Elastomeric proteins

Patent metadata

Field	Value
Publication number	US-10988515-B2
Application number	US-201816477133-A
Country	US
Kind code	B2
Filing date	Jan 16, 2018
Priority date	Jan 13, 2017
Publication date	Apr 27, 2021
Grant date	Apr 27, 2021

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 host cells in a fermentation, wherein said recombinant host cells comprise a vector comprising a secreted resilin coding sequence, and wherein said recombinant host cells secrete a recombinant resilin protein encoded by said secreted resilin coding sequence, and wherein said recombinant host cells are yeast cells; 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 SEQ ID NO: 1 or SEQ ID NO: 4.

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

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

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

7. The method of claim 1 , wherein the yeast cells are methylotrophic yeast cells.

8. The method of claim 1 , wherein the recombinant host cells produce the recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.

9. The method of claim 1 , wherein the recombinant 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 host cells.

10. The method of claim 1 , wherein the recombinant host cells secrete the recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.

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

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

13. 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.

14. The method of claim 13 , 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 recombinant resilin protein from the second supernatant fraction.

15. A vector comprising more than one secreted resilin coding sequence.

16. A recombinant host cell comprising the vector of claim 15 .

17. A fermentation comprising the recombinant host cell of claim 16 and a culture medium suitable for growing the recombinant host cell.

18. A composition comprising recombinant resilin derived from a fermentation of claim 17 .

19. The vector of claim 15 , wherein said secreted resilin coding sequence encodes a recombinant resilin protein comprising the polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 4.

20. The vector of claim 15 , wherein said secreted resilin coding sequence encodes a recombinant resilin protein comprising an alpha mating factor secretion signal or a FLAG-tag.

21. The vector of claim 15 , wherein the vector comprises 3 secreted resilin coding sequences.

22. The recombinant host cell of claim 16 , wherein the recombinant host cell is a yeast cell.

23. The recombinant host cell of claim 22 , wherein the yeast cell is a methylotrophic yeast cell.

24. The recombinant host cell of claim 23 , wherein the recombinant host cell is a species selected from the group consisting of: Pichia ( Komagataella ) pastoris, Hansenula polymorpha, Arxula adeninivorans, Yarrowia lipolytica, Pichia ( Scheffersomyces ) stipitis, Pichia methanolica, Saccharomyces cerevisiae , and Kluyveromyces lactis.

25. The fermentation of claim 17 , wherein the recombinant host cell produces recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.

26. The fermentation of claim 17 , wherein greater than 80% of the recombinant resilin is outside of the recombinant host cells in said fermentation.

27. The fermentation of claim 17 , wherein the fermentation comprises at least 2 g recombinant resilin/L.

28. A method for producing a composition comprising a recombinant resilin protein, comprising:
culturing a population of recombinant host cells in a fermentation, wherein said recombinant host cells comprise a vector comprising a secreted resilin coding sequence, wherein said recombinant host cells secrete a recombinant resilin protein encoded by said secreted resilin coding sequence, and wherein said recombinant host cells produce the recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour; and
purifying said recombinant resilin protein from said fermentation.

29. The method of claim 28 , wherein said recombinant resilin protein is a full-length or truncated native resilin.

30. The method of claim 28 , wherein the recombinant resilin protein comprises SEQ ID NO: 1 or SEQ ID NO: 4.

31. The method of claim 28 , wherein the recombinant resilin protein comprises an alpha mating factor secretion signal.

32. The method of claim 28 , wherein the recombinant resilin protein comprises a FLAG-tag.

33. The method of claim 28 , wherein the vector comprises more than one secreted resilin coding sequence.

34. The method of claim 28 , wherein the recombinant host cells are yeast cells.

35. The method of claim 34 , wherein the yeast cells are methylotrophic yeast cells.

36. The method of claim 28 , wherein the recombinant 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 host cells.

37. The method of claim 28 , wherein the recombinant host cells secrete the recombinant resilin at a rate of greater than 2 mg resilin/g dry cell weight/hour.

38. The method of claim 28 , wherein greater than 80% of the recombinant resilin is outside of the recombinant host cells in said fermentation.

39. The method of claim 28 , 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.

40. The method of claim 39 , 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 recombinant resilin protein from the second supernatant fraction.

41. A method for producing a composition comprising a recombinant resilin protein, comprising:
culturing a popul

Assignees

• Bolt Threads Inc

Inventors

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

Classifications

• C12P21/02 Primary

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

• C07K14/4359

  from fleas · CPC title

• C07K14/43563 Primary

  from insects · CPC title

• C07K14/43577

  from flies · CPC title

• C07K14/43581

  from Drosophila · CPC title