Amphipol

Abstract

To provide complexes which form with membrane proteins and methods for solubilization and / or stabilization of membrane proteins.SOLUTION: The invention relates to polymeric compounds of a formula (I) which allow solubilization and / or stabilization of membrane proteins. The invention relates in particular to complexes which form with membrane proteins and to methods for the solubilization and / or stabilization of membrane proteins, comprising the use of the compounds.SELECTED DRAWING: Figure 7

Description

[Technical field]

[0001] The present invention relates to new amphiphilic polymers called "amphipols". [Background technology]

[0002] These amphiphilic polymers are known in the state of the art to be capable of stabilizing membrane proteins in aqueous solutions when they are extracted from the membrane using detergents.

[0003] Membrane proteins account for about 30% of the proteins encoded by the cellular genome and are involved in many important cellular functions (signaling, secretion, migration, adhesion, homeostasis, energy production, etc.). Membrane proteins can be monomers or oligomers. They are highly structured into α-helices or β-sheets in the transmembrane segments that interact with the alkyl chains of membrane lipids by being integrated into the membrane.

[0004] The numerous functions and enzymatic activities that membrane proteins exhibit make them pharmacological targets or biotechnological tools of choice. However, the study of membrane protein function and structure in solution and their use in the development of therapeutic or biotechnological tools involves their mass production, purification, and maintenance of active conformation, and membrane proteins are generally unstable when extracted from the membrane. This instability in aqueous solutions is important because detergents are required to prevent membrane protein aggregation. In fact, the surface of the extramembrane domains of these proteins is covered mainly with amino acids with hydrophilic side chains, and the transmembrane domains of these proteins are covered with amino acids with hydrophobic chains, so that membrane proteins are highly insoluble in water due to the hydrophobic effect that promotes the aggregation of membrane proteins in solution to minimize the disruption of the hydrogen bond network between water molecules.

[0005] There are many methods to produce sufficient quantities of membrane proteins and a large choice of chemical molecules to purify them (yields are often limited), solubilize them, and stabilize them in aqueous solution.

[0006] Commercially available surfactants can solubilize proteins, but as mentioned above, they cause more or less rapid protein denaturation and inactivation, especially due to the dissociation power of the surfactant. Furthermore, the surfactant needs to be maintained in solution at a concentration above the critical micelle concentration (CMC) of the surfactant. On the other hand, surfactants such as amphipols (Patent Document 1) can solubilize proteins, but offer rather poor solubilizing properties. Amphipols are amphiphilic polymers defined as those capable of maintaining membrane proteins in their native soluble conformation in the form of small complexes (Non-Patent Document 1). The most commonly used and studied amphipol is A8-35. It consists of a polyacrylic acid backbone grafted with octylamine and isopropylamine side chains, 35% non-grafted carboxylic acid groups, 25% carboxylic acid derivatives grafted with octyl chains (octylamine), and 40% carboxylic acid groups grafted with isopropyl groups (isopropylamine). Type A8-35 amphipols generally require prior solubilization of the membrane with a detergent, which is then replaced by the amphipol, either by dilution of the detergent below its CMC (which has the drawback of also diluting the protein) or by its adsorption onto beads. Other amphiphilic polymers have been developed. Among them, mention may be made of SMA (styrene-maleic acid copolymer, US Pat. No. 5,399,433) and their derivatives, which exhibit the feature of being able to solubilize membrane proteins directly from membrane fractions without prior solubilization with detergents. However, unlike amphipols, which are simply purified after synthesis, SMA always contains impurities that preclude the subsequent use of the protein for biotechnological applications.

[0007] Methods for purifying proteins, especially membrane proteins, whether recombinant or not, are generally empirical and difficult to predict, especially depending on the composition of the protein, (micro)organism or membrane, and continuous testing of different agents for solubilizing and / or stabilizing membrane proteins is widely carried out in order to identify the most effective agents under these conditions.

[0008] In this context, it would be desirable to provide new alternatives for the production of membrane proteins, which are of high biotechnological interest. In particular, polymers having both solubilizing and stabilizing properties for membrane proteins would be particularly useful, which do not currently exist. The new amphipols that are the subject of the present invention meet this need. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] WO 98 / 27434 [Patent Document 2] International Publication No. 2011 / 004158 [Non-patent literature]

[0010] [Non-Patent Document 1] Zoonens & Popot, 2014 Summary of the Invention [Problem to be solved by the invention]

[0011] The present invention therefore aims to provide new compounds for solubilizing and stabilizing membrane proteins. More specifically, the present invention relates to new amphipol amphiphilic vinyl polymers that have improved solubilizing properties while allowing sufficient stabilization of membrane proteins. [Means for solving the problem]

[0012] For this purpose, the present invention provides a compound of formula I [ka] The amphiphilic vinyl polymer of the formula M + is an alkali metal, preferably Li + , Na + , K + is selected from R1, R4, R5, and R8 each independently represent a hydrogen atom or a methyl group; R2, R6, and R9 are each independently a group selected from a hydrogen atom and a linear or branched (C1-C8) alkyl group; R 10 is a group selected from linear or branched (C1-C5) alkyl; R3 and R7 are each independently -hydrogen (where X is a single bond), - linear or branched alkyl of at least 6 carbon atoms, linear or branched alkenyl of at least 6 carbon atoms, or linear or branched alkynyl of at least 6 carbon atoms (wherein X is a single bond); - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, and linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenyl, and linear or branched (C2-C8) alkynyl, - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups, (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, where one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene, - polyunsaturated or monounsaturated polycyclic compounds which are unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene and linear or branched (C2-C8) alkynylene, - phenyl, unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; is a group selected from When one of the R3 or R7 groups is a linear or branched alkyl of at least 6 carbon atoms, a linear or branched alkenyl of at least 6 carbon atoms, a linear or branched alkynyl of at least 6 carbon atoms, or a hydrogen atom, and X is a single bond, the other R3 or R7 group is - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene; - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups, (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, where one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene, - polyunsaturated or monounsaturated polycyclic compounds which are unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene and linear or branched (C2-C8) alkynylene, and - phenyl, unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; Selected from Here, w, x, y, and z correspond to the respective proportions of the constituent units, -x is 20% to 90% -y is 0 to 80%; -w is 0 to 80%; -z is 0 to 60%, provided that w+x+y+z=100%, and y or w is at least 10%. According to one optional feature, the amphiphilic vinyl polymer of formula I is such that w=0 and R7 is a group selected from: - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, - phenyl, unsubstituted or substituted by one or more groups, wherein the one or more groups are selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, Here, X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene.

[0013] According to another optional feature, the amphiphilic vinyl polymer of formula I is: R3 and / or R7 are (C6-C8)cycloalkyl as defined above, or - R3 and / or R7 is phenyl, preferably the phenyl is para-substituted with a methyl or ethyl group; Here, X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene.

[0014] According to another optional feature, the amphiphilic vinyl polymer of formula I is: R3 and / or R7 are (C6-C8)cycloalkyl as defined above, where X is preferably a single bond or CH2; -R3 and / or R7 are phenyl, preferably the phenyl is para-substituted with a methyl or ethyl group, where X is a single bond; or -R3 and / or R7 are styrenyl groups, where X is a single bond.

[0015] According to another optional feature, the amphiphilic vinyl polymer of formula I is: -Metal M is Na + or -R 10 is an isopropyl group, -x is less than or equal to 75% -w and / or y is 25% or more, - w and / or y are between 25% and 50% and z is between 0% and 40%, preferably between 15% and 40%, or -functionalized by grafting affinity labels, fluorescent probes, or immunostimulatory molecules.

[0016] According to another optional feature, the vinyl polymer of formula I is: - R1, R2, R4, R5, R6, R8, and R9 are hydrogen atoms; - R3 and / or R7 is (C6-C8)cycloalkyl and X is a single bond; -R 10 is an isopropyl group, -x is 35% or 50%, - y is 25% or more, -w is 0% or 25%, -z is 0%, 15% or 40%, where w+x+y+z=100%.

[0017] According to another optional feature, the vinyl polymer of formula I is selected from the compounds listed in Table 1.

[0018] The vinyl polymers of the invention are amphipols, in other words they are capable of keeping membrane proteins soluble in their native conformation in the form of small complexes.Thus, in another aspect, the invention relates to a water-soluble complex formed of i) a membrane protein or a mixture of membrane proteins and ii) at least one amphiphilic vinyl polymer compound as described above.

[0019] According to one optional feature, the water-soluble complex thus formed further comprises a lipid compound.

[0020] Furthermore, the polymers of the invention are capable of stabilizing membrane proteins in aqueous solutions. An object of the invention is therefore a water-soluble complex formed between i) a membrane protein or a mixture of membrane proteins and ii) at least one amphiphilic vinyl polymer compound of the invention, in which said membrane protein is stabilized in aqueous solution. Another object of the invention is therefore a solution comprising such a complex.

[0021] The present invention also relates to a method for preparing an aqueous solution of one or more membrane proteins, comprising a solubilization and / or stabilization step, in which a protein fraction from a biological or synthetic membrane containing said membrane protein or a mixture of said membrane proteins is bound to a vinyl polymer as described above.

[0022] According to an optional feature, the method is as follows. - the one or more membrane proteins are recombinant proteins expressed in prokaryotic or eukaryotic cells and / or native proteins expressed in specialized membranes with high protein density, - the step of solubilizing said membrane protein or said mixture of membrane proteins has been carried out beforehand using a detergent medium, - the step of fusing the protein fraction containing the membrane protein or the mixture of membrane proteins has been carried out beforehand with vesicles of phospholipids or vesicles of a mixture of phospholipids, preferably with vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), or the solution of said protein fraction containing said membrane proteins is not a solution of said one or more membrane proteins in a detergent medium in micellar form.

[0023] The membrane protein-amphipol complexes of the invention are capable of solubilizing and stabilizing these proteins in their native form, thus allowing the biotechnological use of these proteins. It is therefore also an object of the invention to use the complexes of the invention as reagents in reagent kits, in particular diagnostic kits, which contain at least one complex of the invention as an immunological reagent. The complexes of the invention can also be associated with devices that utilize the activity of said membrane proteins, purified and solubilized as described above by the amphipols of the invention, for diagnostics or activity measurements.

[0024] Other advantages and features of the present invention will become apparent on reading the following description, given by way of illustrative and non-limiting example, with reference to the attached drawings, in which: [Brief description of the drawings]

[0025] [Figure 1] 3. Steric exclusion chromatography analysis of the amphipols of the invention, A8-35, and particles formed with their complexes with bacteriorhodopsin (BR). V0 = dead volume, VT = total volume. [Diagram 2] Complex formation between BR and the amphipol of the invention before (black bars) or after (gray bars) centrifugation [Diagram 3] Thermal denaturation of BR in various environments [Figure 4] Solubilization of H. salinarum purple membrane before (A) or after (B) fusion with DMPC vesicles with A8-35, SMA, or the amphipol of the present invention. [Diagram 5] Quantification of BR extracted by A8-35, SMA, or the polymer of the invention from purple membranes pre-fused to DMPC vesicles (A) and analysis of the thermal stability of these complexes after 6 h incubation at 50° C. (B) The ratio of absorbance values ​​measured at the beginning and end of each experiment is reported. [Figure 6] Purple membrane extraction. (A) Quantification of solubilization (gray bars) and thermal stability (black bars) of purple membrane BR pre-fused to DMPC vesicles by A8-35, SMA, or polymers of the invention, with + / - SD values ​​performed at least three times for each condition. (B) Kinetics of thermal destabilization of complexes formed with SMA or polymers 19 and 23 upon incubation at 50°C for 6 hours. Absorbance values ​​measured at 554 nm were normalized with those measured before incubation of the samples. [Figure 7] Solubilization kinetics of YidC-GFP protein overexpressed in the plasma membrane of E. coli in the presence of SMA, a polymer of the invention, or DDM. (A) Photograph of an electrophoretic gel revealing the fluorescence of GFP fused to the YidC membrane protein, and (B) graphical representation of the quantification of fluorescence from the gel image relative to the fluorescence measured in the presence of DDM after 1 h of incubation. [Figure 8] Solubilization of Torpedo AChR-rich membranes with A8-35, SMA, and amphipols of the invention. Samples corresponding to the supernatant (S) and pellet (C) of each condition were plated on an acrylamide gel, which was stained with Coomassie blue after electrophoresis. Arrows indicate the position of the bands corresponding to Torpedo AChR subunits and Na+ / K+ ATPase. Proteins present in the pellet were not solubilized, whereas proteins in the supernatant represent solubilized proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention relates to new amphipols that allow the solubilization and stabilization of membrane proteins in aqueous media. In particular, the new amphipols of the present invention have improved solubilization and / or stabilization properties compared to standard polymers used for the solubilization of membrane proteins. And the complexes formed with membrane proteins are particularly suitable for the biotechnological use and study of these membrane proteins.

[0027] In the following description, the term "amphipol" refers to a vinyl polymer capable of keeping membrane proteins soluble in their native conformation in the form of small complexes.

[0028] The term "vinyl" in its general sense includes acrylic polymers.

[0029] The term "membrane protein" is used interchangeably in the singular or plural and encompasses mixtures of membrane proteins or preparations containing substantially a single type of protein. In the sense of the present invention, a membrane protein is a protein that crosses the membrane on both sides and therefore contains at least one transmembrane domain or a protein that contains at least one hydrophobic domain that interacts with the membrane. Membrane proteins can be monomeric or oligomeric, with or without cofactors.

[0030] In the sense of the present invention, a "stabilized" membrane protein corresponds to a solubilized membrane protein that retains its native conformation and preferably at least part of its biological activity (even if this activity is temporarily blocked).

[0031] In the sense of the present invention, the term "specialized membrane" includes organelles of eukaryotic cells, such as the cristae of mitochondria, the thylakoids of chloroplasts, the endoplasmic or sarcoplasmic reticulum, etc. "Specialized membrane" also includes membranes with a high protein density, whether integral or not, comprising at least 50% membrane protein, or at least 60%, or at least 70% membrane protein, such as for example the purple membrane of the bacterium Halobacterium salinarum (which may contain up to 75% bacteriorhodopsin), the remainder comprising lipids or other molecules.

[0032] In the sense of the present invention, the term "biomembrane" refers to any lipid membrane extracted or derived from a cell or organism, such as bacteria, fungi, eukaryotic cells, etc. The phrase "synthetic biological membrane" refers to any lipid membrane that is reconstituted, recombinant, or modified with synthetic or naturally occurring lipids. Thus, for example and without limitation, a synthetic membrane can correspond to a lipid membrane extracted from an organism to which one or more synthetic lipids have been added; a membrane made from synthetic lipids; or a lipid membrane reconstituted from deliberately mixed natural and / or synthetic lipids.

[0033] In the sense of the present invention, the term "protein fraction" includes a cell fraction containing the membrane protein to be sought or analyzed. It may therefore be a more or less clarified and / or purified cell homogenate. In the case of membrane proteins, the term "protein fraction" refers in particular to a fraction of this homogenate, such as a membrane fraction or a fraction corresponding to a cellular compartment.

[0034] Thus, the present invention relates to a compound of formula I [ka] The amphiphilic vinyl polymer of the formula M + is an alkali metal, preferably Li + , Na + , K + is selected from R1, R4, R5, and R8 each independently represent a hydrogen atom or a methyl group; R2, R6, and R9 are each independently a group selected from a hydrogen atom and a linear or branched (C1-C8) alkyl group; R 10 is a group selected from linear or branched (C1-C5) alkyl; R3 and R7 are each independently -hydrogen (where X is a single bond), - linear or branched alkyl of at least 6 carbon atoms, linear or branched alkenyl of at least 6 carbon atoms, or linear or branched alkynyl of at least 6 carbon atoms (wherein X is a single bond); - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene; - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups, (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, where one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene, - polyunsaturated or monounsaturated polycyclic compounds which are unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene and linear or branched (C2-C8) alkynylene, or - phenyl, unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; is a group selected from When one of the R3 or R7 groups is a linear or branched alkyl of at least 6 carbon atoms, a linear or branched alkenyl of at least 6 carbon atoms, a linear or branched alkynyl of at least 6 carbon atoms, or a hydrogen atom, and X is a single bond, the other R3 or R7 group is - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene; - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups, (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, where one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene, - polyunsaturated or monounsaturated polycyclic compounds which are unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene and linear or branched (C2-C8) alkynylene, or - phenyl, unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; is selected from Here, w, x, y, and z correspond to the respective proportions of the constituent units, -x is 20% to 90% -y is 0 to 80%; -w is 0 to 80%; -z is 0 to 60%, provided that w+x+y+z=100%, and y or w is at least 10%.

[0035] From the definitions of the previous paragraphs, it is clear that w and y are not 0 when one of the R3 and R7 groups is a linear or branched alkyl of at least 6 carbon atoms, a linear or branched alkenyl of at least 6 carbon atoms, or a linear or branched alkynyl of at least 6 carbon atoms, or a hydrogen atom, where X is a single bond, and the other R3 or R7 group is as specified and defined above.

[0036] In one particular embodiment, the polymer of formula I is: M + is an alkali metal, preferably Li + , Na + , K + is selected from R1, R4, R5, and R8 each independently represent a hydrogen atom or a methyl group; R2, R6, and R9 are each independently a group selected from a hydrogen atom and a linear or branched (C1-C8) alkyl group; R 10 is a group selected from linear or branched (C1-C5) alkyl; R3 and / or R7 each independently represent - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene; - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups, (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, where one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene, - polyunsaturated or monounsaturated polycyclic compounds which are unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene and linear or branched (C2-C8) alkynylene, - phenyl, unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; is a group selected from Here, w, x, y, and z correspond to the respective proportions of the constituent units, -x is 20% to 90% -y is 0 to 80%; -w is 0 to 80%; -z is 0 to 60%, provided that w+x+y+z=100%, and y or w is at least 10%.

[0037] In one particular embodiment, the polymer of formula I is: M + is an alkali metal, preferably Li + , Na + , K + is selected from R1, R4, R5, and R8 each independently represent a hydrogen atom or a methyl group; R2, R6, and R9 are each independently a group selected from a hydrogen atom and a linear or branched (C1-C8) alkyl group; R 10 is a group selected from linear or branched (C1-C5) alkyl; R3 and R7 are each independently -hydrogen (where X is a single bond), - linear or branched alkyl of at least 6 carbon atoms, linear or branched alkenyl of at least 6 carbon atoms, or linear or branched alkynyl of at least 6 carbon atoms (wherein X is a single bond); - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene; - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups, (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, where one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene, - phenyl, unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; is a group selected from When one of the R3 or R7 groups is a linear or branched alkyl of at least 6 carbon atoms, a linear or branched alkenyl of at least 6 carbon atoms, a linear or branched alkynyl of at least 6 carbon atoms, or a hydrogen atom, and X is a single bond, the other R3 or R7 group is - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene; - a polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups, (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, in which one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, in which X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene and linear or branched (C2-C8)alkynylene, or - phenyl, unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; Selected from Here, w, x, y, and z correspond to the respective proportions of the constituent units, -x is 20% to 90% -y is 0 to 80%; -w is 0 to 80%; -z is 0 to 60%, provided that w+x+y+z=100%, and y or w is at least 10%.

[0038] In certain embodiments, R3 and / or R7 are groups selected from linear or branched alkyl of at least 6 carbon atoms, linear or branched alkenyl of at least 6 carbon atoms, or linear or branched alkynyl of at least 6 carbon atoms, where when X is a single bond, the alkyl, alkenyl, or alkynyl has fewer than 18, fewer than 17, fewer than 16, fewer than 15, fewer than 14, fewer than 13, fewer than 12, fewer than 11, fewer than 10, fewer than 9, fewer than 8, or fewer than 7 carbon atoms.

[0039] The average molar mass of the amphipol of the present invention is 100,000 g mol -1 Less than 2000-50,000 g mol -1 It is.

[0040] The following are embodiments of the present invention, which may or may not be combined with one or more of the others.

[0041] In certain embodiments, M + Na + It is.

[0042] In certain embodiments, y and / or w are greater than or equal to 25%. In fact, as shown in the experimental section, polymers grafted at this rate are particularly effective.

[0043] In certain embodiments, R2, R6, and R9 are hydrogen.

[0044] In certain embodiments, w=0 and R7 is a group selected from (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted with one or more groups, where the one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene. Preferably, X is a single bond and R7 is a C6cycloalkyl, preferably a C7cycloalkyl, more preferably a C8cycloalkyl. Alternatively, X is a linear or branched (C1-C8)alkylene, preferably methyl or ethyl.

[0045] In another particular embodiment, w=0 and R7 is phenyl unsubstituted or substituted with one or more groups selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene. Preferably, X is a single bond and said phenyl is para-substituted. More preferably, when mono-substituted, said phenyl is para-substituted with C1-C8 linear alkyl, even more preferably, said para linear alkyl is selected from methyl or ethyl.

[0046] In one embodiment, w=0, R7 is phenyl unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, and X is selected from linear or branched (C1-C8) alkylene, preferably C1 alkylene or C2 alkylene.

[0047] In certain embodiments, w and y are not 0, and R and R are, independently of each other, - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene; - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups, (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, where one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene, - polyunsaturated or monounsaturated polycyclic compounds which are unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene and linear or branched (C2-C8) alkynylene, or - phenyl, unsubstituted or substituted by one or more groups, the one or more groups being selected from linear or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; and Preferably, R3 and R7 are, independently of each other, -C6 cycloalkyl, preferably C7 cycloalkyl, more preferably C8 cycloalkyl; -phenyl, more preferably when monosubstituted, para-substituted by a linear C1-C8 alkyl, more preferably the linear alkyl in the para position is selected from a methyl group or an ethyl group, where X is a single bond; or - phenyl which is unsubstituted or substituted by one or more groups, one or more of which are substituted by one or more groups selected from linear or branched (C1-C8) alkyl, linear, cyclic or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, and X is selected from linear or branched (C1-C8) alkylene, preferably C1 alkylene or C2 alkylene; and More preferably, R3 and R7 are different from each other.

[0048] In another particular embodiment, R3 and / or R7 are groups selected from (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, where the one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkyl, preferably methyl or ethyl.

[0049] In another particular embodiment, R3 and / or R7 is phenyl, unsubstituted or substituted with one or more groups, selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, preferably methyl or ethyl. Preferably, X is a single bond and said phenyl is para-substituted. More preferably, in the case of mono-substitution, said phenyl is para-substituted with C1-C8 linear alkyl, more preferably the para linear alkyl is selected from a methyl or ethyl group.

[0050] In another embodiment, z is not 0 and is preferably R 10 is then an isopropyl group, more preferably R 10 is an isopropyl group, and for the constitutional unit containing R3 and / or R7, X is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene, and R3 and / or R7 are - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, where one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, linear or branched (C1-C8)alkynyl, - polyunsaturated or monounsaturated polycyclic compounds which are unsubstituted or substituted by one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, or - phenyl, unsubstituted or substituted by one or more groups, wherein the one or more groups are selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, is selected from.

[0051] In certain embodiments, the amphiphilic vinyl polymer of formula I is selected from the compounds listed in Table 1 below, where M + is an alkali metal, preferably Li + , Na + , K + In certain embodiments, M + Na + It is. [Table 1] TIFF2024180675000005.tif147152

[0052] As mentioned above, in the vinyl polymer of the present invention, the proportions w, x, y, and z of the various constitutional units may vary, provided that w+x+y+z=100%, where the proportions are specified to an accuracy of plus or minus 10%. Thus, for example, if the proportion for a certain constitutional unit is specified as 50%, it is understood that this constitutional unit may be present in the polymer of the present invention at 45% to 55%.

[0053] In certain embodiments, x is less than or equal to 75%.

[0054] In another embodiment, w and / or y is between 25% and 50%, and z is between 0% and 40%, preferably between 15% and 40%.

[0055] In certain embodiments, w+y is greater than or equal to 25%, preferably less than 50%.

[0056] In another particular embodiment, the polymer according to the invention is selected from the compounds of Table 1 having the ratios w, x, y, and z as specified in Table 2 below. [Table 2] TIFF2024180675000007.tif237168TIFF2024180675000008.tif87168

[0057] As demonstrated in the experimental section, the amphiphilic vinyl polymers of the invention are new amphipols that are particularly suitable for solubilizing and stabilizing membrane proteins, especially compared to other polymers such as A8-35 or SMA. And the complexes formed with membrane proteins are of particular interest for the study of membrane proteins and the biotechnological use of these proteins. Stabilization of the protein in its native state and / or at least partial maintenance of its activity is essential for these applications, and as demonstrated in the experimental section, the polymers of the invention allow the solubilization of membrane proteins and their stabilization. In particular, the solubilization can be carried out without the use of detergents, which is a particular advantage.

[0058] There is no limitation regarding the type of membrane protein in the complex of the present invention. From a biotechnological point of view, membrane proteins known to be associated with pathology, membrane proteins as targets for drugs, or membrane proteins likely to be immunogenic are particularly preferred.

[0059] Possible uses for these complexes are thus, for example: -NMR, electron microscopy in liquid or solid media; -Diagnosis: detection of circulating antibodies, either soluble or carried by lymphocytes; - Vaccination or antibody production: presentation of membrane proteins as immunogens, - detection / use of the enzymatic activity of a membrane protein, where the membrane protein is an enzyme; - In pharmacology, the measurement of the affinity of cellular receptors for molecules of pharmaceutical interest

[0060] In this respect, functionalized polymers of the invention are particularly advantageous. The skilled artisan will be able to identify functionalization means relevant to the intended use of the polymers of the invention. By way of example and without limitation, such means can be selected from those given by Le Bon et al. (2014).

[0061] As mentioned above, the object of the present invention is a complex formed by the polymer of the invention as described in this application and a membrane protein. In certain embodiments, for the above mentioned uses, the complex of the invention can be present in aqueous solution, in lyophilized form or immobilized in a matrix or on a surface by means of functional groups grafted onto the polymer of the invention.

[0062] The skilled artisan will know how to experimentally determine the optimal mass ratio between the polymer of the invention and the membrane protein to obtain sufficient solubilization and / or stabilization of the membrane protein. In a particular embodiment of the detergent exchange for amphipol A8-35, the protein:amphipol ratio is 1:0.1 to 1:10, preferably 1:1 to 1:7.5, more preferably 1:1.5 to 1:5.

[0063] As explained in the experimental section, these compounds are amphipols, which are capable of stabilizing and / or solubilizing membrane proteins without the use of detergents, which are known to adversely affect protein stability and therefore activity, and thus advantageously, in one embodiment, the complexes are substantially free of detergents.

[0064] In another embodiment, the water-soluble complexes of the invention contain a lipid, or a mixture of lipids, which may be useful for diluting membrane proteins as described in the experimental section, or which may be derived from the membrane of the organism or organelle from which the solubilized and stabilized membrane protein in the complex is derived. In a particular embodiment, the lipid is 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC).

[0065] Advantageously, the water-soluble complex may contain all subunits of a multimeric protein associated with a membrane protein or any cofactor, as shown by the experiments on solubilization and stabilization of bacteriorhodopsin presented in the experimental section.

[0066] As mentioned above, the present invention also relates to a method for preparing an aqueous solution of one or more stabilized membrane proteins present in a complex with a polymer of the present invention.

[0067] Methods for preparing membrane protein complexes with prior art amphipols such as A8-35 are known in the prior art and include a prior solubilization of the membrane protein by combining it with a detergent medium. The method includes a subsequent step of replacing the detergent with an amphipol by reducing the detergent concentration to a concentration below the critical micelle concentration. The reduction in detergent concentration can be done by dilution, for example by adsorption of the detergent onto Bio-Beads® SM-2, dialysis, separation on molecular sieves, or gradients. However, this additional step is usually performed at the expense of the yield or final concentration of the membrane protein-containing complex.

[0068] The new amphipols of the invention have the ability to solubilize membrane proteins without this prior solubilization step with detergent preparations.An object of the invention is therefore the use of the polymers of the invention for the solubilization and / or stabilization of membrane proteins.

[0069] In another particular embodiment, the method for preparing a membrane protein solution or a mixture of membrane proteins according to the invention does not comprise a step of solubilization with a detergent preparation, and solubilization is carried out with one of the polymers of the invention, which in a further particular embodiment is selected from the polymers of formula I selected as follows: -w=0, R7 is a group selected from (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, where the one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C1-C8)alkenyl, and linear or branched (C1-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene, and linear or branched (C2-C8)alkynylene. Preferably, X is a single bond and R7 is C6cycloalkyl, preferably C7cycloalkyl, more preferably C8cycloalkyl. -w=0 and R7 is phenyl which is unsubstituted or substituted with one or more groups, the one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene. Preferably, X is a single bond and said phenyl is para-substituted. More preferably, in the case of monosubstitution, said phenyl is para-substituted with C1-C8 linear alkyl, even more preferably, said para linear alkyl is selected from methyl or ethyl.

[0070] In one embodiment, w=0, R7 is phenyl unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C1-C8) alkenyl, linear or branched (C1-C8) alkynyl, and X is selected from linear or branched (C1-C8) alkylene, preferably C1 alkylene or C2 alkylene.

[0071] The amphipols of the present invention have polyacrylic acid as a precursor. Thus, the synthesis of the amphipols of the present invention is very similar to the synthesis of other amphipols described in Gohon et al. (2006). Those skilled in the art will know without undue difficulty how to adapt this protocol to randomly graft the above-specified groups onto polyacrylic acid precursors by the formation of amide bonds between the primary amines carried by the groups and some of the carboxylate groups carried by the precursors. Preferably, the reaction is carried out in N-methylpyrrolidone (NMP) in the presence of dicyclohexylcarbodiimide (DCC). The synthesis can be carried out in succession, with each step consisting of the grafting of a certain building block. EXAMPLES

[0072] The following abbreviations are used in the examples below: -OTG: Octylthioglucoside -DMPC: Dimyristoylphosphatidylcholine -BR: bacteriorhodopsin -SERCA1a: Sarcoplasmic reticulum / endoplasmic reticulum Ca 2+ -ATPase 1a -SMA: styrene-maleic acid copolymer of the formula [ka] SMA(2:1) is two-thirds styrene and one-third maleic acid. SMA(3:1) is three-quarters styrene and one-quarter maleic acid. -A8-35: Amphipol of the following formula [ka] where x=~35%, y=~40%, and z=~25%. -A8-75: Same as above, but x=~75%, y=~25%, and z=0%. -E.coli: Escherichia coli -DDM: n-dodecyl-β-D-maltopyranoside -GFP: Green fluorescent protein -AChR: Acetylcholine receptor -EDTA: Ethylenediaminetetraacetic acid -CHAPS: 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate hydrate -SDS: Sodium dodecyl sulfate -Tris: Tris(hydroxymethyl)aminomethane

[0073] The following experimental data was obtained using membrane fractions and / or proteins to study the new amphipols of the invention. These examples are non-limiting, in particular the relative efficiency of the amphipols of the invention may vary depending on the membrane fraction or membrane protein considered. Nevertheless, the improved solubilization and / or stabilization properties of the amphipols of the invention relative to the reference polymers apply generally.

[0074] 1. Example 1 Synthesis of amphipol of the present invention As mentioned above, the amphipols of the invention have polyacrylic acid as a precursor. The synthesis of the amphipols of the invention is therefore very similar to the synthesis of A8-35 described in Gohon et al. (2006). It consists of randomly grafting a group onto a polyacrylic acid precursor by the formation of an amide bond between the primary amine carried by the group to be grafted and part of the carboxylate groups carried by the precursor. This reaction is carried out in N-methylpyrrolidone (NMP) in the presence of dicyclohexylcarbodiimide (DCC). The grafting and obtaining of different building blocks can be carried out in a sequential manner. In particular, when z is not 0, the grafting of the group onto part of the carboxylate groups of the precursor is carried out in a second step in NMP in the presence of 1-hydroxybenzotriazole (HOBt) / DCC.

[0075] The amphipol is then obtained in its acid form. After synthesis, the polymer was purified by three cycles of precipitation at pH = 3, followed by solubilization at pH = 10. The polymer was then dialyzed against deionized water and lyophilized.

[0076] The chemical composition of the polymer was elucidated by proton and carbon NMR analysis as well as pH measurement studies.

[0077] Example of synthesis of compound 23 In a 100 mL two-neck flask, 1.00 g of polyacrylic acid (1 eq., 13.89 mmol) is dissolved in 20 mL of N-methyl-2-methylpropane (NMP). The medium is left under stirring at 60° C. for 4 hours. 884.3 mg of cyclooctylamine (0.50 eq., 6.94 mmol) are dissolved in 2 mL of NMP and the resulting solution is added dropwise to the reaction medium while stirring the reaction medium. After the addition of the amine is complete, stirring is continued for 10 minutes, after which 1.58 g of DCC (0.55 eq., 7.64 mmol), previously dissolved in 4 mL of NMP, is added dropwise to the reaction medium. After all the reagents have been added, the reaction medium is maintained at 60° C. with stirring for 1 hour and then left under stirring without heating for 4 hours.

[0078] Once at room temperature, the reaction medium is filtered through a sintered filter (pore size 4) and 1.5 g of sodium methanolate (2 equivalents, 27.8 mmol) are added to the filtrate. Stirring is continued for approximately 15 minutes.

[0079] The reaction medium is then poured into 260 mL (10 equivalent volumes) of purified deionized water (Milli-Q®) and the pH is adjusted to 10 (pH paper) with sodium hydroxide (11 N). The solution is maintained under vigorous stirring for about 30 minutes and then filtered through a sintered filter (pore size 4). The filtrate obtained is manually added dropwise to 21 mL of 2 N hydrochloric acid (3 equivalents, 42 mmol) under vigorous stirring. The addition is carried out by means of a syringe connected at the tip to a Millipore filter system (0.45 μm PES and 0.2 μm cellulose acetate filters in series).

[0080] Once the addition is complete, the resulting suspension is filtered through a sintered filter (pore size 4) and the precipitate is dissolved in 50 mL of "Milli-Q" water after the addition of 1.9 mL (1.5 equivalents, 21 mmol) of 11 N sodium hydroxide. After four repetitions of this precipitation / solubilization cycle, the final basic solution is 4.5×10 -3 The mixture was dialyzed against N sodium hydroxide solution (molecular weight cut-off: 8,000 g mol -1The membrane was then freeze-dried.

[0081] One skilled in the art will have no difficulty in adapting this protocol to obtain other amphipols of the invention, particularly by varying the choice of amine that will react with the carboxylate groups of the polyacrylic acid.

[0082] 2. Example 2 Analysis of Amphipol Particles of the Invention by Steric Exclusion Chromatography (SEC) Materials and Methods Superose 12 / HR or 12 10 / 300 GL columns (GE Healthcare) were used.

[0083] The column was equilibrated in 20 mM Tris (pH 8.0)-100 mM NaCl buffer (FIG. 1A) or 20 mM sodium phosphate (pH 7.0)-100 mM NaCl buffer (FIG. 1B).

[0084] For BR / amphipol complexes (FIG. 1C), purple membranes were solubilized in OTG (see Example 2) and then BR was complexed with amphipol at a BR / amphipol mass ratio of 1:5. The column was equilibrated in 20 mM sodium phosphate (pH 7.0)-100 mM NaCl buffer.

[0085] Elution was carried out in 20 mM sodium phosphate (pH 7.0)-100 mM NaCl buffer.

[0086] result The elution profiles of the polymers of the invention obtained by SEC were compared with those of A8-35 (Figure 1A and B). In solution, A8-35 is known to form homogeneous, dense, spherical particles of 40 kDa containing about 9-10 molecules (Giusti et al., 2014; Gohon et al., 2006). Compared to A8-35, the tested polymers of the invention have a broader elution peak. This result can be explained by the presence of rings in the amphipols of the invention, which are less flexible than the linear octyl chains of A8-35 and therefore less favorable for the formation of spherical particles, resulting in the formation of larger and less homogeneous particles.

[0087] Also, the number of carbon atoms between the amide bond and the cycloalkyl group of the amphipols of the present invention appears to affect the size and homogeneity of the particles they form. Indeed, the addition of a carbon between the amide bond and the cycloalkyl ring (see elution profiles of compounds 3 and 11 in FIG. 1A) results in more homogeneous particles with an elution profile similar to that of amphipol A8-35. However, the presence of two carbon atoms (compound 17 in FIG. 1A) results in larger and less homogeneous particles. For amphipols grafted with phenyl groups (e.g., compounds 2 or 4), the elution profiles show that the particles formed by these polymers have a size of more than 40 kDa and are less homogeneous compared to A8-35. The homogeneity is greatly improved by the addition of ethyl groups (e.g., elution profile of compound 8), resulting in a particle size more consistent with that of A8-35.

[0088] Interestingly, the elution profiles of the complexes formed by compounds 11 and 8 with bacteriorhodopsin (BR) have similar homogeneity and size as the BR / A8-35 complex (FIG. 1C).

[0089] Thus, the polymers of the invention constitute customizable tools that exhibit particle self-assembly properties in aqueous media that are compatible with protein purification processes with respect to homogeneity, and these particles are comparable to those formed by the art standard Amphipol A8-35.

[0090] 3. Example 3 Study of stabilizing effect The membrane protein stabilization effect of the amphipols of the present invention was examined using two model membrane proteins, SERCA1a (sarcoplasmic reticulum / endoplasmic reticulum Ca 2+ -ATPase 1a) and bacteriorhodopsin from Halobacterium salinarum were tested.

[0091] Materials and Methods Model membrane protein used SERCA1a is a calcium pump that requires the hydrolysis of ATP molecules to perform its transport function. The protein belongs to the "P-type" ATPase family, and its transmembrane domain exhibits large amplitude conformational changes to allow the passage of calcium ions.

[0092] The purple color of BR is indicative of its native conformation. In its folded form, BR reversibly binds covalently to retinal (the chromophore) through the formation of a Schiff base. Protonation of this base shifts the absorption peak of retinal from ∼380 nm (free form) to 570 nm or ∼555 nm (bound form), depending on whether RB is embedded in a membrane or solubilized in aqueous solution. And the absorbance at 554 nm is indicative of the native conformation of BR.

[0093] SERCA1a production and activity measurement Rabbit sarcoplasmic reticulum vesicles were prepared from muscle tissue according to the protocol described by Champeil et al. (1985). These membranes are highly enriched for SERCA1a.

[0094] The ATPase activity of SERCA1a was indirectly measured by coupling it to multiple enzymatic reactions. The assay was performed in the presence of pyruvate kinase (0.1 mg / mL) and lactate dehydrogenase (0.1 mg / mL) to maintain a constant ATP concentration in the samples and to observe the consumption of ATP by measuring the decrease in absorbance at 340 nm of NADH over time. The sample buffer was 100 mM KCl, 1 mM MgCl, 0.2 mM Na ... 2+ , 0.1 mM Ca 2+ , 50 mM Tes-Tris (pH 7.5 at 20° C.), and 1-5 mM ATP. To perform the test, the reaction medium also contains 1 mM phosphoenolpyruvate and 0.15 mM NADH.

[0095] Formation of purple membrane and preparation of BR / amphipol complex. The preparation of the BR / amphipol complex is carried out in the following steps. i) Halobacterium salinarum was cultured and the membrane fraction corresponding to the purple membrane was purified according to the protocol previously described by Oesterhelt & Stoeckenius (1971). ii) Purple membranes were solubilized in 10 mM sodium phosphate-100 mM NaCl-100 mM OTG buffer for incubation in a cold room in the dark with stirring for 48 hours. Samples were centrifuged at 200,000 g for 20 minutes at 6° C. The supernatant was diluted with buffer without OTG to reduce the detergent concentration from 100 mM to 18 mM. iii) Surfactant replacement and preparation of BR / amphipol complexes The concentration of BR was determined by measuring the absorbance at 554 nm. The amount of amphipol added was calculated based on the mass of BR present in the sample, corresponding to a BR / amphipol mass ratio of 1:5. After 20 min of incubation at 4° C., polystyrene beads (Bio-Beads® SM2, BioRad) were added in a mass 20 times that of the surfactant present in the sample. After 2 h of incubation at 4° C. with agitation, the beads were removed.

[0096] The formation of BR / A8-35 or BR / A8-75 complexes (positive control) and BR complexes with amphipols of the invention was carried out according to the protocol of replacement and removal of the detergent by adsorption to polystyrene beads (Bio-Beads®, BioRad).

[0097] After 20 min of incubation in the presence of OTG and amphipol, Bio-Beads® were added to the mixture at a Bio-Beads® / OTG mass ratio of 20:1 and incubated for 2 h at 4° C. The absorbance at 554 nm of each sample was measured before and after 20 min of centrifugation at 100,000 g. The percentage of BR was calculated with respect to the absorbance measured with OTG before centrifugation.

[0098] Thermal denaturation of BR The ratio of BR protein to the various solubilizing agents tested is 1:5 in 20 mM sodium phosphate (pH 7.0)-100 mM NaCl buffer. Samples were incubated for 30 min at each temperature and cooled on ice before reading the absorbance at 554 nm. For BR samples in detergent (OTG, octylthioglucoside), a centrifugation step was performed to remove protein aggregates. The absorbance of the samples at 554 nm was measured in a UV-visible spectrophotometer using protein-free buffer as a reference. The absorbance values ​​are normalized to the absorbance measured at 4 °C.

[0099] result It was observed that compound 11 appeared to inhibit the ATPase activity of SERCA1a to a greater extent than A8-35 (not shown). Since the inhibitory effect of amphipols may be related to their stabilizing effect, compound 11 is likely to stabilize SERCA1a more effectively than A8-35.

[0100] The thermal stability of BR complexed with A8-35, A8-75, or the amphipols of the invention was analyzed. First, the ability of these polymers to maintain soluble BR in its native form was analyzed by measuring the absorbance of the supernatant at 554 nm.

[0101] The percentage of BR in the native form in the samples after complexation with an amphipol of the invention containing 25% hydrophobic cyclic groups and 75% unmodified carboxylate groups, after removal of the detergent with Bio-Beads®, is higher than that observed with Apol A8-75 as a reference, which is particularly poor in terms of stabilizing BR (Figure 2A and B). This result indicates that the cyclic group increases the stabilizing effect of the amphipol. Compounds 7 and 1 are "gentler" than A8-75 in terms of denaturation. However, the stability of BR decreases with increasing number of carbons between the ring and the amide bond, until it reaches a level of destabilization similar to that of A8-75 (e.g. BR complexed with compound 17). This effect is more pronounced for amphipols grafted with cycloalkyl groups than with phenyl groups (Figure 2B, compounds 2, 12, 22).

[0102] Effect of Amphipol Addition The effect of the addition of amphipols of the invention was studied by increasing the grafting rate of the polymer with isopropyl groups, which increase results in a reduction in the overall addition of amphipols. The reduction in addition thus obtained (by a grafting rate of isopropyl groups of up to 40%) improves the percentage of BR present in the sample in its native form after replacement of the surfactant with amphipols in the presence of Bio-Beads® (compare for example the absorbance of compounds 7 and 13). Conversely, when the addition density is reduced, a reduction in the amount of BR found in the supernatant is observed (compare for example compounds 11 and 15), which may be due to aggregation of BR.

[0103] For amphipols with higher grafting rates (50% cycloalkyl and / or phenyl groups, 50% unmodified carboxylate groups), the destabilizing effect of increasing the number of carbons between the ring and the amide bond is greatly reduced (Figure 2C).

[0104] The amphipols of the present invention grafted with 50% cycloalkyl and / or phenyl groups have loading densities comparable to or higher than SMA (2:1) (Figure 2). However, with the exception of compound 4, all amphipols tested provide more native BR than these two SMAs (Figure 2).

[0105] The thermal stability of BR complexed with the amphipols of the invention was tested on the fraction of BR that remained in the native state after detergent exchange with amphipols using Bio-Beads®. The absorbance at 554 nm of the samples was monitored as a function of temperature. A drop in absorbance indicates the denaturation of the protein (Figure 3).

[0106] In the case of the BR / A8-35 complex, protein denaturation occurs within a very narrow temperature range as shown by the resulting sigmoidal slope, where the molecular melting temperature (Tm) is approximately 63°C (Figure 3A). The Tm of the protein in OTG detergent is significantly lower (~47°C, Figure 3A), indicating weaker stabilization of the protein in OTG. Similar Tms are observed for the protein complexed with SMA (3:1) or SMA (2:1), ~45°C and ~49°C, respectively. Conversely, the Tms obtained with the amphipols of the invention are significantly higher than those with SMA, and in some cases are comparable or even higher than those of the BR / A8-35 complex (e.g., compounds 4 and 3, with Tms of ~57°C and ~66°C, respectively, Figure 3B).

[0107] Thus, with regard to stabilization, the amphipols of the invention are more effective than SMA and may be at least as effective as the reference amphipol A8-35 or even more effective, as suggested by the results obtained with SERCA1.

[0108] 4. Example 4 Evaluation of the solubilizing effect of the amphipol of the present invention on membranes with high membrane protein density Unlike detergents, A8-35 is known not to effectively solubilize SERCA1 from sarcoplasmic reticulum vesicles (Champeil et al., 2000). In general, membrane proteins are always solubilized with detergents, which are then exchanged for A8-35 to stabilize the membrane proteins.

[0109] Conversely, styrene-maleic acid (SMA) copolymers are polymers known to enable the solubilization of membrane proteins without the need for a detergent solubilization step, leading to the formation of lipid particles containing membrane proteins and lipids surrounded by SMA (SMA lipid particles, SMALPs for short). However, as mentioned above, the stability of BR in SMA is relatively poor compared to the same protein complexed with A8-35.

[0110] The solubilization properties of SMA, A8-35, and the amphipols of the present invention were compared on different types of membranes that contain more or less abundant membrane proteins (E. coli, Electric ray electric organ, Chlamydomonas reinhardtii thylakoid membrane, and Halobacterium salinarum purple membrane). The results obtained are specific to the membranes tested and indicate different properties of the different polymers. Thus, a polymer that is less effective on one of these model membranes may be quite effective on another type of membrane due to its specific protein and / or lipid composition.

[0111] Materials and Methods Preparation of fractions of Halobacterium salinarum purple membrane, Chlamydomonas reinhardtii thylakoid membrane, Torpedo electric organ membrane, or Escherichia coli membrane As described above, Halobacterium salinarum purple membrane is prepared according to the protocol described by Oesterhelt & Stoeckenius (1971).

[0112] Membranes extracted from the electric organ of Torpedo Ray, which are naturally rich in acetylcholine receptors (AChRs), are prepared according to the protocol described by Sobel et al. (1977).

[0113] Chlamydomonas reinhardtii thylakoid membranes are prepared according to the protocol described by Pierre et al. (1995).

[0114] E. coli membranes overexpressing the YidC protein C-terminally fused to GFP are prepared after mechanical lysis of the bacteria using a cell disrupter (Constant Systems). Cell debris and unbroken cells are removed by low-speed centrifugation. The supernatant is collected and the membranes are pelleted by high-speed centrifugation (100,000 g for 1 h). The protocol for the preparation of E. coli membranes is described in Angius et al. (2018).

[0115] Dilution of purple membrane fraction with DMPC vesicles DMPC vesicles (50 mg / mL) were extruded 10 times through a polycarbonate membrane (Whatman) with pores of 100 nm diameter. DMPC vesicles and purple membrane were mixed to obtain a BR:DMPC mass ratio of 1:5 and then incubated in a sonication bath for 30 min according to a protocol previously described in Knowles et al. (2009). Sample buffer contained 20 mM sodium phosphate (pH 7.0), 100 mM NaCl.

[0116] Solubilization of purple membrane in OTG Solubilization in OTG (100 mM) was performed as described above. Samples were incubated for 1 h at 4° C. before centrifugation (20 min, 100,000 g) and the supernatant was collected for absorbance measurement at 544 nm.

[0117] Solubilization of purple membrane by SMA or amphipol of the present invention For each condition, the BR / SMA or amphipol mass ratio is 1:6.25 and the BR / DPMC mass ratio is 1:5. The buffer used is 20 mM sodium phosphate (pH 7.0)-100 mM NaCl as described above.

[0118] The samples were incubated overnight at room temperature with agitation, then centrifuged at 100,000 g for 20 min and absorbance measurements were performed on the supernatants.

[0119] The absorbance was normalized to the absorbance value measured before the ultracentrifugation step.

[0120] Solubilization of E. coli membranes overexpressing recombinant membrane proteins with amphipols, SMA, or DDM of the present invention YidC is an evolutionarily conserved membrane protein with six transmembrane domains in Gram-negative bacteria. A recombinant version fused to GFP is used as a membrane-solubilized "reporter" protein to study the properties of the amphipols of the present invention.

[0121] The total membrane proteins present in the E. coli membrane extract are tested using a colorimetric test. The protein concentration is adjusted to 2 mg / mL using 20 mM Tris (pH 8)-150 mM NaCl buffer.

[0122] For solubilization, the total protein / amphipol or SMA mass ratio is 1:1. For control samples in the presence of detergent, the total protein / DMD mass ratio is 1:5. As mentioned above, the buffer used in all conditions consisted of 20 mM Tris (pH 8.0), 150 mM NaCl.

[0123] The samples are incubated with agitation for 2 hours at 4° C. Aliquots are taken from each tube every 15 minutes during the first hour of incubation and every 30 minutes for the next hour to monitor the solubilization kinetics.

[0124] At each sampling, the samples are centrifuged at 250,000 g for 10 min. The pellet separated from the supernatant is resuspended in the initial volume of solubilization using 20 mM Tris (pH 8), 150 mM NaCl buffer containing 5% SDS.

[0125] These samples are loaded with a blue loading dye corresponding to fluorescence measurement on an acrylamide gel, prepared according to the protocol described in Drew et al. (2008). Samples taken at the times mentioned above, corresponding to the supernatant and pellet of each condition, are plated on a 12% acrylamide gel.

[0126] Electrophoresis is carried out in a running buffer containing Tris-glycine (pH 8.3) in the presence of SDS. The proteins are run on the gel by applying a current of 100 V and 20 mA per gel for 1 hour.

[0127] After completion of the run, each gel was placed in “Milli-Q®” water and scanned through a laser scanner (λ excitation = 495 nm, λ emission GFP fluorescence can be measured by taking a fluorescent photograph of the gel at a fluorescence wavelength of 519 nm (λ=519 nm).

[0128] The ImageJ software allows integration of the area of ​​each band to quantify the fluorescence. Results were normalized to the fluorescence obtained with detergent (DDM, 1 h).

[0129] Solubilization of AChR-rich Torpedo Membranes with Amphipol, SMA, or CHAPS of the Invention - Solubilization of AChR AChR is an integral membrane protein. The electric organ of the Torpedo Ray is particularly rich in this protein, and is therefore an important source and model for studying acetylcholine receptors.

[0130] The total membrane proteins present in the Torpedo membrane extract are tested using a colorimetric test. The protein concentration is adjusted to 1 mg / mL using 5 mM NaP (pH 7.2)-100 mM NaCl-1 mM EDTA buffer.

[0131] For solubilization, the total protein / amphipol or SMA mass ratio is 1:5. In the case of the control sample, ie in the presence of detergent, the total protein / CHAPS mass ratio is 1:4.

[0132] The samples are incubated with stirring for 1 h at 4° C. and then centrifuged at 100,000 g for 20 min. The pellet separated from the supernatant is resuspended in the initial volume of solubilization using 5 mM NaPi (pH 7.2)-100 mM NaCl-1 mM EDTA buffer.

[0133] Samples corresponding to the supernatant and pellet of each condition are mixed with blue loading dye according to the protocol described by Laemmli (1970) and then plated on a 10% acrylamide gel.

[0134] Electrophoresis is carried out in a running buffer containing Tris-glycine pH 8.3 in the presence of SDS. The proteins are run on the gel for 1 hour by applying a current of 100 V and 20 mA per gel.

[0135] After the run is complete, each gel is stained with Coomassie blue and a photograph of the gel is taken under white light.

[0136] result It was observed that the solubilization efficiency of the polymers is closely related to the composition (both in terms of proteins and lipids) of the membranes from which the membrane proteins are extracted. In fact, none of the tested polymers, whether A8-35, the amphipols of the invention or even SMA, is able to solubilize membranes rich in proteins such as the purple membrane of H. salinarum (Figure 4A) or the membranes of Chlamydomonas reinhardtii thylakoids (not shown), whereas most of them are able to solubilize the membranes of E. coli overexpressing recombinant proteins such as YidC-GFP (see Figure 7). Nevertheless, the amphipols of the invention are generally more effective than the A8-35 polymer or SMA.

[0137] The purple membrane of H. salinarum contains 75% BR for 25% lipid. Due to this unique composition, its solubilization by SMA is only possible if the membrane is previously fused with DPMC vesicles in order to "dilute" the BR protein in lipid (Knowles et al., 2009; Orwick-Rydmark et al., 2012). Under these conditions, our amphipols are also able to solubilize BR, whereas A8-35 remains ineffective (Figure 4B).

[0138] For the amphipols of the present invention that contain grafts with phenyl groups, it is observed that the addition of an ethyl group to the aromatic moiety can improve the solubilization properties (compare the percentage of solubilized BR for compounds 4, 6, and 8). Note that the placement of the ethyl group at the para (4-position) or ortho (1-position) position does not appear to be important, as compounds 8 and 24 provide comparable solubilization (FIG. 4B).

[0139] The amphipols of the invention grafted with cycloalkyl are particularly effective in solubilizing BR. In particular, compound 23 has been found to be as effective as SMA(2:1) (compare pellet size), but the amount of BR present in the supernatant in native form is higher than that obtained with SMA(2:1) or SMA(3:1). Compound 21 is significantly more effective than SMA(2:1).

[0140] Furthermore, steric exclusion chromatography analysis of the BR / lipid / amphipol complexes according to the present invention showed that the complexes were much more homogeneous in size than those obtained with SMA, which is particularly advantageous in protein characterization techniques (not shown).

[0141] The efficiency of the polymers of the invention to extract the membrane protein YidC-GFP overexpressed in E. coli membranes was also tested and compared with A8-35 and SMA. By setting the protein / polymer ratio at 1:1, i.e. at low polymer concentrations, the amphipol polymers of the invention have a better extraction efficiency of the recombinant protein of interest than A8-35 and SMA. In fact, after 2 hours of incubation, the amount of protein extracted in the presence of the amphipol polymers of the invention is about 80% compared to about 35% in the presence of A8-35 or SMA. Furthermore, the extraction kinetics of the membrane protein of interest is faster (Figure 7). Thus, the amphipols of the invention are effective at low concentrations and allow for a more rapid extraction, which is particularly advantageous, especially in the context of recombinant membrane protein production.

[0142] The efficiency of the polymers of the invention to extract acetylcholine receptors (AChR) from the membrane of the electric organ of Torpedo was also tested. At a protein / polymer ratio of 1:5, the polymers of the invention show a better extraction efficiency of the membrane protein of interest than A8-35 and SMA (2:1) and comparable to SMA (3:1) (Figure 8, compare the columns marked with S). ​​Surprisingly, a control polymer (A8-50) containing octylamine chains and thus having the same number of carbon atoms as cyclohexylethylamine groups (as in compound 19) and cyclooctylamine groups (as in compound 23) grafted in the same proportion (50%) is less effective than the polymers of the invention in extracting AchR. This result indicates that the molecular structure of the hydrophobic group may play an important role in the amphipols of the invention.

[0143] Notably, the amphipols of the invention are also much more effective than A8-50 or A8-35 at solubilizing the sodium-potassium pump from the membrane of Torpedo electric organ.

[0144] At lower polymer concentrations, the amphipols of the invention were also found to be more effective than A8-35, A8-50 and SMA3-1 in solubilizing AChR or the sodium-potassium pump (data not shown).

[0145] 5. Example 5 Study of the stabilizing effect of amphipols on membrane proteins after direct extraction from membranes The stabilizing effect of the amphipols of the invention on BR was tested immediately after solubilization of purple membranes without a solubilization step with OTG and a subsequent exchange step of OTG with amphipols. Two independent sets of experiments were performed. The results of the first experiment are shown in Figures 5A and B. For the second series of experiments, each condition was performed at least three times (Figure 6).

[0146] Materials and Methods Solubilization of purple membrane after fusion with preformed DMPC vesicles (i) As described above, purple membranes were fused to DMPC vesicles according to the protocol described in Orwick-Rydmark et al. (2012) before the solubilization step with amphipols. After 24 h of incubation at room temperature, samples were centrifuged at 100,000 × g for 20 min. The supernatant was separated from the pellet. (ii) The absorbance of the supernatant at 554 nm, which reflects the amount of native BR, was measured and plotted against the value measured before centrifugation to quantify the solubilization efficiency (Figure 5A and gray bar in Figure 6A), which provides the percentage of native BR solubilized in the supernatant.

[0147] Assessment of BR stability After solubilization, the samples were incubated at 50° C. for 6 h, after which the absorbance at 554 nm was measured. The absorbance values ​​were normalized to the initial absorbance values ​​measured before incubation (FIGS. 5B and 6B). The black bars in FIG. 6A correspond to the amount of stabilized BR, normalized to the initial amount of BR before centrifugation (i.e. before the solubilization step). To observe the kinetics of BR denaturation, the experiment was repeated by measuring the absorbance at 554 nm of the samples every hour (FIG. 6B).

[0148] Comparing the benchmark polymers, A8-35 and the two SMAs, the solubilizing ability of SMA correlates with its low stabilizing ability for BR. Conversely, A8-35, which solubilizes BR to a limited extent, effectively stabilizes BR (Figures 5A and 5B and 6A).

[0149] The polymers of the invention tested are able to solubilize BR as effectively as SMA (3:1), while stabilizing the protein at least as effectively as A8-35 (compare absorbance of compounds 19 and 23 with the reference polymers, Figures 5A, B and 6).

[0150] 6. Conclusion The experimental data presented therefore show that the new amphipols of the invention are new polymers with particular advantages. In fact, the purification of membrane proteins requires their solubilization while maintaining their conformation and stability, and these two steps may seem to be in opposition, since solubilization in particular involves the use of detergents with dissociation forces, which generally lead to at least partial denaturation of the membrane protein over time. Based on model membrane proteins, the results show that the new amphipols of the invention have interesting solubilization properties for the purification of membrane proteins, while maintaining the stability of these proteins, and thus their conformation and biological activity, which is essential in the study or use of membrane proteins in biotechnological applications and has not been observed with the polymers of the prior art. Furthermore, the polymers of the invention are effective even at low concentrations, which is particularly advantageous in these applications.

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Claims

1. A water-soluble membrane protein-amphiphilic vinyl polymer conjugate, the amphiphilic vinyl polymer being of formula I, 【Chemistry 1】 Here, M ＋ is an alkali metal, R １ , R ４ , R ５ , R ８ each independently represents a hydrogen atom or a methyl group; R ２ , R ６ , R ９ are each independently a group selected from a hydrogen atom and a linear or branched (C1-C8) alkyl; R １０ is a group selected from linear or branched (C1-C5) alkyl; R ３ and R ７ are each independently -hydrogen (where X is a single bond), - linear or branched alkyl of at least 6 carbon atoms, linear or branched alkenyl of at least 6 carbon atoms, or linear or branched alkynyl of at least 6 carbon atoms (wherein X is a single bond); - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein the one or more groups are selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, and linear or branched (C2-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, wherein the one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C2-C8)alkenyl, linear or branched (C2-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene and linear or branched (C2-C8)alkynylene, - polyunsaturated or monounsaturated polycyclic compounds, unsubstituted or substituted by one or more groups, selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, linear or branched (C2-C8) alkynyl, where X is a single bond or selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene and linear or branched (C2-C8) alkynylene, or phenyl, unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, linear or branched (C2-C8) alkynyl, where X is a single bond or selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; is a group selected from R ３ Or R ７ When one of the groups is a linear or branched alkyl of at least 6 carbon atoms, a linear or branched alkenyl of at least 6 carbon atoms, or a linear or branched alkynyl of at least 6 carbon atoms, or a hydrogen atom, and X is a single bond, the other R ３ Or R ７ The base is - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein the one or more groups are selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, and linear or branched (C2-C8) alkynyl, where X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; - polyunsaturated or monounsaturated, unsubstituted or substituted by one or more groups (C3-C10)cycloalkenyl or (C3-C10)(hetero)cycloalkenyl, wherein the one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C2-C8)alkenyl, linear or branched (C2-C8)alkynyl, where X is a single bond or is selected from linear or branched (C1-C8)alkylene, linear or branched (C2-C8)alkenylene and linear or branched (C2-C8)alkynylene, - polyunsaturated or monounsaturated polycyclic compounds, unsubstituted or substituted by one or more groups, selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, linear or branched (C2-C8) alkynyl, where X is a single bond or selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene and linear or branched (C2-C8) alkynylene, or phenyl, unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, linear or branched (C2-C8) alkynyl, where X is a single bond or selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene; is selected from Here, w, x, y, and z correspond to the respective proportions of the structural units. -x is 20 to 90%, -y is 0 to 80%, -w is 0 to 80%, -z is 0 to 60%, provided that w+x+y+z=100%, and y or w is at least 10%. A water-soluble membrane protein-amphiphilic vinyl polymer complex, comprising:

2. In the amphiphilic vinyl polymer of formula I, M ＋ Li ＋ , Na ＋ , K ＋ The water-soluble membrane protein-amphiphilic vinyl polymer complex according to claim 1, wherein the water-soluble membrane protein-amphiphilic vinyl polymer complex is selected from the group consisting of:

3. In the amphiphilic vinyl polymer of formula I, M ＋ Na ＋ 2. The water-soluble membrane protein-amphiphilic vinyl polymer complex according to claim 1, wherein

4. 2. The water-soluble membrane protein-amphiphilic vinyl polymer conjugate according to claim 1, characterized in that in the amphiphilic vinyl polymer of formula I, w and / or y is 25% or more.

5. In the amphiphilic vinyl polymer of formula I, R １０ 5. The water-soluble membrane protein-amphiphilic vinyl polymer complex according to claim 1, wherein is an isopropyl group.

6. 6. A water-soluble membrane protein-amphiphilic vinyl polymer conjugate according to any one of claims 1 to 5, characterized in that in the amphiphilic vinyl polymer of formula I, x is 75% or less.

7. A water-soluble membrane protein-amphiphilic vinyl polymer complex according to any one of claims 1 to 6, characterized in that in the amphiphilic vinyl polymer of formula I, w and / or y are 25% to 50% and z is 0% to 40%.

8. In said amphiphilic vinyl polymer of formula I, -R ３ and / or R ７ is selected from those described in claim 1 (C ６ ~C ８ ) cycloalkyl; or -R ３ and / or R ７ is phenyl, The water-soluble membrane protein-amphiphilic vinyl polymer conjugate according to any one of claims 1 to 7, characterized in that X is a single bond or is selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene.

9. In said amphiphilic vinyl polymer of formula I, -R １ , R ２ , R ４ , R ５ , R ６ , R ８ , R ９ is a hydrogen atom, -R ３ and / or R ７ But (C ６ ~C ８ ) cycloalkyl and X is a single bond; -R １０ is an isopropyl group, -x is 35% or 50%, - y is 25% or more, - w is 0% or 25%, -z is 0%, 15% or 40%, with the proviso that w+x+y+z=100% A water-soluble membrane protein-amphiphilic vinyl polymer complex according to any one of claims 1 to 8.

10. The amphiphilic vinyl polymer is 【Table 1】 10. The water-soluble membrane protein-amphiphilic vinyl polymer complex according to claim 1, wherein the water-soluble membrane protein-amphiphilic vinyl polymer complex is selected from the group consisting of:

11. A water-soluble membrane protein-amphiphilic vinyl polymer conjugate according to any one of claims 1 to 10, characterized in that the amphiphilic vinyl polymer of formula I is functionalized by grafting an affinity label, a fluorescent probe, or an immunostimulatory molecule.

12. 12. A water-soluble membrane protein-amphiphilic vinyl polymer complex according to any one of claims 1 to 11, further comprising a lipid compound.

13. 13. An aqueous solution comprising a complex according to any one of claims 1 to 12, characterized in that one or more membrane proteins are stabilized.

14. 14. A method for preparing a membrane protein solution according to claim 13, comprising a solubilisation and / or stabilisation step in which a protein fraction derived from a biological or synthetic membrane, containing said membrane protein or a mixture of said membrane proteins, is incorporated into said vinyl polymer of formula I.

15. 15. The method for preparing a membrane protein solution or a mixture of membrane proteins according to claim 14, characterized in that the one or more membrane proteins are one or more recombinant proteins expressed in prokaryotic or eukaryotic cells and / or one or more native proteins expressed in specialized membranes with high protein density.

16. 16. A method for preparing a membrane protein solution or a mixture of membrane proteins according to claim 15, characterized in that it first comprises a step of solubilising the membrane protein or the mixture of membrane proteins by combining the protein fraction or the mixture of membrane proteins with a detergent medium.

17. A method for preparing a membrane protein solution or a mixture of membrane proteins as described in claim 15, characterized in that it first comprises a step of fusing the protein fraction containing the membrane protein or the mixture of membrane proteins with vesicles of phospholipids or vesicles of a mixture of phospholipids.

18. A method for preparing a membrane protein solution or a mixture of membrane proteins according to claim 15 or 17, characterized in that the solution of the protein fraction containing the membrane protein is not an aqueous solution of membrane protein in a detergent medium in micellar form.

19. 11. A polymer of formula I according to any one of claims 1 to 10, wherein w=0 and R ７ but, - (C3-C10)cycloalkyl or (C3-C10)(hetero)cycloalkyl, unsubstituted or substituted by one or more groups, wherein the one or more groups are selected from linear, cyclic or branched (C1-C8)alkyl, linear or branched (C2-C8)alkenyl, and linear or branched (C2-C8)alkynyl, or - phenyl substituted by one or more groups, said one or more groups being selected from linear, cyclic or branched (C1-C8) alkyl, linear or branched (C2-C8) alkenyl, linear or branched (C2-C8) alkynyl, is a group selected from wherein X is a single bond or a polymer selected from linear or branched (C1-C8) alkylene, linear or branched (C2-C8) alkenylene, and linear or branched (C2-C8) alkynylene.

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