Immobilization of membrane proteins onto supports via an amphiphile

• US 8,207,263 B2
• Filed: 11/13/2007
• Issued: 06/26/2012
• Est. Priority Date: 11/13/2006
• Status: Active Grant

• Current Assignee: Centre National De La Recherche Scientifique, Universite Paris CITE
• Original Assignee: Centre National De La Recherche Scientifique, Universite Paris Diderot Paris 7
• Inventors: Popot, Jean-Luc, Charvolin, Delphine, Giusti, Fabrice
• Primary Examiner(s): HEINCER, LIAM J
• Application Number:
  US12/514,656
• Publication Number:
  US 20090275066A1
• Time in Patent Office: 1,687 Days
• Field of Search: 525/54.1, 524/17, 526/303.1, 526/306, 526/317.1, 526/287, 435/4
• US Class Current: 525/54.1
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 17/06 attached to the carrier via a bridging agent

Abstract

The invention pertains to the field of membrane protein immobilization onto supports. It relates to a product comprising a support and at least one membrane protein attached to the surface thereof, characterized in that said membrane protein is attached to said support using an amphiphilic molecule with which said membrane protein is complexed. It also relates to a process for preparing such product, as well as to various applications in the fields of diagnosis, drug design and biotechnologies. It further relates to a kit, together with a functionalized amphiphilic molecule, for preparing a product according to the invention comprising a support and an amphiphilic molecule, wherein the amphiphilic molecule and the support interact through a hydrophobic bond, an ionic bond, a specific bond or a covalent bond.

Claims

1. A product comprising a support and at least one membrane protein attached to the surface thereof,

wherein said membrane protein is attached to said support through an amphiphilic molecule with which said membrane protein is complexed, the binding to the support being done through the amphiphilic molecule,

wherein the amphiphilic molecule is a vinyl polymer of the formula (I):

2. The product according to claim 1 wherein n is 3 and the vinyl polymer has the formula (II):

3. The product according to claim 2 wherein:

Ra₁, Ra₂ and Ra₃ are the same or different, and represent a hydrogen atom or a methyl radical;

Rb₁ represents COO⁻

M⁺, wherein M⁺is a cationic counter-ion;

Rb₂ represents CONRq1Rq2, wherein Rq1 and Rq2 represent independently a linear or branched and/or cyclic alkyl, alkynyl or alkenyl radical containing from 3 to 50 carbon atoms, and further either one of them can represent a hydrogen atom;

Rb₃ represents CONRkRl, wherein Rk and Rl represent independently a (C₁-C₅) alkyl radical, an alkylsulfonate, a sugar moiety, a primary, secondary or tertiary hydroxyalkyl —

(CH₂)mOH, wherein m is within the range of 1 to 4, a polyoxyalkylene having 4 to 10 alkylene oxide units, a zwitterionic radical, a (CH₂)t-NRf1Rf2 radical, wherein t is an integer from 1 to 5, Rf1, Rf2 are the same or different, and represent a hydrogen atom or a (C₁-C₄) alkyl radical, and further either one of Rk and Rl can represent a hydrogen atom.

4. The product according to claim 1 wherein the support is a solid support.

5. The product according to claim 4 wherein the support is chosen from a chip, a bead, a membrane, a fiber, a nanotube, and a soluble macromolecule or particle chosen from a polymer, a dendrimer, a vesicle, and a micelle.

6. The product according to claim 1 wherein the receptor-ligand pair (functional group in the amphiphilic molecule / functional group attached to the support) is chosen from the following pairs:

(biotin/avidin), (glutathion/glutathion S-transferase, glutathion-binding proteins, or fusion proteins including glutathion S-transferase), (calmoduline/ATPase, protein kinase, phosphodiesterase, or neurotransmitter), (L-arginine or p-aminobenzamidine/serine protease), (L-lysine/plasminogen (and activator) or rRNA), (AMP, ADP, or ATP/Cofactor enzyme), (lectin/glucanated protein, glucolipid, or polysaccharide), (heparin/growth and coagulation factor, steroid receptor, endonuclease, lipoprotein, or lipase), or (Cibacron blue®

/NAD or NADP cofactor enzymes, albumin, coagulation factor, or interferon), (antigen/antibody), (hapten/antibody), (antibody/antigen), (antibody/hapten), (nitrilotriacetic acid (NTA)/transition metal), (EDTA/transition metal), (phenylboronic acid (APB)/salicylhydrox-amic acid (ASH)), and (oligonucleotide/complementary oligonucleotide), and the corresponding reversed pairs.

7. The product according to claim 1 wherein the chemical reactive pair (reactive functional group in the amphiphilic molecule/reactive functional group attached to the support) is selected from the following pairs:

(cyclopentadienyl group / quinone derivative), (alkynyl group/azide group), (alkoxylamine group/carbonyl group), and (carbonyl group/alkoxylamine group).

8. The product according to claim 1 wherein said at least one membrane protein is chosen from an antigen, an antibody, an enzyme, a cell receptor, an ion channel, and a membrane protein of viral, bacterial, or eukaryotic origin.

9. A process for preparing a product according to claim 1 comprising:

providing at least one membrane protein, a support, and an amphiphilic molecule according to claim 1;

forming a complex between the amphiphilic molecule and the membrane protein; and

attaching the amphiphilic molecule of the complex to the support via a hydrophobic bond, an ionic bond, a specific receptor-ligand binding between at least one functional group in the amphiphilic molecule and at least one functional group at the support surface, or a covalent bond between at least one reactive functional group in the amphiphilic molecule and at least one reactive functional group at the support surface.

10. A method for detecting the presence or absence in a biological specimen of a ligand of at least one membrane protein comprising preparation of a product according to claim 1 with said membrane protein and contacting said product with said biological specimen.

11. The method according to claim 10 wherein said at least one membrane protein is a membrane antigen of a pathogenic agent, said biological specimen is a serum sample, and said ligand to be detected is an antibody raised against said antigen.

12. A method for screening a bank of compounds for ligands of at least one membrane protein, comprising preparation,of a product according to claim 1 with said membrane protein and contacting said product with said bank of compounds.

13. A method for transforming an enzyme substrate under controlled conditions, comprising preparation of a product according to claim 1 with said membrane protein which is an enzyme and contacting said enzyme substrate.

14. A process for assaying a membrane protein in a sample, comprising:

a) solubilizing a membrane protein in a detergent;

b) preparing a product according to claim 1 by;

i) complexing the membrane protein with an amphiphilic molecule and removing the detergent; and

ii) immobilizing the membrane protein onto a support through the amphiphilic molecule,

c) extensively washing, and adding a protease for generating protein fragments of the membrane protein, wherein the transmembrane domain remains complexed with the amphiphilic molecule attached to the support; and

d) removing the support to which the transmembrane domain still complexed with the amphiphilic molecule remains bound, and analyzing the protein fragments by mass spectrometry.