Methods for isolating spider silk proteins via high shear solubilization

Patent metadata

Field	Value
Publication number	US-2022372086-A1
Application number	US-202017636721-A
Country	US
Kind code	A1
Filing date	Sep 16, 2020
Priority date	Sep 16, 2019
Publication date	Nov 24, 2022
Grant date	—

Abstract

The present disclosure relates to methods of isolating and purifying synthetic block copolymer proteins via the application of solvents and high shear microfluidization methods.

First claim

1 . A method of isolating a recombinant spider silk protein from a host cell, comprising:
a. providing an insoluble mass comprising a recombinant spider silk protein; b. adding the insoluble mass to an aqueous solution comprising a solvent; and c. applying a shear force to the aqueous solution comprising the insoluble mass thereby solubilizing the recombinant spider silk protein in the aqueous solution.

2 . The method of claim 1 , wherein the shear force is applied via microfluidization.

3 . The method of claim 2 , wherein the microfluidization generates shear rates of about 6×10 6 s −1 to 10×10 6 s −1 .

4 .- 5 . (canceled)

6 . The method of claim 2 , wherein the microfluidization is performed at between 20,000 psi to 30,000 psi.

7 .- 8 . (canceled)

9 . The method of claim 2 , wherein the microfluidizer an M-110P or an LM10 microfluidizer.

10 .- 11 . (canceled)

12 . The method of claim 1 , wherein the shear force is applied at least twice.

13 .- 15 . (canceled)

16 . The method of claim 1 , wherein the insoluble mass is derived from a cell culture comprising a host cell, wherein the host cell expresses the recombinant spider silk protein, and the method further comprises collecting the insoluble mass derived from the cell culture.

17 . (canceled)

18 . The method of claim 1 , wherein the solvent is a chaotropic agent.

19 . The method of claim 18 , wherein the chaotropic agent is urea, guanidine thiocyanate (GdnSCN), or guanidine chloride (GdnHCL).

20 . The method of claim 1 , wherein the insoluble mass is added to the aqueous solution at about a 5%, 10%, 15%, 20%, 25%, or 30% insoluble mass to solvent volume.

21 . The method of claim 19 , wherein the chaotropic agent is present in the aqueous solution at a concentration from 0.1-10M.

22 . The method of claim 19 , wherein the aqueous solution comprises about 10M urea, about 4M-8M GdnHCl, or about 3M-6M GdnSCN.

23 .- 24 . (canceled)

25 . The method of claim 20 , wherein the aqueous solution comprises about a 15% insoluble portion mass to an 85% volume of 3M GdnSCN.

26 . The method of claim 20 , wherein the aqueous solution comprises about a 15% insoluble portion mass to an 85% volume of 4M GdnHCI.

27 . The method of claim 20 , wherein the aqueous solution comprises about a 15% insoluble portion mass to an 85% volume of 10M urea.

28 . The method of claim 1 , wherein the insoluble mass is incubated between 20° C. and 30° C.

29 .- 30 . (canceled)

31 . The method of claim 1 , wherein the insoluble portion is incubated in the aqueous solution comprising the solvent for 60 to 120 minutes.

32 .- 35 . (canceled)

36 . The method of claim 1 , wherein the insoluble mass comprises a cell pellet, and method further comprises:
a. incubating the cell pellet with a solution comprising 4M urea at a 10:1 urea volume to pellet mass ratio; and b. centrifuging the solution comprising 4M urea to obtain a second cell pellet prior to incubating the second cell pellet in the aqueous solution comprising a solvent.

37 . The method of claim 1 , further comprising isolating the recombinant spider silk protein from the aqueous solution, thereby producing an isolated recombinant spider silk protein.

38 . The method of claim 1 , wherein the recombinant spider silk protein is a highly crystalline silk protein, a high beta sheet content silk protein, or a low solubility silk protein.

39 . The method of claim 38 , wherein the recombinant spider silk protein comprises the Uloborus diversus MiSP protein as set forth in SEQ ID NO: 23.

40 . The method of claim 38 , wherein the recombinant spider silk protein has a solubility threshold of less than 90%, 80%, 70%, 60%, or 50% in a non-chaotropic solvent.

41 . (canceled)

42 . The method of claim 1 , wherein the cell is Escherichia coli.

43 .- 44 . (canceled)

45 . The method of claim 1 , wherein the isolated recombinant spider silk protein is full-length recombinant spider silk protein.

46 .- 48 . (canceled)

49 . The method of claim 1 , wherein the purity of the isolated recombinant spider silk protein is 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 09-95%, or 95-100%.

50 . A method of isolating a recombinant spider silk protein from a host cell, comprising:
a. providing an insoluble mass comprising a recombinant spider silk protein; b. adding the insoluble mass to an aqueous solution comprising a solvent, wherein the aqueous solution comprises 15% (w/v) insoluble portion in a final 10M urea concentration; c. applying a shear force via microfluidization to the aqueous solution comprising the insoluble mass thereby solubilizing the recombinant spider silk protein in the aqueous solution; and d. isolating the recombinant spider silk protein from the aqueous solution, thereby producing an isolated recombinant spider silk protein.

51 . A method of isolating a recombinant spider silk protein from a host cell, comprising:
a. providing an insoluble mass comprising a recombinant spider silk protein; b. adding the insoluble mass to an aqueous solution, wherein the aqueous solution comprises about a 15% (w/v) insoluble portion in a final 10M urea concentration; c. applying a shear force via microfluidization, wherein the shear force is about 10×10 6 s −1 , to the aqueous solution thereby solubilizing the recombinant spider silk protein in the aqueous solution; and d. isolating the recombinant spider silk protein from the aqueous solution, thereby producing an isolated recombinant spider silk protein.

52 . A composition comprising a recombinant spider silk protein produced by the method of claim 1 .

53 . (canceled)

54 . A silk fiber comprising a recombinant spider silk protein produced by the method of claim 1 .

Assignees

• Bolt Threads Inc

Inventors

• Wheatley Robert William

Classifications

• D10B2211/04

  Silk · CPC title

• C07K14/43518 Primary

  from spiders · CPC title

• D01F4/00

  Monocomponent artificial filaments or the like of proteins; Manufacture thereof · CPC title

• A61L27/36

  containing ingredients of undetermined constitution or reaction products thereof {, e.g. transplant tissue, natural bone, extracellular matrix (isolated natural compounds, e.g. collagen A61L27/24)} · CPC title

• C07K2319/21

  containing a His-tag · CPC title