Purification of triple helical proteins

Patent metadata

Field	Value
Publication number	US-10053501-B2
Application number	US-201414778845-A
Country	US
Kind code	B2
Filing date	Mar 21, 2014
Priority date	Mar 21, 2013
Publication date	Aug 21, 2018
Grant date	Aug 21, 2018

Abstract

The present invention relates to a method for purifying triple-helical or collagen-like proteins recombinantly produced from a bacterial, yeast or plant host cell.

First claim

The invention claimed is:

1. A method for the purification of a recombinantly expressed triple-helical protein contained within a non-mammalian host cell culture extract or homogenate, the method comprising:
(i) precipitating host cell materials in the host cell culture extract or homogenate from the triple-helical protein under acidic conditions and at a temperature in which the triple-helical protein remains thermally stable; followed by
(ii) digesting host cell materials present in the precipitated host cell culture extract or homogenate by addition of a protease which is functional under said acidic conditions, wherein the triple-helical protein is resistant to the protease; and
(iii) collecting the purified triple-helical protein;

wherein the triple-helical protein remains in-solution throughout at least steps (i) and (ii).

2. The method according to claim 1 , wherein the triple-helical protein remains soluble throughout steps (i) to (iii).

3. The method according to claim 1 , wherein the digestion is carried out using a protease selected from the group consisting of pepsin, papain, or papain-like enzymes selected from bromelain, ficin or actinidin, Aspergillus saitoi acid protease, trypsin or chymotrypsin.

4. The method according to claim 1 , wherein the host cell is a bacterial, yeast or plant host cell.

5. The method according to claim 1 , wherein acid conditions refers to a pH less than 7.

6. The method according claim 1 , wherein the precipitation step is conducted at a temperature that is less than the melting temperature of the triple-helical protein.

7. The method according to claim 1 , further comprising an additional separation step between the precipitating step and the digesting step of physically separating the triple-helical protein from precipitated host cell materials.

8. The method according to claim 7 , wherein the intermediary separation step is selected from one or more of centrifugation, filtration, cross flow filtration, or sedimentation.

9. The method according to claim 1 , wherein the expressed triple-helical protein is produced intracellularly within the host cell.

10. The method according to claim 1 , wherein the expressed triple-helical protein is secreted from the host cell.

11. The method according to claim 1 , comprising an additional step prior to step (i) of producing the host cell culture extract or homogenate which contains the triple-helical protein.

12. The method according to claim 1 , wherein the method is carried out at a temperature which is the melting temperature (Tm) of the recombinant triple-helical protein.

13. The method according to claim 1 , wherein the temperature is at least 10° C. or more below the Tm of the recombinant triple-helical protein.

14. The method according to claim 5 , wherein the pH is between 2 and 4 and the host cell is a bacterial host cell.

15. The method according to claim 5 , wherein the pH is between 4 and 6 and the host cell is a yeast host cell.

16. The method according to claim 5 , wherein the pH is between 2 and 4.5 and the host cell is a plant host cell.

17. The method according to claim 3 , wherein the triple-helical protein is proteolytically stable.

18. The method according to claim 3 , wherein the method selectively purifies proteolytically stable protein over proteolytically unstable protein.

19. The method according to claim 1 , wherein host cell nucleic acid is removed from the collected triple-helical protein.

20. The method according to claim 1 , wherein collecting the purified triple helical protein is performed by precipitation or diafiltration.

21. The method according to claim 1 , wherein the collected triple-helical protein is stabilised by a stabilising agent.

22. The method according to claim 1 wherein the triple-helical protein comprises a repeating (Gly-X-Y)n motif, where n is between 5 and 600.

23. The method according to claim 1 , wherein the triple-helical protein is collagen.

24. The method according to claim 1 , wherein the triple-helical protein sequence is derived from a bacteria, yeast, plant, insect, or silkworm.

25. The method according to claim 20 , wherein precipitation of the collected protein is achieved by addition of ammonium sulphate, by adjustment of pH or adjustment of temperature, and/or by use of polyethylene glycol.

Assignees

• Commonwealth Scient And Industrial Research Organization
• Commw Scient Ind Res Org

Inventors

• Ramshaw John Alan Maurice
• Werkmeister Jerome Anthony
• Peng Yong Yi

Classifications

• C07K1/145 Primary

  by extraction or solubilisation · CPC title

• C07K14/195

  from bacteria · CPC title

• C07K14/315

  from Streptococcus (G), e.g. Enterococci · CPC title

• C07K14/78 Primary

  Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG] · CPC title

• C07K1/30

  by precipitation · CPC title