Modified fibroin

Patent metadata

Field	Value
Publication number	US-12319718-B2
Application number	US-202017421243-A
Country	US
Kind code	B2
Filing date	Jan 9, 2020
Priority date	Jan 9, 2019
Publication date	Jun 3, 2025
Grant date	Jun 3, 2025

Abstract

The present invention relates to a modified fibroin including a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) n motif, and having a serine residue content rate of less than 5.5%.
[In Formula 1 and Formula 2, the (A) n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues, and the number of alanine residues with respect to the total number of amino acid residues in the (A) n motif is 80% or more. REP represents an amino acid sequence consisting of 10 to 200 amino acid residues. m represents an integer of 10 to 300. The plurality of (A) n motifs may be the same amino acid sequence or different amino acid sequences. A plurality of REPs may be the same amino acid sequence or different amino acid sequences.]

First claim

The invention claimed is:

1. A modified fibroin comprising a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) n motif, wherein a serine residue content rate is less than 5.5%, and wherein:
in Formula 1 and Formula 2, the (A) n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A) n motif is 80% or more, REP represents an amino acid sequence consisting of 10 to 200 amino acid residues, m represents an integer of 10 to 300, the plurality of (A) n motifs may be the same amino acid sequence or different amino acid sequences, and the plurality of REPs may be the same amino acid sequence or different amino acid sequences.

2. The modified fibroin according to claim 1 , wherein a threonine residue content rate is 9% or less.

3. The modified fibroin according to claim 1 , wherein a content rate of serine residue and threonine residue is 9% or less.

4. The modified fibroin according to claim 1 , wherein the modified fibroin comprises: an amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 28; or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, or SEQ ID NO: 28.

5. The modified fibroin according to claim 1 , further comprising a tag sequence at either or both of an N-terminal and a C-terminal.

6. The modified fibroin according to claim 5 , wherein the tag sequence includes an amino acid sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

7. The modified fibroin according to claim 1 , wherein the modified fibroin comprises: an amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 25; or an amino acid sequence having 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 25.

8. A nucleic acid encoding the modified fibroin according to claim 1 .

9. A nucleic acid hybridizing with a complementary strand of the nucleic acid according to claim 8 under hybridization conditions, wherein there is at least 85% or more identity between the sequences, and include at 42° C. using 5×SSC containing 0.5% SDS, and encoding a modified fibroin including a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A)˜motif-REP]m-(A) n motif, and wherein: in Formula 1 and Formula 2, the (A) n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A) n motif is 80% or more, REP represents an amino acid sequence consisting of 10 to 200 amino acid residues, m represents an integer of 10 to 300, the plurality of (A) n motifs may be the same amino acid sequence or different amino acid sequences, and the plurality of REPs may be the same amino acid sequence or different amino acid sequences.

10. A nucleic acid having 90% or more sequence identity with the nucleic acid according to claim 8 and encoding a modified fibroin including a domain sequence represented by Formula 1: [(A) n motif-REP] m or Formula 2: [(A) n motif-REP] m -(A) n motif, and wherein:
in Formula 1 and Formula 2, the (A) n motif represents an amino acid sequence consisting of 4 to 27 amino acid residues and the number of alanine residues with respect to the total number of amino acid residues in the (A) n motif is 80% or more, REP represents an amino acid sequence consisting of 10 to 200 amino acid residues, m represents an integer of 10 to 300, the plurality of (A) n motifs may be the same amino acid sequence or different amino acid sequences, and a plurality of REPs may be the same amino acid sequence or different amino acid sequences.

11. An expression vector comprising: the nucleic acid sequence according to any of one of claims 8-10 , and one or more regulatory sequences operably linked thereto.

12. A host transformed with the expression vector according to claim 11 .

13. The host according to claim 12 , which is a prokaryote.

14. The host according to claim 12 , which is a eukaryote.

15. An artificially modified fibroin composition comprising the modified fibroin according to claim 1 .

16. The artificially modified fibroin composition according to claim 15 , which is a protein powder.

17. The artificially modified fibroin composition according to claim 15 , which is a doping liquid.

18. The artificially modified fibroin composition according to claim 15 , which is a fiber.

19. The artificially modified fibroin composition according to claim 15 , which is a film.

20. A method for producing a modified fibroin, the method comprising
a step in which a host transformed with an expression vector including a nucleic acid sequence encoding a modified fibroin and one or a plurality of regulatory sequences operably linked to the nucleic acid sequence expresses the nucleic acid, wherein
the modified fibroin is the modified fibroin according to claim 1 .

21. A method for producing an artificially modified fibroin composition containing a modified fibroin, the method comprising
a step of preparing a modified fibroin, wherein
the modified fibroin is the modified fibroin according to claim 1 .

22. The production method according to claim 20 , further comprising a step of bringing the modified fibroin into contact with a carboxylic acid.

23. The production method according to claim 21 , further comprising a step of adjusting a modified fibroin solution containing the modified fibroin with a carboxylic acid.

24. A product comprising the modified fibroin according to claim 1 ,
the product being selected from the group consisting of a fiber, a yarn, a film, a foam, a grain, a nanofibril, a gel, and a resin.

Assignees

• Spiber Inc

Inventors

• Morita Keisuke
• ABE Yunosuke
• Nakamura Hiroyuki
• TOGASHI Shota
• Asakura Tetsuo

Classifications

• D01F4/02

  from fibroin · CPC title

• C12P21/02

  having a known sequence of two or more amino acids, e.g. glutathione · CPC title

• C12N15/63

  Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression · CPC title

• D01D5/00

  Formation of filaments, threads, or the like · CPC title

• C12R2001/19

  Escherichia coli · CPC title