Efficient production of peptides

Patent metadata

Field	Value
Publication number	US-2016208304-A1
Application number	US-201615002259-A
Country	US
Kind code	A1
Filing date	Jan 20, 2016
Priority date	Nov 9, 2009
Publication date	Jul 21, 2016
Grant date	—

Abstract

The present invention relates to processes for the production of peptides, and the peptides produced accordingly. Peptides produced according to the invention may be produced more efficiently than peptides produced according to prior art processes. The production process of the invention may lead to advantages in yield, purity, and/or price. Methods of marketing peptides are also disclosed.

First claim

1 .- 22 . (canceled)

23 . A method for producing a target peptide, the method comprising:
expressing a heterologous fusion peptide in a genetically modified cell, the heterologous fusion peptide comprising an affinity tag, a cleavable tag, and the target peptide, wherein the cleavable tag is tryptophan (Trp), and wherein the affinity tag or the cleavable tag is heterologous to the target peptide; binding the heterologous fusion peptide to an affinity material via the affinity tag; and cleaving the heterologous fusion peptide with DMSO while bound to the affinity material to release the target peptide, thereby producing the target peptide.

24 . The method of claim 23 , wherein the target peptide of the heterologous fusion peptide is expressed in native form.

25 . The method of claim 23 , further comprising the step of solubilizing the heterologous fusion peptide after the step of expressing a heterologous fusion peptide.

26 . The method of claim 23 , wherein the target peptide is selected from amyloid beta, calcitonin, enfuvirtide, epoetin, epoetin delta, erythropoietin, exenatide, factor VIII, factor X, glucocerebrosidase, glucagon-like peptide-1 (GLP-1), granulocyte-colony stimulating factor (G-CSF), human growth hormone (hGH), insulin, insulin A, insulin B, insulin-like growth factor 1 (IGF-1), interferon, liraglutide, somatostatin, teriparatide, or tissue plasminogen activator (TPA).

27 . The method of claim 23 , wherein the step of expressing a heterologous fusion peptide in a genetically modified cell is performed in a bacterial expression system.

28 . The method of claim 27 , wherein the bacterial expression system is an Escherichia coli expression system.

29 . The method of claim 23 , wherein the step of expressing a heterologous fusion peptide in a genetically modified cell is performed in a yeast expression system.

30 . The method of claim 23 , wherein the heterologous fusion peptide further comprises an inclusion-body directing peptide.

31 . The method of claim 28 , wherein prior to binding the heterologous fusion peptide to an affinity material, the method further comprises removal of inclusion bodies containing the heterologous fusion peptide from the Escherichia coli expression system and solubilization of the heterologous fusion peptide.

32 . The method of claim 28 , wherein the inclusion-body directing peptide is selected from a ketosteroid isomerase or a BRCA2 peptide.

33 . The method of claim 23 , wherein subsequent to binding the heterologous fusion peptide to the affinity material, the method further comprises washing the affinity material to remove unbound material.

34 . The method of claim 23 , wherein the affinity tag is selected from poly-histidine, poly-lysine, poly-aspartic acid, or poly-glutamic acid.

35 . The method of claim 23 , wherein the target peptide is present in its native form after releasing the target peptide.

36 . The method of claim 23 , wherein the heterologous fusion peptide is secreted from the cell after expressing the heterologous fusion peptide.

37 . The method of claim 23 , further comprising lysing the cell after expressing the heterologous fusion peptide.

38 . The method of claim 23 , wherein the target peptide that is produced is greater than 95% pure.

39 . The method of claim 23 , wherein the target peptide that is produced is greater than 99% pure.

40 . The method of claim 23 , wherein the target peptide is refolded after the target peptide is released.

41 . The method of claim 23 , wherein the rate of cleavage of the cleavable tag is modified by adjusting the concentration of the DMSO.

Assignees

• Univ Colorado Regents
• AmideBio LLC

Inventors

• Stowell Michael H B
• Caruthers Jonathan
• Nemkov Travis
• Hiester Brian
• Boux Leslie
• Plam Mikhail

Classifications

• C07K14/4711

  Alzheimer's disease; Amyloid plaque core protein · CPC title

• C12P21/06 Primary

  produced by the hydrolysis of a peptide bond, e.g. hydrolysate products (preparing foodstuffs by protein hydrolysis A23J3/00) · CPC title

• C07K1/12

  by hydrolysis {, i.e. solvolysis in general} · CPC title

• C07K1/04

  on carriers {(C07K1/003, C07K1/006 take precedence)} · CPC title

• C12Y503/03001

  Steroid DELTA-isomerase (5.3.3.1) · CPC title