Method for the purification of biological macromolecular complexes

Patent metadata

Field	Value
Publication number	US-10829515-B2
Application number	US-201716307200-A
Country	US
Kind code	B2
Filing date	Jun 6, 2017
Priority date	Jun 6, 2016
Publication date	Nov 10, 2020
Grant date	Nov 10, 2020

Abstract

The present invention relates in a first aspect to a method for the purification of biological macromolecular complexes. Typically, no chromatography steps are applied. That is, the present invention relates to a method for the purification of biological macromolecular complexes Furthermore, the present invention relates to a method for crystallization of biological macromolecular complexes comprising the step of purification as described followed by crystallization in a reservoir solution containing a water-soluble polymer. Furthermore, purified biological macromolecular complexes obtainable by the method according to the present invention are provided as well as crystallized biological macromolecular complexes. Finally, a method for determining the suitability of a candidate compound for inhibiting the 20S proteasome of an individual is provided. Said method is particularly useful in personalized medicine identifying suitable inhibitors of the 20S proteasome in individuals for treating, ameliorating or preventing a cancer, an autoimmune disease, a muscular dystrophy, emphysema or cachexia accompanying cancer or AIDS.

First claim

The invention claimed is:

1. A method for the purification of biological macromolecular complexes comprising:
a) providing a crude sample containing the biological macromolecular complexes;
b) conducting a first centrifugation step for separation of cell debris at 25.000 to 35.000×g;
c) supplementing the supernatant obtained from the first centrifugation step with an osmolyte in an amount of from 0% to 25% (w/v) and compounds allowing thiol-alkylation of cysteines;
d) conducting a second centrifugation step by centrifugation at 50.000 to 150.000×g;
e) treating the supernatant obtained from the second centrifugation step with a water-soluble polymer for precipitation;
f) conducting a density gradient centrifugation using the osmolyte with a polymer-based precipitate, after resuspension thereof in a buffer not containing said water-soluble polymer;
g) optionally repeating once or multiple times steps e) and f);
h) concentration by water-soluble polymer based precipitation of the biological macromolecular complexes for obtaining a purified biological macromolecular complexes.

2. The method according to claim 1 wherein the water soluble polymer is polyethylene glycol 400 to 20.000.

3. The method according to claim 1 wherein the first centrifugation step is a centrifugation to obtain a S30 fraction and/or the second centrifugation step is a centrifugation obtaining a S100 fraction.

4. The method according to claim 1 wherein the osmolyte gradient is a sucrose-based gradient of from 10 to 40% (w/v) sucrose.

5. The method according to claim 1 wherein after the second centrifugation step the supernatant is subject to a differential precipitation the water-soluble polymer comprising a first precipitation step with a lower concentration of the water-soluble polymer whereby the biological macromolecular complexes are maintained in the supernatant and with a second precipitation step with a higher concentration of the water-soluble polymer for precipitating the biological macromolecular complexes.

6. The method according to claim 1 wherein the biological macromolecular complexes are proteasomes.

7. The method according to claim 1 wherein the providing the crude sample step includes obtaining a cytosolic extract obtained by hypotonic lysis of cells or by freeze grinding.

8. The method according to claim 7 wherein the biological macromolecular complexes are immunoproteasomes and the method comprises cultivating the cells with cytokines for a time sufficient to induce expression of the immunoproteasome.

9. A method for crystallization of biological macromolecular complexes comprising:
obtaining purified biological macromolecular complexes according to claim 1 ; and
crystallization of the purified macromolecular complexes in a reservoir solution containing a water-soluble polymer wherein the water-soluble polymer in the reservoir solution may be the same or different as the water-soluble polymer used in the treating step.

10. The method according to claim 9 wherein a protein concentration of the purified macromolecular complexes in the reservoir solution is at least 5 mg/ml for crystallization.

11. The method according to claim 9 wherein the step of crystallization is first at a temperature above 15° C. and, thereafter, at a temperature of equal or below 8° C.

12. The method according to claim 9 further comprising stabilization and dehydration of the crystal at a temperature equal to or below 8° C.

13. The method of claim 1 wherein the water soluble polymer is a nonionic polymer or a polymer with zero net charge.

14. The method of claim 13 wherein the water soluble polymer is selected from the group consisting of polyalkylene glycol, polyamine, and polycarboxylate.

15. The method of claim 6 wherein the proteasomes are selected from the group consisting of a 20S proteasome and a 26S proteasome fatty acid synthase.

16. The method of claim 8 wherein the cytokines include IFN y.

Assignees

• Max Planck Gesellschaft

Inventors

• Chari Ashwin
• Stark Holger
• Schrader Jil
• Henneberg Fabian

Classifications

• C07K1/30

  by precipitation · CPC title

• C07K1/32

  as complexes · CPC title

• C12Y203/01085

  Fatty-acid synthase (2.3.1.85) · CPC title

• C07K1/306

  by crystallization · CPC title

• C07K1/36 Primary

  by a combination of two or more processes of different types · CPC title