# US 2019/0169600 A1 · Ribosome display complex and production method therefor

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- Number: US 2019/0169600 A1
- Document ID: US20190169600A1
- Status: Published application
- Kind: A1
- Application number: 16/213,454
- Filed: 2018-12-07
- Published: 2019-06-06
- Earliest claimed: 2016-06-07
- Assignees: Kaneka Corporation
- Applicants: Kaneka Corporation
- Inventors: Hiroshi Kita; Toshihiro Shikakura; Hirofumi Maeda; Keishi Takatsu
- Web page: https://patentgrep.com/patent/US20190169600A1

## CPC

- C07K 1/1077: {by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids}
- C12N 15/1041: {Ribosome/Polysome display, e.g. SPERT, ARM}
- C07K 2/00: Peptides of undefined number of amino acids; Derivatives thereof
- C12N 15/09: Recombinant DNA-technology
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C40B 40/10: Libraries containing peptides or polypeptides, or derivatives thereof
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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)}
- C40B: COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES (in silico combinatorial libraries of nucleic acids, proteins or peptides G16B35/00; in silico combinatorial chemistry G16C20/60)

## Abstract

A method for producing a ribosome display complex includes obtaining a ribosome complex including an unmodified polypeptide chain, an mRNA molecule and a ribosome by initiating translation of the mRNA molecule in a cell-free peptide synthesis system including the ribosome, and modifying the unmodified polypeptide chain by reacting a side chain reactive functional group in the unmodified polypeptide chain with a modifying reagent to produce a ribosome display complex including a modified polypeptide chain, the mRNA molecule and the ribosome. The unmodified polypeptide chain includes at least one reactive amino acid residue selected from the group consisting of a cysteine residue, a lysine residue, a histidine residue and a tryptophan residue. The at least one reactive amino acid residue includes the side chain reactive functional group, and the mRNA molecule includes a base sequence encoding an amino acid sequence of the polypeptide chain.

## Claims

### Claim 1

A method for producing a ribosome display complex, comprising:
obtaining a ribosome complex comprising an unmodified polypeptide chain, an mRNA molecule and a ribosome by initiating translation of the mRNA molecule in a cell-free peptide synthesis system comprising the ribosome; and
modifying the unmodified polypeptide chain by reacting a side chain reactive functional group in the unmodified polypeptide chain with a modifying reagent to produce the ribosome display complex comprising a modified polypeptide chain, the mRNA molecule and the ribosome,
wherein the unmodified polypeptide chain comprises at least one reactive amino acid residue selected from the group consisting of a cysteine residue, a lysine residue, a histidine residue and a tryptophan residue,
wherein the at least one reactive amino acid residue comprises the side chain reactive functional group, and
wherein the mRNA molecule comprises a base sequence encoding an amino acid sequence of the polypeptide chain.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the modifying reagent is a compound represented by the following formula:
(A)b-B-(C)c
wherein A is a group capable of forming a linkage by reacting with a side chain of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue, B is a linker group or a single bond, C is a functional group, a is an integer of 1 or more, c is 0 or an integer of 1 or more, provided that when a is an integer of 2 or more, a plurality of A are the same as or different from each other.

### Claim 3 (depends on 2)

The method according to claim 2, wherein A is a halogenated alkyl group, an activated carbonyl group, an unsaturated hydrocarbon group, an epoxy group, a sulfonyl-containing group, an isocyanate group, an isothiocyanate group, a carbene-precursor group, a carbene-containing group, a disulfide bond-containing group or a thiol group.

### Claim 4 (depends on 2)

The method according to claim 2, wherein
B is the linker group and has one or more selected from the group consisting of: a hetero atom-containing polar group; a chained or cyclic aliphatic hydrocarbon group optionally having the hetero atom-containing polar group between carbon atoms and optionally having a substituent group; and an aromatic ring optionally having a substituent group,
the hetero atom-containing polar group is —O—, —S—, —NR1—, —CO—, —COO—, —CONR2—, —N═N— or —SO2—, wherein R1 is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group, and R2 is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group,
the substituent group on the aliphatic hydrocarbon group is a halogeno group, an aryl group, a carboxy group, an alkoxycarbonyl group or a hydroxy group, and
the substituent group on the aromatic ring is a halogeno group, an alkyl group, an aralkyl group, a carboxy group, an alkoxycarbonyl group, a hydroxyalkyl group or a carboxyalkyl group.

### Claim 5 (depends on 4)

The method according to claim 4, wherein
the linker group B has -B1- unit, -B2- unit, -B2-B1- unit or -B2-B1-B3- unit,
B1 is a group having one or more selected from the group consisting of the chained or cyclic aliphatic hydrocarbon group and the aromatic ring,
B2 and B3 are each independently the hetero atom-containing polar group, and
B1 or B2 is bound to A.

### Claim 6 (depends on 5)

The method according to claim 5, wherein c is 0, and the modifying reagent is any one of compounds represented by the following formulae:
wherein A, B1, B2 and B3 have the same meanings as the above, n is an integer of 1 or more, one or more of B1, B2 and B3 may be bound by the one or more C.

### Claim 7 (depends on 2)

The method according to claim 2, wherein
A is a halogenated alkyl group, and
a carbon atom bound by a halogeno group in the halogenated alkyl group is a carbon atom at α position of a carbonyl group or a carbon atom directly binding to an aromatic ring.

### Claim 8 (depends on 2)

The method according to claim 2, wherein
the unmodified polypeptide chain comprises two or more of the reactive amino acid residues,
a in the formula is an integer of 2 or more, and
the unmodified polypeptide chain and the modifying reagent form a ring by the reaction of the side chain reactive functional group and the modifying reagent.

### Claim 9 (depends on 1)

The method according to claim 1, wherein the unmodified polypeptide chain has 100 to 5000 amino acid residues.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the reactive amino acid residue is located at a position selected from the group consisting of the 2nd position from the N-terminal to the 30th position from the C-terminal in the unmodified polypeptide chain.

### Claim 11 (depends on 1)

The method according to claim 1, wherein the unmodified polypeptide comprises a random sequence having 1 to 30 amino acid residues within a region ranging from the 2nd position from the N-terminal to the 30th position from the C-terminal.

### Claim 12 (depends on 1)

The method according to claim 1, wherein the ribosome is originated from Escherichia coli.

### Claim 13

A ribosome display complex,
comprising a polypeptide chain, an mRNA molecule, and a ribosome, wherein
the polypeptide chain comprises at least, one reactive amino acid, residue selected from the group consisting of a cysteine residue, a lysine residue, a histidine residue and a tryptophan residue,
the at least one reactive amino acid, residue comprises a side chain reactive functional group that is modified with a modifying reagent, and
the mRNA molecule comprises a base sequence encoding an amino acid sequence of the polypeptide chain.

### Claim 14 (depends on 13)

The ribosome display complex according to claim 13, wherein the side chain reactive functional group comprises a modification structure represented by the following formula:
(Ax)a-B(C)c
wherein Ax is a binding group formed by reacting the modifying reagent with a side chain of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue, B is a linker group or a single bond, C is a functional group, a is an integer of 1 or more, c is 0 or an integer of 1 or more, provided that when a is an integer of 2 or more, a plurality of Ax are the same as or different from each other.

### Claim 15 (depends on 14)

The ribosome display complex according to claim 14, wherein Ax is a chemical bond formed by reacting a halogenated alkyl group, an activated carbonyl group, an unsaturated hydrocarbon group, an epoxy group, a sulfonyl-containing group, an isocyanate group, an isothiocyanate group, a carbene-precursor group, a carbene-containing group, a disulfide bond-containing group or a thiol group with the side chain of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue.

### Claim 16 (depends on 15)

The ribosome display complex according to claim 15, wherein
B is the linker group and has one or more selected from the group consisting of: a hetero atom-containing polar group; a chained or cyclic aliphatic hydrocarbon group optionally having the hetero atom-containing polar group between carbon atoms and optionally having a substituent group; and an aromatic ring optionally having a substituent group,
the hetero atom-containing polar group is —O—, —S—, —NR1—, —CO—, —COO—, —CONR2—, —N═N— or —SO2—, wherein R1 is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group, and R2 is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group,
the substituent group on the aliphatic hydrocarbon group is a halogeno group, an aryl group, a carboxy group, an alkoxycarbonyl group or a hydroxy group, and
the substituent group on the aromatic ring is a halogeno group, an alkyl group, an aralkyl group, a carboxy group, an alkoxycarbonyl group, a hydroxyalkyl group or a carboxyalkyl group.

### Claim 17 (depends on 16)

The ribosome display complex according to claim 16, wherein
the linker group B has -B1- unit, -B2- unit, -B2-B1- unit or -B2-B1-B3- unit,
B1 is a group having one or more selected from, the group consisting of the chained or cyclic aliphatic hydrocarbon group and the aromatic ring,
B2 and B3 are each independently the hetero atom-containing polar group, and
B1 or B2 is bound to Ax.

### Claim 18 (depends on 13)

The ribosome display complex according to claim 13, wherein the side chain reactive functional group comprises a modification structure represented by any one of the following formulae:
wherein
Ax is a binding group formed by reacting the modifying reagent with a side chain, of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue,
B1 is a group having one or more selected from the group consisting of: a chained or cyclic aliphatic hydrocarbon group optionally having a hetero atom-containing polar group between carbon atoms and optionally having a substituent group; and an aromatic ring optionally having a substituent group,
the hetero atom-containing polar group is —O—, —S—, —NR1—, —CO—, —COO—, —CONR2—, —N═N— or —SO2—, wherein R1 is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group, and R2 is a hydrogen atom, a hydrocarbon group or an atomic bonding at a terminal of the linker group,
the substituent group on the aliphatic hydrocarbon group is a halogeno group, an aryl group, a carboxy group, an alkoxycarbonyl group or a hydroxy group,
the substituent group on the aromatic ring is a halogeno group, an alkyl group, an aralkyl group, a carboxy group, an alkoxycarbonyl group, a hydroxyalkyl group or a carboxyalkyl group,
B2 and B3 are each independently the hetero atom-containing polar group,
B1 or B2 is bound to Ax,
n is an integer of 1 or more,
optionally one or more of B1, B2 and B3 are bound to one or more functional groups.

### Claim 19 (depends on 14)

The ribosome display complex according to claim 14, wherein
Ax is a chemical bond formed between a halogenated alkyl group and the side chain of the cysteine residue, the lysine residue, the histidine residue or the tryptophan residue, and
a carbon atom bound by the halogeno group in the halogenated alkyl group is a carbon atom at a position of a carbonyl group or a carbon atom directly binding to an aromatic ring.

### Claim 20 (depends on 14)

The ribosome display complex according to claim 14, wherein
the polypeptide chain comprises two or more of the reactive amino acid residues,
a in the formula is an integer of 2 or more,
the polypeptide chain and the modification structure form a ring.

### Claim 21 (depends on 13)

The ribosome display complex according to claim 13, wherein the polypeptide chain consists of 100 to 5000 amino acid residues.

### Claim 22 (depends on 13)

The ribosome display complex according to claim 13, wherein the reactive amino acid residue is located at a position selected from the group consisting of the 2nd position from the N-terminal to the 30th position from the C-terminal in the polypeptide chain.

### Claim 23 (depends on 13)

The ribosome display complex according to claim 13, wherein the polypeptide chain comprises a random sequence having 1 to 30 amino acid residues within a region of the 2nd position from the N-terminal to the 30th position from the C-terminal.

### Claim 24 (depends on 13)

The ribosome display complex according to claim 13, wherein the ribosome is originated from Escherichia coli.