitution Y306A, the double substitution Y306A/Y384F and the double substitution L309A/C348V, in the amino acid sequence of SEQ ID NO:2.