Immobilization of membrane proteins onto supports via an amphiphile

• US 8,674,044 B2
• Filed: 05/24/2012
• Issued: 03/18/2014
• 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
• Inventors: Popot, Jean-Luc, Charvolin, Delphine, Giusti, Fabrice
• Primary Examiner(s): HEINCER, LIAM J
• Application Number:
  US13/479,890
• Publication Number:
  US 20130023026A1
• Time in Patent Office: 663 Days
• Field of Search: 526/303.1, 526/306, 526/317.1, 526/287
• US Class Current: 526/303.1
• 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. An amphiphilic molecule comprising a functional group providing the first half of a receptor-ligand molecule pair or of a chemical reactive pair,

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

2. The amphiphilic molecule according to claim 1 wherein the amphiphilic molecule is a polymer of the formula (II)

3. The amphiphilic molecule according to claim 2, wherein the functional group providing a first half of a receptor-ligand pair is biotin, avidin, glutathion, glutathion S-transferase, calmoduline, an ATPase, a protein kinase, a phosphodiesterase, a neurotransmitter, L-arginine, p-aminobenzamidine, a serine protease, L-lysine, plasminogen (and activator), an rRNA, AMP, ADP, ATP, a cofactor enzyme, a lectin, a glucanated protein, a glucolipid, a polysaccharide, heparin, a growth and coagulation factor, a steroid receptor, an endonuclease, a lipoprotein, a lipase, Cibacron Blue®

, a NAD or NADP cofactor enzyme, albumin, a coagulation factor, an interferon, an antigen, a hapten, an antibody, nitrilotriacetic acid (NTA), EDTA, polyhistidine, Ni-nitrilotriacetic acid (NTA), or an oligonucleotide.

4. The amphiphilic molecule according to claim 2, wherein the functional group providing a first half of a chemical reactive pair is a cyclopentadienyl group, an alkynyl group, an alkoxylamine group, or a carbonyl group.

5. The amphiphilic molecule 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⁺, where M⁺ is Na⁺ or K⁺;

Rb₂ represents CONRq1Rq2, where Rq1 is n-octyl, and Rq2 is H;

x1 is between 70 and 80%, x2 is between 20 and 30%, and x3 is 0%; and

the weight average molecular weight (Mw) is between 2,000 and 50,000 g·

mol<sup>−

1</sup>.

6. The amphiphilic molecule 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⁺, where M⁺ is a cationic counter-ion;

Rb₂ represents CONRq1Rq2, where Rq1 is n-octyl, and Rq2 is H;

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

(CH₂)mOH, where 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, where 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;

x1 is between 30 and 40%, x2 is between 20 and 30%, and x3 is between 30 and 50%; and

the weight average molecular weight (Mw) is between 2,000 and 50,000 g·

mol<sup>−

1</sup>.

7. The amphiphilic molecule according to claim 1, wherein the functional group providing a first half of a receptor-ligand pair is biotin, avidin, glutathion, glutathion S-transferase, calmoduline, an ATPase, a protein kinase, a phosphodiesterase, a neurotransmitter, L-arginine, p-aminobenzamidine, a serine protease, L-lysine, plasminogen (and activator), an rRNA, AMP, ADP, ATP, a cofactor enzyme, a lectin, a glucanated protein, a glucolipid, a polysaccharide, heparin, a growth and coagulation factor, a steroid receptor, an endonuclease, a lipoprotein, a lipase, Cibacron Blue®

, a NAD or NADP cofactor enzyme, albumin, a coagulation factor, an interferon, an antigen, a hapten, an antibody, nitrilotriacetic acid (NTA), EDTA, polyhistidine, Ni-nitrilotriacetic acid (NTA), or an oligonucleotide.

8. The amphiphilic molecule according to claim 1, wherein the functional group providing a first half of a chemical reactive pair is a cyclopentadienyl group, an alkynyl group, an alkoxylamine group, or a carbonyl group.

9. A kit for preparing a product comprising a support and at least one membrane protein attached to the surface thereof through an amphiphilic molecule with which said membrane protein is complexed, the binding to the support being done through the amphiphilic molecule, comprising:

a support; and

an amphiphilic molecule;

wherein;

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

10. The kit according to claim 9 wherein the amphiphilic molecule is a polymer of the formula (II)

11. The kit according to claim 10 wherein:

the amphiphilic molecule further comprises at least one functional group providing the first half of a receptor-ligand molecule pair;

the support further comprises at least one functional group attached to the surface thereof providing the second half of said receptor-ligand molecule pair; and

the binding of the amphiphilic molecule to the support is mediated through a specific receptor-ligand binding between the functional group(s) in the amphiphilic molecule and the functional group(s) attached to the support.

12. The kit according to claim 11 wherein the receptor-ligand pair (functional group of the amphiphilic vinyl polymer/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), and (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)/salicylhydroxamic acid (ASH)), (oligonucleotide/complementary oligonucleotide), (polyhistidine/Ni-nitrilotriacetic acid (NTA)), and the corresponding reversed pairs.

13. The kit according to claim 9 wherein:

the amphiphilic molecule further comprises at least one functional group providing the first half of a receptor-ligand molecule pair;

the support further comprises at least one functional group attached to the surface thereof providing the second half of said receptor-ligand molecule pair; and

the binding of the amphiphilic molecule to the support is mediated through a specific receptor-ligand binding between the functional group(s) in the amphiphilic molecule and the functional group(s) attached to the support.

14. The kit according to claim 13 wherein the receptor-ligand pair (functional group of the amphiphilic vinyl polymer/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), and (Cibacron Blue®

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

15. The kit according to claim 9 wherein:

the amphiphilic molecule further comprises at least one functional group providing the first half of a chemical reactive pair;

the support further comprises at least one functional group attached to the surface thereof providing the second half of said chemical reactive pair; and

the binding of the amphiphilic molecule to the support is carried out by chemically reacting the functional group(s) in the amphiphilic molecule with the functional group(s) attached to the support.

16. The kit according to claim 15 wherein the chemical reactive pair (functional group of the amphiphilic vinyl polymer/functional group attached to the support) is chosen from the following pairs:

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

17. The kit according to claim 15 wherein:

the amphiphilic molecule further comprises at least one functional group providing the first half of a chemical reactive pair;

the support further comprises at least one functional group attached to the surface thereof providing the second half of said chemical reactive pair; and

the binding of the amphiphilic molecule to the support is carried out by chemically reacting the functional group(s) in the amphiphilic molecule with the functional group(s) attached to the support.

18. The kit according to claim 17 wherein the chemical reactive pair (functional group of the amphiphilic vinyl polymer/functional group attached to the support) is chosen from the following pairs:

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

19. The kit according to claim 10, wherein:

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

Rb₁ represents COO⁻

M⁺, where M⁺ is Na⁺ or K⁺;

Rb₂ represents CONRq1Rq2, where Rq1 is n-octyl, and Rq2 is H;

x1 is between 70 and 80%, x2 is between 20 and 30%, and x3 is 0%; and

the weight average molecular weight (Mw) is between 2,000 and 50,000 g·

mol<sup>−

1</sup>.

20. The kit according to claim 10, wherein:

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

Rb₁ represents COO⁻

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

Rb₂ represents CONRq1Rq2, where Rq1 is n-octyl, and Rq2 is H;

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

(CH₂)mOH, where 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, where 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;

x1 is between 30 and 40%, x2 is between 20 and 30%, and x3 is between 30 and 50%; and

the weight average molecular weight (Mw) is between 2,000 and 50,000 g·

mol<sup>−

1</sup>.