Method for producing peptide library, peptide library, and screening method

Patent metadata

Field	Value
Publication number	US-10711268-B2
Application number	US-201414889868-A
Country	US
Kind code	B2
Filing date	May 12, 2014
Priority date	May 10, 2013
Publication date	Jul 14, 2020
Grant date	Jul 14, 2020

Abstract

An object of the present invention is to provide a method for producing a peptide library capable of incorporating an arbitrary number of arbitrary proteinogenic and/or non-proteinogenic amino acids in an arbitrary site. The invention provides a method for producing a peptide library including 1×10 6 or more kinds of peptides containing amino acids encoded by N 1 N 2 N 3 , including a step of preparing an mRNA library including mRNAs which encode peptides of the peptide library and each contain at least one N 1 N 2 N 3 ; and a step of translating each mRNA of the mRNA library in a cell-free translation system added with tRNA containing an anticodon to any one of N 1 N 2 N 3 codons and charged with an amino acid corresponding to the codon (wherein, N 1 , N 2 , and N 3 are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U) and an arbitrary amino acid is reassigned to each N 1 N 2 N 3 ).

First claim

The invention claimed is:

1. A method for producing a peptide library including 1×10 6 or more peptides containing amino acids encoded by N 1 N 2 N 3 , comprising:
a step of preparing an mRNA library including mRNAs which encode peptides of the peptide library, respectively, and each contain a plurality of N 1 N 2 N 3 s; and
a step of translating each of the mRNAs of the mRNA library in a cell-free translation system added with a tRNA containing an anticodon to any of N 1 N 2 N 3 codons and charged with an amino acid corresponding to the codon,
wherein N 1 , N 2 , and N 3 are each independently selected from adenine (A), guanine (G), cytosine (C), and uracil (U); and an arbitrary amino acid is reassigned to each N 1 N 2 N 3 and N 1 N 2 U codons are reassigned to non-proteinogenic amino acids, and
wherein the amino acids encoded by N 1 N 2 N 3 contain a non-proteinogenic amino acid selected from cyclic N-alkyl amino acids represented by chemical structures 2 to 22:

2. The method according to claim 1 , wherein each of the mRNAs included in the mRNA library is represented by the following formula (I):
X 1 —(N 1 N 2 N 3 ) n -X 2   (I)

wherein, X 1 and X 2 each represent an mRNA encoding a peptide composed of an arbitrary number of amino acids; and n stands for an arbitrary integer selected from 4 to 20.

3. The method according to claim 1 ,
wherein the peptide library is a peptide-mRNA complex library in which each peptide is complexed with an mRNA encoding the peptide,
wherein the mRNA library is a puromycin-bound mRNA library each mRNA has puromycin bound to a downstream region of ORF in the mRNA; and
the step of translating produces a peptide-mRNA complex library.

4. The method according to claim 3 , wherein each of the mRNAs included in the puromycin-bound mRNA library is represented by the following formula (I):
X 1 —(N 1 N 2 N 3 ) n -X 2   (I)

wherein, X 1 and X 2 each represent an mRNA encoding a peptide having an arbitrary number of amino acids and n stands for an arbitrary integer selected from 4 to 20.

5. The method according to claim 1 , wherein the N 3 is either the following (i) or (ii) in one translation system:
(i) cytosine (C) or uracil (U);
(ii) adenine (A) or guanine (G).

6. The method according to claim 1 , wherein 16 kinds of the N 1 N 2 N 3 s are present in one translation system.

7. The method according to claim 1 , wherein, of the tRNAs charged with an amino acid corresponding to the N 1 N 2 N 3 codon, elongator tRNAs have base sequences having 85% or more sequence homology with each other, respectively.

8. The method according to claim 1 , wherein, of the tRNAs charged with an amino acid corresponding to the N 1 N 2 N 3 codon, elongator tRNAs have RNA sequences identical to each other except for an anticodon loop, respectively.

9. The method according to claim 1 , further comprising, after the translation step, a peptide macrocyclization step.

10. A screening method for identifying a peptide to be bound to a target substance, comprising:
a step of producing the peptide library according to claim 1 and bringing the peptide library into contact with the target substance, followed by incubation; and
a step of selecting the peptide bound to the target substance.

11. A screening method for identifying a peptide to be bound to a target substance, comprising:
a step of producing the peptide library according to claim 3 and subjecting the peptide-mRNA complex library to a reverse transcription reaction to obtain a peptide-DNA complex library;
a step of bringing the peptide-DNA complex library into contact with the target substance, followed by incubation;
a step of selecting a peptide-DNA complex group bound to the target substance;
a step of amplifying DNA of the selected peptide-DNA complex group by PCR; and
a step of transcribing the amplified DNA to produce an mRNA library, binding puromycin to a downstream region of ORF in the mRNA to produce a puromycin-bound mRNA library, and translating it to produce a peptide-mRNA complex library,
wherein the steps from the reverse transcription reaction to the production of the peptide-mRNA complex library is repeated twice or more to select a peptide having high affinity for the target substance.

12. The screening method according to claim 11 , wherein the N 3 is either the following (i) or (ii) in one translation system:
(i) cytosine (C) or uracil (U);
(ii) adenine (A) or guanine (G).

13. The screening method according to claim 11 , wherein 16 kinds of the N 1 N 2 N 3 s are present in one translation system.

14. The screening method according to claim 11 , wherein, of the tRNAs charged with an amino acid corresponding to the N 1 N 2 N 3 codon, elongator tRNAs have base sequences having 85% or more sequence homology with each other, respectively.

15. The screening method according to claim 11 , wherein, of the tRNAs charged with an amino acid corresponding to the N 1 N 2 N 3 codon, elongator tRNAs have RNA sequences identical to each other except for an anticodon loop, respectively.

16. The screening method according to claim 11 , further comprising, after the translation step, a peptide macrocyclization step.

17. The screening method for identifying a peptide to be bound to a target substance according to claim 10 , comprising:
a step of bringing the peptide library into contact with the target substance, followed by incubation; and
a step of selecting the peptide bound to the target substance, wherein the mRNA library is represented by the following formula (I):
X 1 —(N 1 N 2 N 3 ) n -X 2   (I)

wherein X 1 and X 2 each represent an mRNA encoding a peptide having an arbitrary number of amino acids; and n stands for an arbitrary integer selected from 4 to 20.

18. The screening method for identifying a peptide to be bound to a target substance according to claim 17 , comprising:
a step of subjecting the peptide-mRNA complex library, wherein each peptide is complexed with an mRNA encoding the peptide, to a reverse transcription reaction to obtain a peptide-DNA complex library;
a step of bringing the peptide-DNA complex library into contact with the target substance, followed by incubation;
a step of selecting a peptide-DNA complex group bound to the target substance;
a step of amplifying DNA of the selected peptide-DNA complex group by PCR; and
a step of transcribing the amplified DNA to produce an mRNA library, binding puromycin to a downstream region of ORF in the mRNA to produce a puromycin-bound mRNA library, and translating it to produce a peptide-mRNA complex library,
wherein the steps from the reverse transcription reaction to the production of the peptide-mRNA complex library is repeated twice or more to select a peptide having high affinity for the target substance.

Assignees

• Univ Tokyo
• Peptidream Inc

Inventors

• Murakami Hiroshi
• Kawakami Takashi
• Reid Patrick
• Sasaki Toru

Classifications

• C12N15/67

  General methods for enhancing the expression · CPC title

• C12N15/1062 Primary

  mRNA-Display, e.g. polypeptide and encoding template are connected covalently · CPC title