Nanolipoprotein particles and related methods and systems for protein capture, solubilization, and/or purification

Patent metadata

Field	Value
Publication number	US-9458191-B2
Application number	US-201414536513-A
Country	US
Kind code	B2
Filing date	Nov 7, 2014
Priority date	Jan 11, 2008
Publication date	Oct 4, 2016
Grant date	Oct 4, 2016

Abstract

Provided herein are methods and systems for assembling, solubilizing and/or purifying a membrane associated protein in a nanolipoprotein particle, which comprise a temperature transition cycle performed in presence of a detergent, wherein during the temperature transition cycle the nanolipoprotein components are brought to a temperature above and below the gel to liquid crystalling transition temperature of the membrane forming lipid of the nanolipoprotein particle.

First claim

What is claimed is:

1. A method for solubilizing a membrane associated protein from a cell membrane, the method comprising
contacting the cell membrane with a scaffold protein and a membrane forming lipid to provide an admixture, the membrane forming lipid having a membrane forming lipid gel-crystalline transition temperature; and
subjecting the admixture to at least three temperature transition cycles in presence of a detergent, for a time and under condition to allow assembly of a nanolipoprotein particle comprising a solubilized target protein, the temperature transition cycle comprising:
a temperature increase step wherein the admixture is brought to a temperature above said membrane forming lipid gel crystalline transition temperature and
a temperature decrease step wherein the admixture is brought to a temperature below said membrane forming lipid gel crystalline transition temperature.

2. The method of claim 1 , wherein the cell membrane contacted with the membrane forming lipid and the scaffold protein, is in a crude cell pellet or a membrane fraction.

3. The method of claim 1 , wherein the cell membrane is an inner membrane of gram-negative bacteria, an outer membrane of gram-negative bacteria, a single bilayer membrane of gram-positive bacteria, or a plasma membrane of a eukaryotic cell.

4. The method of claim 1 , further comprising incubating the admixture subjected to the temperature transition cycle at the membrane forming lipid gel-crystalline transition temperature for a predetermined amount of time that is functional to a desired amount of assembled nanolipoprotein particles.

5. The method of claim 1 , further comprising removing the detergent from the admixture subjected to the temperature transition cycle.

6. The method of claim 1 , wherein the membrane forming lipid is contacted with the membrane associated protein and the scaffold protein at a temperature above the membrane forming lipid gel-crystalline transition temperature.

7. The method of claim 1 , wherein subjecting the admixture to a temperature transition cycle in presence of a detergent is performed by subjecting the admixture to multiple temperature transition cycles.

8. The method of claim 1 , wherein the temperature increase step and/or the temperature decrease step are performed for a time and under conditions such that at least a fraction of the membrane forming lipid molecules in the admixture changes state.

9. The method of claim 1 , wherein the membrane associated protein contacted with the membrane forming lipid and with the scaffold protein is comprised in a cell membrane.

Assignees

• L Livermore Nat Security Llc

Inventors

• Chromy Brett A
• Henderson Paul
• Hoeprich Jr Paul D

Classifications

• Y10S977/799

  Containing biological material · CPC title

• C12P3/00

  Preparation of elements or inorganic compounds except carbon dioxide {(recovery of carbon dioxides as by-products C12F3/02)} · CPC title

• C12N11/02 Primary

  Enzymes or microbial cells immobilised on or in an organic carrier · CPC title

• C07K1/145 Primary

  by extraction or solubilisation · CPC title