Method for the purification of biological macromolecular complexes

Patent metadata

Field	Value
Publication number	US-11401301-B2
Application number	US-202017060627-A
Country	US
Kind code	B2
Filing date	Oct 1, 2020
Priority date	Jun 6, 2016
Publication date	Aug 2, 2022
Grant date	Aug 2, 2022

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 determining the suitability of a candidate compound for inhibiting the 20S proteasome of an individual comprising:
obtaining purified biological macromolecular complexes by
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); and
h) concentration by water-soluble polymer based precipitation of the biological macromolecular complexes for obtaining purified biological macromolecular complexes;

crystallization of the purified biological 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; and
determining a crystal structure of a crystallized biological macromolecular complex wherein the crystallized biological macromolecular complex is a 20S proteasome, wherein determining is performed by diffraction analysis with resolution of 2.2 Å or below; and
determining suitability of the candidate compound as an inhibitor of the 20S proteasome of an individual based on said analysis.

2. The method according to claim 1 wherein a protein concentration of the purified biological macromolecular complexes in the reservoir solution is at least 5 mg/ml for crystallization.

3. The method according to claim 1 wherein the step of crystallization is first at a temperature above 15° C. and, thereafter, at a temperature of equal or below 8° C.

4. The method according to claim 1 further comprising stabilization and dehydration of the crystal at a temperature equal to or below 8° C.

5. The method according to claim 1 wherein the individual is an individual not responding to a first inhibitor of the 20S proteasome.

6. The method of claim 5 wherein the first inhibitor is selected from the group consisting of bortezomib, carfilzomib, dihydroeponemycin, eponomycin, marizomib, ixazomib, delanzomib, ONX-912, and ONX-0914.

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