Abstract: A technique for efficiently culturing cells such as stem cells is provided. Cells such as stem cells are cultured using a cell culture vessel coated with a fibroin-like protein inserted with a cell adhesion sequence containing RGD (Arg-Gly-Asp). [Figure 1]
1. A protein comprising a repetitive structure and a cell adhesion sequence, wherein the repetitive structure consists of a 3 to 15 times repeat of a repeating unit, wherein each repeating unit consists of an Ala-rich region and an Ala-non-rich region mutually linked, wherein each Ala-rich region consists of an amino acid sequence having a length of 8 to 54 residues, wherein the ratio of Ala residue in each Ala-rich region is 30% or more, wherein each Ala-rich region contains at least one Ala residue in any continuous four amino acid residues, wherein each Ala-non-rich region consists of an amino acid sequence having a length of 4 residues or longer, wherein the ratio of Ala residue in each Ala-non-rich region is 20% or less, wherein the cell adhesion sequence is contained in one or more repeating units selected from repeating units constituting the repetitive structure, and wherein each cell adhesion sequence consists of an amino acid sequence containing Arg-Gly-Asp and having a length of 3 to 18 residues.
2. The protein according to claim 1, wherein the cell adhesion sequence is contained in two or more repeating units selected from repeating units constituting the repetitive structure.
3. The protein according to claim 1 or 2, wherein the total number of the cell adhesion sequence contained in the protein is 1 to 50.
4. The protein according to any one of claims 1 to 3, wherein each cell adhesion sequence has been inserted in the Ala-non-rich region, or in the linkage site between the Ala-rich region and the Ala-non-rich region
5. The protein according to any one of claims 1 to 4, wherein each cell adhesion sequence consists of an amino acid sequence of (a), (b), or (c) shown below: (a) the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 30, or 31; (b) the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 30, or 31, including substitution, deletion, insertion, and/or addition of 1 to 3 amino acid residues at position(s) other than Arg-Gly-Asp; (c) an amino acid sequence showing an identity of 80% or higher to the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 30, or 31, provided that Arg-Gly-Asp is conserved.
6. The protein according to any one of claims 1 to 5, wherein each Ala-rich region consists of an amino acid sequence having a length of 8 to 15 residues.
7. The protein according to any one of claims 1 to 6, wherein each Ala-rich region consists of an amino acid sequence having a length of 9 residues.
8. The protein according to any one of claims 1 to 7, wherein each Ala-rich region contains one or more Gly residues and one or more Ser residues.
9. The protein according to any one of claims 1 to 8, wherein the ratio of Ala residue in each Ala-rich region is 50% or more.
10. The protein according to any one of claims 1 to 9, wherein the ratio of Ala, Gly, and Ser residues in each Ala-rich region is 90% or more in total.
11. The protein according to any one of claims 1 to 10, wherein each Ala-rich region consists of an amino acid sequence of (a), (b), or (c) shown below: (a) the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10; (b) the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10, including substitution, deletion, insertion, and/or addition of 1 to 3 amino acid residues; (c) an amino acid sequence showing an identity of 80% or higher to the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10.
12. The protein according to any one of claims 1 to 11, wherein each Ala-non-rich region consists of an amino acid sequence having a length of 15 to 100 residues.
13. The protein according to any one of claims 1 to 12, wherein the ratio of Ala residue in each Ala-non-rich region is 5% or less.
14. The protein according to any one of claims 1 to 13, wherein the ratio of Gly, Ser, Gln, Pro, and Tyr residues in each Ala-non-rich region is 90% or more in total.
15. The protein according to any one of claims 1 to 14, wherein each Ala-non-rich region comprises one or more kinds of amino acid sequences selected from amino acid sequences of (a), (b), and (c) shown below: (a) the amino acid sequences of SEQ ID NOS: 11 to 21; (b) the amino acid sequences of SEQ ID NOS: 11 to 21, including substitution, deletion, insertion, and/or addition of 1 to 3 amino acid residues; (c) amino acid sequences showing an identity of 80% or higher to the amino acid sequences of SEQ ID NOS: 11 to 21.
16. The protein according to any one of claims 1 to 15, wherein each Ala-non-rich region comprises an amino acid sequence of (a), (b), or (c) shown below: (a) the amino acid sequence of amino acid numbers of 62 to 91, 101 to 120, 130 to 151, 161 to 185, 195 to 218, 228 to 257, 267 to 291, 301 to 325, 335 to 369, 379 to 438, 448 to 497, or 507 to 536 of SEQ ID NO: 2, or a partial sequence thereof; (b) the amino acid sequence of amino acid numbers of 62 to 91, 101 to 120, 130 to 151, 161 to 185, 195 to 218, 228 to 257, 267 to 291, 301 to 325, 335 to 369, 379 to 438, 448 to 497, or 507 to 536 of SEQ ID NO: 2, or a partial sequence thereof, including substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues; (c) an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of amino acid numbers of 62 to 91, 101 to 120, 130 to 151, 161 to 185, 195 to 218, 228 to 257, 267 to 291, 301 to 325, 335 to 369, 379 to 438, 448 to 497, or 507 to 536 of SEQ ID NO: 2, or a partial sequence thereof.
17. A composition containing the protein according to any one of claims 1 to 16.
18. The composition according to claim 17, wherein the composition is a cell scaffold material.
19. The composition according to claim 18, wherein the cell scaffold material is for culturing a stem cell.
20. The composition according to claim 19, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
21. The composition according to claim 17, wherein the composition is a coating agent for a cell culture vessel.
22. The composition according to claim 21, wherein the cell culture vessel is for culturing a stem cell.
23. The composition according to claim 22, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
24. The composition according to any one of claims 17 to 23, wherein the composition further contains another protein, which is a protein other than the proteins according to claims 1 to 16.
25. The composition according to claim 24, wherein the other protein is any one of proteins (1) to (8) shown below: (1) a protein that stabilizes a component in a culture medium; (2) a protein comprising an amino acid sequence that stabilizes a component in a culture medium; (3) a protein having a cell adhesion ability; (4) a protein comprising an amino acid sequence having a cell adhesion ability; (5) a protein that promotes proliferation of a cell; (6) a protein comprising an amino acid sequence that promotes proliferation of a cell; (7) a protein that binds to any one of the proteins (1) to (6); (8) a protein comprising an amino acid sequence that binds to any one of the proteins (1) to (6).
26. An apparatus comprising the protein according to any one of claims 1 to 16.
27. The apparatus according to claim 26, wherein the apparatus is an apparatus for medical or research use.
28. The apparatus according to claim 26 or 27, wherein the apparatus is a cell culture vessel.
29. The apparatus according to claim 26 or 27, wherein the apparatus is a cell scaffold material.
30. The apparatus according to claim 28, wherein a cell culture surface of the apparatus has been coated with the protein.
31. The apparatus according to any one of claims 26 to 30, wherein the apparatus is for culturing a stem cell.
32. The apparatus according to claim 31, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
33. The apparatus according to any one of claims 26 to 32, wherein the apparatus further comprises another protein, which is a protein other than the proteins according to of claims 1 to 16.
34. The apparatus according to claim 33, wherein the other protein is any one of proteins (1) to (8) shown below: (1) a protein that stabilizes a component in a culture medium; (2) a protein comprising an amino acid sequence that stabilizes a component in a culture medium; (3) a protein having a cell adhesion ability; (4) a protein comprising an amino acid sequence having a cell adhesion ability; (5) a protein that promotes proliferation of a cell; (6) a protein comprising an amino acid sequence that promotes proliferation of a cell; (7) a protein that binds to any one of the proteins (1) to (6); (8) a protein comprising an amino acid sequence that binds to any one of the proteins (1) to (6).
35. A method for producing a cultured cell, the method comprising: culturing a cell by using the apparatus according to any one of claims 26 to 34.
36. The method according to claim 35, wherein the cell to be cultured is a stem cell.
37. The method according to claim 36, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
38. The method according to any one of claims 35 to 37, wherein another protein, which is a protein other than the proteins according to claims 1 to 16, is further used in combination.
39. The method according to claim 38, wherein the other protein is any one of proteins (1) to (8) shown below: (1) a protein that stabilizes a component in a culture medium; (2) a protein comprising an amino acid sequence that stabilizes a component in a culture medium; (3) a protein having a cell adhesion ability; (4) a protein comprising an amino acid sequence having a cell adhesion ability; (5) a protein that promotes proliferation of a cell; (6) a protein comprising an amino acid sequence that promotes proliferation of a cell; (7) a protein that binds to any one of the proteins (1) to (6); (8) a protein comprising an amino acid sequence that binds to any one of the proteins (1) to (6).
40. A gene encoding the protein according to any one of claims 1 to 16.
41. A vector having the gene according to claim 40.
42. A host having the gene according to claim 40.
WE CLAIM:
1. A protein comprising a repetitive structure and a cell adhesion sequence,
wherein the repetitive structure consists of a 3 to 15 times repeat of a repeating
unit,
wherein each repeating unit consists of an Ala-rich region and an Ala-non-rich region mutually linked,
wherein each Ala-rich region consists of an amino acid sequence having a length of 8 to 54 residues,
wherein the ratio of Ala residue in each Ala-rich region is 30% or more,
wherein each Ala-rich region contains at least one Ala residue in any continuous four amino acid residues,
wherein each Ala-non-rich region consists of an amino acid sequence having a length of 4 residues or longer,
wherein the ratio of Ala residue in each Ala-non-rich region is 20% or less,
wherein the cell adhesion sequence is contained in one or more repeating units selected from repeating units constituting the repetitive structure, and
wherein each cell adhesion sequence consists of an amino acid sequence containing Arg-Gly-Asp and having a length of 3 to 18 residues.
2. The protein according to claim 1, wherein the cell adhesion sequence is contained in two or more repeating units selected from repeating units constituting the repetitive structure.
3. The protein according to claim 1 or 2, wherein the total number of the cell adhesion sequence contained in the protein is 1 to 50.
4. The protein according to any one of claims 1 to 3, wherein each cell adhesion sequence has been inserted in the Ala-non-rich region, or in the linkage site between the Ala-rich region and the Ala-non-rich region
5. The protein according to any one of claims 1 to 4, wherein each cell adhesion sequence consists of an amino acid sequence of (a), (b), or (c) shown below:
(a) the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 30, or 31;
(b) the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 30, or 31, including substitution, deletion, insertion, and/or addition of 1 to 3 amino acid residues at position(s) other than Arg-Gly-Asp;
(c) an amino acid sequence showing an identity of 80% or higher to the amino acid sequence of SEQ ID NO: 22, 23, 24, 25, 30, or 31, provided that Arg-Gly-Asp is conserved.
6. The protein according to any one of claims 1 to 5, wherein each Ala-rich region consists of an amino acid sequence having a length of 8 to 15 residues.
7. The protein according to any one of claims 1 to 6, wherein each Ala-rich region consists of an amino acid sequence having a length of 9 residues.
8. The protein according to any one of claims 1 to 7, wherein each Ala-rich region contains one or more Gly residues and one or more Ser residues.
9. The protein according to any one of claims 1 to 8, wherein the ratio of Ala residue in each Ala-rich region is 50% or more.
10. The protein according to any one of claims 1 to 9, wherein the ratio of Ala, Gly, and Ser residues in each Ala-rich region is 90% or more in total.
11. The protein according to any one of claims 1 to 10, wherein each Ala-rich region consists of an amino acid sequence of (a), (b), or (c) shown below:
(a) the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10;
(b) the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10, including substitution, deletion, insertion, and/or addition of 1 to 3 amino acid residues;
(c) an amino acid sequence showing an identity of 80% or higher to the amino acid sequence of SEQ ID NO: 7, 8, 9, or 10.
12. The protein according to any one of claims 1 to 11, wherein each Ala-non-rich
region consists of an amino acid sequence having a length of 15 to 100 residues.
13. The protein according to any one of claims 1 to 12, wherein the ratio of Ala residue in each Ala-non-rich region is 5% or less.
14. The protein according to any one of claims 1 to 13, wherein the ratio of Gly, Ser, Gln, Pro, and Tyr residues in each Ala-non-rich region is 90% or more in total.
15. The protein according to any one of claims 1 to 14, wherein each Ala-non-rich region comprises one or more kinds of amino acid sequences selected from amino acid sequences of (a), (b), and (c) shown below:
(a) the amino acid sequences of SEQ ID NOS: 11 to 21;
(b) the amino acid sequences of SEQ ID NOS: 11 to 21, including substitution, deletion, insertion, and/or addition of 1 to 3 amino acid residues;
(c) amino acid sequences showing an identity of 80% or higher to the amino acid sequences of SEQ ID NOS: 11 to 21.
16. The protein according to any one of claims 1 to 15, wherein each Ala-non-rich
region comprises an amino acid sequence of (a), (b), or (c) shown below:
(a) the amino acid sequence of amino acid numbers of 62 to 91, 101 to 120, 130 to 151, 161 to 185, 195 to 218, 228 to 257, 267 to 291, 301 to 325, 335 to 369, 379 to 438, 448 to 497, or 507 to 536 of SEQ ID NO: 2, or a partial sequence thereof;
(b) the amino acid sequence of amino acid numbers of 62 to 91, 101 to 120, 130 to 151, 161 to 185, 195 to 218, 228 to 257, 267 to 291, 301 to 325, 335 to 369, 379 to 438, 448 to 497, or 507 to 536 of SEQ ID NO: 2, or a partial sequence thereof, including substitution, deletion, insertion, and/or addition of 1 to 10 amino acid residues;
(c) an amino acid sequence showing an identity of 90% or higher to the amino acid sequence of amino acid numbers of 62 to 91, 101 to 120, 130 to 151, 161 to 185, 195 to 218, 228 to 257, 267 to 291, 301 to 325, 335 to 369, 379 to 438, 448 to 497, or 507 to 536 of SEQ ID NO: 2, or a partial sequence thereof.
17. A composition containing the protein according to any one of claims 1 to 16.
18. The composition according to claim 17, wherein the composition is a cell
scaffold material.
19. The composition according to claim 18, wherein the cell scaffold material is for culturing a stem cell.
20. The composition according to claim 19, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
21. The composition according to claim 17, wherein the composition is a coating agent for a cell culture vessel.
22. The composition according to claim 21, wherein the cell culture vessel is for culturing a stem cell.
23. The composition according to claim 22, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
24. The composition according to any one of claims 17 to 23, wherein the composition further contains another protein, which is a protein other than the proteins according to claims 1 to 16.
25. The composition according to claim 24, wherein the other protein is any one of proteins (1) to (8) shown below:
(1) a protein that stabilizes a component in a culture medium;
(2) a protein comprising an amino acid sequence that stabilizes a component in a culture medium;
(3) a protein having a cell adhesion ability;
(4) a protein comprising an amino acid sequence having a cell adhesion ability;
(5) a protein that promotes proliferation of a cell;
(6) a protein comprising an amino acid sequence that promotes proliferation of a cell;
(7) a protein that binds to any one of the proteins (1) to (6);
(8) a protein comprising an amino acid sequence that binds to any one of the proteins (1) to (6).
26. An apparatus comprising the protein according to any one of claims 1 to 16.
27. The apparatus according to claim 26, wherein the apparatus is an apparatus for medical or research use.
28. The apparatus according to claim 26 or 27, wherein the apparatus is a cell culture vessel.
29. The apparatus according to claim 26 or 27, wherein the apparatus is a cell scaffold material.
30. The apparatus according to claim 28, wherein a cell culture surface of the apparatus has been coated with the protein.
31. The apparatus according to any one of claims 26 to 30, wherein the apparatus is for culturing a stem cell.
32. The apparatus according to claim 31, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
33. The apparatus according to any one of claims 26 to 32, wherein the apparatus further comprises another protein, which is a protein other than the proteins according to of claims 1 to 16.
34. The apparatus according to claim 33, wherein the other protein is any one of proteins (1) to (8) shown below:
(1) a protein that stabilizes a component in a culture medium;
(2) a protein comprising an amino acid sequence that stabilizes a component in a culture medium;
(3) a protein having a cell adhesion ability;
(4) a protein comprising an amino acid sequence having a cell adhesion ability;
(5) a protein that promotes proliferation of a cell;
(6) a protein comprising an amino acid sequence that promotes proliferation of a cell;
(7) a protein that binds to any one of the proteins (1) to (6);
(8) a protein comprising an amino acid sequence that binds to any one of the proteins (1) to (6).
35. A method for producing a cultured cell, the method comprising:
culturing a cell by using the apparatus according to any one of claims 26 to 34.
36. The method according to claim 35, wherein the cell to be cultured is a stem cell.
37. The method according to claim 36, wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell.
38. The method according to any one of claims 35 to 37, wherein another protein, which is a protein other than the proteins according to claims 1 to 16, is further used in combination.
39. The method according to claim 38, wherein the other protein is any one of proteins (1) to (8) shown below:
(1) a protein that stabilizes a component in a culture medium;
(2) a protein comprising an amino acid sequence that stabilizes a component in a culture medium;
(3) a protein having a cell adhesion ability;
(4) a protein comprising an amino acid sequence having a cell adhesion ability;
(5) a protein that promotes proliferation of a cell;
(6) a protein comprising an amino acid sequence that promotes proliferation of a cell;
(7) a protein that binds to any one of the proteins (1) to (6);
(8) a protein comprising an amino acid sequence that binds to any one of the proteins (1) to (6).
40. A gene encoding the protein according to any one of claims 1 to 16.
41. A vector having the gene according to claim 40.
42. A host having the gene according to claim 40.
| # | Name | Date |
|---|---|---|
| 1 | 201847039183.pdf | 2018-10-16 |
| 2 | 201847039183-TRANSLATIOIN OF PRIOIRTY DOCUMENTS ETC. [16-10-2018(online)].pdf | 2018-10-16 |
| 3 | 201847039183-STATEMENT OF UNDERTAKING (FORM 3) [16-10-2018(online)].pdf | 2018-10-16 |
| 4 | 201847039183-SEQUENCE LISTING(PDF) [16-10-2018(online)].pdf | 2018-10-16 |
| 5 | 201847039183-SEQUENCE LISTING [16-10-2018(online)].txt | 2018-10-16 |
| 6 | 201847039183-PROOF OF RIGHT [16-10-2018(online)].pdf | 2018-10-16 |
| 7 | 201847039183-PRIORITY DOCUMENTS [16-10-2018(online)].pdf | 2018-10-16 |
| 8 | 201847039183-POWER OF AUTHORITY [16-10-2018(online)].pdf | 2018-10-16 |
| 9 | 201847039183-FORM 1 [16-10-2018(online)].pdf | 2018-10-16 |
| 10 | 201847039183-DRAWINGS [16-10-2018(online)].pdf | 2018-10-16 |
| 11 | 201847039183-DECLARATION OF INVENTORSHIP (FORM 5) [16-10-2018(online)].pdf | 2018-10-16 |
| 12 | 201847039183-COMPLETE SPECIFICATION [16-10-2018(online)].pdf | 2018-10-16 |
| 13 | 201847039183-CLAIMS UNDER RULE 1 (PROVISIO) OF RULE 20 [16-10-2018(online)].pdf | 2018-10-16 |
| 14 | abstract 201847039183.jpg | 2018-10-17 |
| 15 | Correspondence by Agent_Assignment_22-10-2018.pdf | 2018-10-22 |
| 16 | 201847039183-FORM 3 [04-04-2019(online)].pdf | 2019-04-04 |