Elastomeric proteins

Patent metadata

Field	Value
Publication number	US-11858971-B2
Application number	US-202117219277-A
Country	US
Kind code	B2
Filing date	Mar 31, 2021
Priority date	Jan 13, 2017
Publication date	Jan 2, 2024
Grant date	Jan 2, 2024

Abstract

This invention relates to elastomeric protein and elastomeric protein production. In particular, the invention is directed to elastomeric protein sequences, including methods and compositions for production of elastomeric protein sequences, such as expression constructs, and host cells, and including compositions generated from the elastomeric protein sequences.

First claim

What is claimed is:

1. A method for producing a composition comprising a recombinant resilin protein, comprising:
culturing a population of recombinant yeast host cells in a fermentation, wherein said recombinant yeast host cells comprise a vector comprising a secreted resilin coding sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 7, 8, 9, 10, 11, and 12, and wherein said recombinant yeast host cells secrete a recombinant resilin protein encoded by said secreted resilin coding sequence; and
purifying said recombinant resilin protein from said fermentation.

2. The method of claim 1 , wherein said recombinant resilin protein is a full-length or truncated native resilin.

3. The method of claim 1 , wherein the recombinant resilin protein comprises an alpha mating factor secretion signal.

4. The method of claim 1 , wherein the recombinant resilin protein comprises a FLAG-tag.

5. The method of claim 1 , wherein the vector comprises more than one secreted resilin coding sequence.

6. The method of claim 1 , wherein the recombinant yeast host cells are methylotrophic yeast cells.

7. The method of claim 1 , wherein the recombinant yeast host cells produce a secreted fraction of the recombinant resilin that is greater than 50% as compared to the total recombinant resilin protein expressed by the recombinant yeast host cells.

8. The method of claim 1 , wherein greater than 80% of the recombinant resilin is outside of the recombinant host cells in said fermentation.

9. The method of claim 1 , wherein the fermentation comprises at least 2 g recombinant resilin/L.

10. The method of claim 1 , wherein purifying said recombinant resilin protein comprises
generating a first pellet fraction and a first supernatant fraction by centrifuging the fermentation; and
isolating the recombinant resilin protein from the first pellet fraction.

11. The method of claim 10 , wherein purifying said recombinant resilin protein further comprises:
adding a chaotrope to the first pellet fraction to generate a solution in which the recombinant resilin protein is soluble;
generating a second supernatant fraction and a second pellet fraction by centrifuging the first pellet fraction comprising said chaotrope; and
isolating the soluble full-length resilin from the second supernatant fraction.

12. A vector comprising a secreted resilin coding sequence selected from the group consisting of SEQ ID NOs: 2, 3, 5, 6, 7, 8, 9, 10, 11, and 12.

13. A recombinant host cell comprising the vector of claim 12 .

14. A fermentation comprising the recombinant host cell of claim 13 and a culture medium suitable for growing the recombinant host cell.

15. A composition comprising recombinant resilin derived from a fermentation of claim 14 .

16. A composition comprising a gel comprising a recombinant resilin selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12.

17. The composition of claim 16 , wherein the recombinant resilin comprises the sequence set forth in SEQ ID NO: 1.

18. The composition of claim 16 , wherein the recombinant resilin comprises the sequence set forth in SEQ ID NO: 4.

19. The composition of claim 16 , wherein the gel comprises full-length resilin.

20. The composition of claim 16 , wherein the gel comprises a plurality of cross-linked recombinant resilins.

21. The composition of claim 20 , wherein the cross-linking comprises enzymatic cross-linking, photochemical cross-linking, or chemical cross-linking.

Assignees

• Bolt Threads Inc

Inventors

• Kittleson Joshua
• Lee Michael
• Breslauer David N
• Widmaier Daniel M

Classifications

• C07K14/43563 Primary

  from insects · CPC title

• C07K14/4359

  from fleas · CPC title

• C07K14/43577

  from flies · CPC title

• C07K14/43581

  from Drosophila · CPC title

• C12P21/02 Primary

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