Copolymers based on poly(alpha-olefin-co-maleic acid) for the solubilization, isolation and molecular labelling of membrane proteins in aqueous media

Abstract

The present disclosure relates to specifically defined copolymers, a method for their preparation, a complex containing this copolymer, the use of the copolymer in particular for solubilizing and stabilizing membrane proteins as well as a kit containing the polymer according to the present disclosure in particular for this use containing the polymers according to the present disclosure.

Description

[0001] CUB-PA12-PCT

[0002] Copolymers Based on Poly(a-olefin-co-maleic acid) for the Solubilization, Isolation and Molecular Labelling of Membrane Proteins in Aqueous Media

[0003] TECHNICAL FIELD

[0004] The present disclosure relates to specifically defined copolymers, a method for their preparation, a complex containing this copolymer, the use of the copolymer in particular for solubilizing and stabilizing membrane proteins as well as a kit containing the polymer according to the present disclosure in particular for this use containing the polymers according to the present disclosure.

[0005] TECHNICAL BACKGROUND

[0006] Membrane proteins are of great relevance for the biomedical research. About one third of human genes code for membrane proteins which are strongly involved in cellular communication, intermembrane transport of molecules and enzymatic reactions and thus can be targets for drugs. Due to their natural interaction with the alkyl chains of the cell membrane lipids and their structure with helices and sheets inserted into the hydrophobic membrane, the isolation and stabilization of membrane proteins is challenging. Amphiphilic detergents are needed for the disintegration and later mimicking the cell membrane. Commonly, the surface of membrane proteins is characterized by the localization of hydrophobic amino acids in the regions inserted in the cell membrane and the localization of hydrophilic amino acids in the peripheral parts of membrane proteins. This Janus-like structure is the origin of the reduced solubility and thus stability of membrane proteins in aqueous media.

[0007] However, the function of cell membrane proteins often relies on their natural lipid environment which is disrupted by solubilization using detergents. Furthermore, the structural stability of membrane proteins is disturbed after solubilization using common detergents (e.g., sodium dodecyl sulfate (SDS) or n-Dodecyl-beta-maltoside (DDM)), as they tend to aggregate especially during their expression and purification in large quantities.

[0008] The commercially available detergents (e.g. SDS) allow the dissolution of the membrane as well as the solubilization of the membrane protein of interest with high yield. [1] However, the detergent solubilization of membrane proteins is often followed by denaturation and inactivation. Moreover, the detergent concentration must be always kept above the critical micelle concentration (CMC). Detergent concentrations in the regime of the CMC can have detrimental effects on the membrane protein functionCUB-PA12-PCT

[0009] as they poorly mimic the lipids of the cell membrane. Beside this, the presence of detergence in membrane protein solutions can have detrimental effects in further analysis or application of the membrane proteins (e.g., cryo transmission electron spectroscopy or crystallization). Below the CMC the solubility of the membrane protein cannot be ensured due to the not fully detergent-covered hydrophobic patches of the protein's transmembrane part.

[0010] Classic amphiphilic polymers called amphipols are used to overcome the challenges as they enable the stabilization of solubilized membrane proteins. The amphiphilic polymer directly binds with their hydrophobic parts to the likewise hydrophobic parts of the protein transmembrane region. This makes the use of detergent redundant since solubilization and stabilization of the membrane protein can be performed in a unique one-step process. The amphipol prototype A8-35 as described in WO1998027434A1 consists of a polyacrylic acid backbone grafted with octylamine (~25%), isopropylamine (~40%) and ~35% remaining carboxylic acid groups. However, the use of A8-35 requires in general the detergent-based solubilization of the membrane protein which can lead to a loss of function of the protein as the amphiphilic polymer surrounding the membrane protein does not mimic the unique natural environment of the cell membrane surrounding the membrane protein.

[0011] A new class of amphiphilic polymers published and patented are SMAs (Styrene Maleic-Acid, WO2006129127, W02011004158), CyclApols (US20220119558A1) and AASTYs (Poly(acrylic acid-co-styrene).[2-4] The stabilization of these new polymers is based on their ability to mimic the natural environment of membrane proteins by the formation of small complexes called nanodiscs. [2, 5].

[0012] The ring-like nanometer-sized nanodisc complexes consist of one or more amphiphilic polymer molecules which encircle a patch of cell membrane including the membrane protein. [6, 7] Additionally, SMAs, CyclApols and AASTYs allow the solubilization and stabilization of membrane proteins without the use of detergents. However, the described polymers are limited with respect of either controlling the polymer length, the polydispersity, the monomer sequency, the flexibility of varying the hydrophobic or hydrophilic sidechains of the polymer and the ability of further functionalization of the polymer chain with fluorophores or biomolecular tags without affecting the polymerization efficiency itself.

[0013] The solubilization efficiency as well as the stability of the formed membrane protein containing nanodisc strongly depends on the characteristics of the used polymer. Polymers, like SMAs, CyclApols and AASTYsCUB-PA12-PCT

[0014] are stabilized by the electrostatic repulsion of the negatively charged polymer chain. The charges are generated by carboxylic groups which are implemented in the polymer by using monomers like acrylic acid or maleic acid and copolymerization with styrene in the case of SMA and AASTY.[3, 8-10] These nanodisc forming polymers bear the risk of aggregation as they are highly sensitive to the presence of divalent ions, low pH values (<6.5) and high ionic strength due to the covered negative charges of the stabilizing carboxylic acid group. In contrast, polymers with highly charged polar groups like phosphates, sulfates, quaternary amines as well as flexible non-polar side chains, like described in this disclosure, enhance both the solubilization efficiency as well as the stability of the nanodiscs.

[0011] Besides the described drawbacks of current copolymers for membrane protein solubilization, new polymer backbones allow the implementation of functionalities in the polymer chain such as fluorophores or biomolecular tags as already shown in different fields.

[0012] Furthermore, new backbones show different backbone flexibilities, which can strongly influence the migration of the copolymer in the membrane during solubilization and can limit the downstream applications.

[0015] The length of the polymer as well as the polydispersity and the homogenous sequence of co-polymerized monomers like acrylic acid and styrene are critical factors for the solubilization itself or for the field of use like cryo transmission electron microscopy.

[0013] Highly pure and homogenous samples are typically required to obtain a high-resolution structure from Cryo-TEM. For detergent-solubilized membrane proteins, the heterogeneity of the detergent micelle is known to be a key factor determining the success of Cryo-TEM work.

[0014] For polymer-stabilized membrane proteins, the polymer nanodisc region represents one of the regions with the highest structural heterogeneity, potentially hindering successful structure determination. Therefore, controlling polymer backbone flexibility and length can significantly affect the resolution, and feasibility, of a Cryo-TEM project. A common issue with Cryo-TEM sample preparation, is that proteins tend to attach to the harsh environment found at the interface between the sample and the air / vacuum, leading to preferential orientation of the proteins or protein denaturation. A solution to this is to use grids with a support layer that would specifically recognize the protein of interest either through its His-tag or other affinity labels, bringing them away for the air-water interface.

[0016] [W02020041202A1, US20210041388A1,

[0015] ] Adding an affinity label (e.g. biotin) to the co-polymers would create a general tool affinity-grid-based Cryo-TEM work with membrane proteins bringing two key advantages: (i) it would bring the proteins away from the air-water interface and (ii) it should impose the correct orientation distribution of the sample, independent of the protein's identity. Thus, it is tremendously important to search for new polymer backbones with different combinations of hydrophilicCUB-PA12-PCT

[0017] and hydrophobic side chain combinations to enable protein structure analysis of membrane proteins using cryo-TEM.

[0018] SUMMARY OF THE DISCLOSURE

[0019] To overcome the drawbacks of current copolymers for membrane solubilization, the technical problem underlying the present disclosure is to provide a copolymer, which bears a polymer backbone adaptive with respect of the used hydrophilic and hydrophobic side chains, is stable at high ionic strength, is resistant against divalent ions (e.g., Ca2+, >5 mM), can be chemically modified, is efficiently solubilizing membrane proteins, and is efficiently stabilizing membrane proteins.

[0020] This has been achieved by the subject-matter of the independent claims. Preferred embodiments are defined in the dependent claims.

[0021] According to the present disclosure, there is provided a poly(a-olefine-co-maleic acid) copolymer comprising repeating units of formulas (1) and (2)

[0022]

[0023] formula (2)

[0024] formula (1)

[0025] or comprising repeating units of formulas (1) and (2a)CUB-PA12-PCT

[0026]

[0027] formula (1) formula (2a)

[0028] wherein

[0029] Xi and X2are independently NH, (Cl-C5-alkyl)N, in particular (CH3)N, O, O', S, S';

[0030] X3is N;

[0031] a is an integer of 1 to 20;

[0032] Ri, R2, R3, and R4, can be independently derived from the following:

[0033] a (C3-C10) cycloalkane or a (C3-C10) (hetero)cycloalkan, which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, and linear or branched (C1-C8) alkynyls;

[0034] a (C3-C10) cycloalken or a (C3-C10) (hetero)cycloalken, which can be poly or monounsaturated, and which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, and linear or branched (C1-C8) alkynyls; a poly or monounsaturated polycycle, which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, and linear or branched (C1-C8) alkenyls, linear or branched (C1-C8) alkynyls;

[0035] a linear C2 to CIO alkyl-amine, wherein one primary and / or mono-, di- or trimethylated aminegroup is present , wherein in case of the trimethylated amine a quaternary amine with a positive charge is present, the linear alkyl amine e.g. dimethylaminopropylamine can be further sulfonated by using 1,3 propane sultone, carboxylated by using exemplary a halo-(C2-C7)alkanoic acid (e.g. chloroacetic acid) or phosphorylated

[0036] an polyol, wherein a polyol is any compound having at least two hydroxyl groups;

[0037] a linear or cyclic mono-, di- or polysaccharide;

[0038] a linear or branched polyethylene oxide;

[0039] an linear or branched alkylated quaternary trimethyl ammonium chlorides;CUB-PA12-PCT

[0040] a group selected from biological tags, fluorophores, peptides, biotin and functional groups for click chemistry;

[0041] H, or an alkali metal ion for example Na+, and

[0042] R3can be further selected, with the provisio that the above definitions are not included in this definition of R3, from the group consisting of a linear alkane chain, optionally, containing or terminating with a cyclic carbon; a linear chain alkoxy alkane of the formula -(CH2)qO(CH2)rCH3where q is 1 to 5 and r is 1 to 15; an alkoxy alkane containing or terminating with a cyclic carbon chain; a halogenated alkane; a halogenated cycloalkane; a halogenated arene; a chain containing a repeating sequence of (CH2CH2O)tterminating with -OR6whereintequals a value of 1 to 50 and R6is hydrogen, a linear alkane, a cyclic alkane, trifluoromethyl, a halogenated alkane, a halogenated cycloalkane, or a halogenated arene; a hydrogen, and a mixture thereof

[0043] and salts thereof,

[0044] provided that R1 and R2 are not simultaneously O' or HO

[0045] The group R3 can also be bound to the back bone of the polymer via an N-, O-, or S-containing group

[0046] The term "derived" as used herein means that the compounds specifically list in connection with Rl, R2, R3, and R4 are used to provide the groups as present in the copolymer according to the present disclosure. That is, they are present in the copolymer according to the present disclosure in that one atom, in particular H is omitted to provide the linkage to Xi, X2and X3.

[0047] The copolymer according to the present disclosure does also envisage the presence of further repeating units, that is also terpolymers are envisaged according to the present disclosure.

[0048] The synthesis and use of poly(a-olefin-co-maleic acid) and poly(a-olefin-alt-maleic acid) polymers was described in literature before (WO2021165227A1, W02023108069A1,

[0016] )

[0049] Surprisingly it was found that the modification of poly(a-olefin-co-maleic acid) with respect to their hydrophilic and hydrophobic moieties dramatically increase their beneficial properties for the isolation, stabilization and application of membrane proteins in copolymer nanodiscs.CUB-PA12-PCT

[0050] The copolymer of the present disclosure generally comprises of a hydrophobic region comprising a hydrophobic aliphatic chain having a length of 3 to 23, preferably 3 to 16, most preferably 4 to 12 carbon atoms.

[0051] The copolymer of the present disclosure generally comprises of a hydrophilic region comprising a hydrophilic region based on maleic acid anhydrides functionalized with one or more different functionalities or additionally unfunctionalized maleic acid.

[0052] That is, the copolymer according to the present disclosure comprises repeating units derived from hydrophobic monomers (the a-olefin) and repeating units derived from hydrophilic monomers (the maleic acid residue). The ratio of the hydrophobic and hydrophilic monomers can be in the range of 1:1 - 3:1, preferred in the range of 2:1 - 1.5:1 and most preferred in the range between 1.1:1 and 1.5:1.

[0053] Ther term "functionality" or "functionalized" as used herein means that the basic a-olefin-maleic acid copolymer known from WO 2023 / 108069 Al is provided with the functional residues Xi_Ri, X2-R2, R3, and X4-R4. The terms "functionality" or "functionalized" if used in connection with chemical compounds and residues of the formulas does mean that the chemical compound and residue do bear a functional group.

[0054] The hydrophilic region of the amphiphilic polymer may be net negatively, neutral or positively charged in an aqueous solution.

[0055] The copolymer can spontaneously form nanoparticles in solution which may include lipids and biomolecules like proteins and more preferably membrane proteins.

[0056] The number average molecular weight (Mw) of the copolymer can be 24.000 g / mol or less, for example 20.000 g / mol or less, preferably 10.000 g / mol or less, or 8.000 g / mol or less. The lower value of the Mw can be 1.000 g / mol or more, for example 2.000 g / mol, or 3.000 g / mol. In particular, the range of the Mw can be 1.000 g / mol to 8.000 g / mol, for example 3.000 to 8.000 g / mol.

[0057] The n umber average molecular weight may be determined using gel permeation chromatography (GPC), preferably using poly(methyl acrylate) as calibration standard.CUB-PA12-PCT

[0058] The copolymer according to the present disclosure can be a random copolymer or a block copolymer.

[0059] The copolymer may be an alternating copolymer or non-alternating copolymer. An alternating copolymer is a copolymer comprising two species of monomeric units distributed in alternating sequence.

[0060] The copolymer may be a non-alternating copolymer with more than two species of monomeric units distributed in randomized sequence over the polymer chain.

[0061] The copolymer can be derived from a copolymer of maleic anhydride and at least one alkene used as repeating units.

[0062] The alkene, which can be used in the production of the copolymer, may be selected from one or more of 1-penten, 1-hexen, 1-hepten, 1-octen, 1-nonen, 1-decene, 1-undecene, 1-dodecene, 1-tridecene, 1-tetradecene, 1-pentadecene, 1-hexadecene, 1-heptadecene, 1-octadecene, and preferably the alkene is 1-octen.

[0063] The copolymer may comprise the following building blocks:

[0064] the hydrophobic building block is a linear alkyl group, preferably a linear C5 to C18 alkyl group, most preferably the linear alkyl group is a hexyl, heptyl or octyl group;

[0065] the hydrophilic building block is a maleic acid group, preferably functionalized with polar molecules.

[0066] The added functionality of the hydrophilic building blocks can be chosen from hydroxyl, amino, ether, carboxylic acid, carboxylate, phosphate, phosphonate, phosphocholine, carboxylic ether, carboxylic ester, phosphate ester, amide, phosphonamide, ammonium, or their respective salts, ammonium cations (e.g., alkylammonium cations such as mono-, di-, tri-, or tetra-alkylammonium cations). Negatively charged hydrophilic groups may include, but are not limited to sulfates, carboxylate or phosphate groups; linear alkyl primary or mono-, di- or trimethylated amines, wherein in case of the trimethylated amine a quaternary amine with a positive charge may be present. In the case of a zwitterionic hydrophilic side chain, the linear alkyl amine (e.g. dimethylaminopropylamine) can be further sulfonated by using exemplary 1,3 propane sultone, carboxylated by using exemplary a halo-(C2-C7)alkanoic acid (e.g. chloroacetic acid) or phosphorylated;CUB-PA12-PCT

[0067] molecules from the class of polyols like tris(hydroxymethyl)aminomethane, l-amino-2,3-propandiol, 2-amino-2-methyl-l,3-propandiol, l-amino-2,3-butanediol, 3-amino-l,2-propanediol, 1-amino-l,2-ethanedio or 2-amino-2-deoxy-D-glucitol;

[0068] molecules from the class of amine functionalized linear or circular mono-, di- or polysaccharides like N-methyl-D-glucamine, 2-amino-2-deoxy-glucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxy-galactose, 2-amino-2-deoxy-mannose, 2-amino-2-deoxy-ribose, 2-amino-2-deoxy-arabinose, 2-amino-2-deoxy-xylose;

[0069] molecules from the class of amine functionalized linear or branched polyethylene oxides like 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol;

[0070] molecules from the class of amine functionalized linear or branched alkylated quaternary trimethyl ammonium chlorides like (2-aminoethyl)trimethylammoniumchlorid

[0071] The hydrophilic building block of the polymer can contain one, two or more different functionalities.

[0072] The functionality, additionally to the defined above can be chosen from the class of biological tags (e.g., Biotin-PEG4-Amine) or fluorophores (e.g. Fluoresceine amine) or peptides (e.g., Rho tag) to implement a polymer intrinsic functionalization like target specificity, binding to affinity resins or labeling with chromophores for spectroscopic detection.

[0073] Definitions

[0074] The term "amphipol" refers to vinyl polymers which are capable of solubilizing membrane proteins and keep them stable in solution in their native form.

[0075] The term "nanodisc" refers to a small nanometer-sized complex formed by an amphiphilic polymer and lipids and is capable of stabilizing membrane proteins.CUB-PA12-PCT

[0076] The term "membrane protein" comprises proteins which contain a hydrophobic transmembrane domain or are associated with the membrane by at least one hydrophobic domain. The membrane proteins can be monomeric or oligomeric and can be associated with cofactors.

[0077] The term "grafting" describes in the context of the present disclosure the functionalization of maleic acid anhydride units within the polymer chain with other amine, thio or hydroxy containing molecules.

[0078] The prefix "sulfo-" refers to an polymer grafted with varying percentages of a zwitterionic sidechain to enhance the solubility of the said polymer and enhance the resulting solubilization and stabilization efficiency during membrane protein solubilization, handling and downstream processes.

[0079] The prefix "glyco-" refers to an polymer grafted with varying percentages of meglumine sidechain to reduce the net-charge of the said polymer and enhance the resulting solubilization and stabilization efficiency during membrane protein solubilization, handling and downstream processes.

[0080] The term "affinity chromatography" is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. Affinity chromatography is useful for its high selectivity and resolution of separation, compared to other chromatographic methods. Examples for affinity chromatography are the purification of his- or rho-tagged proteins. These proteins have a poly-his (for example his6or hisio) called Rho-Tag on the C or N terminus, allowing the purification from a mixture via selective binding onto Ni-NTA agarose (for his-tagged protein) or anti Rho-1D4 antibody agarose for rho-tagged proteins. Protocols for these purification procedures can be found on the Cube Biotech web site and in the literature of Hochuli et al (Crowe J, Dobeli H, Gentz R, Hochuli E, Stuber D, Henco K., 6xHis-Ni-NTA chromatography as a superior technique in recombinant protein expression / purification. Methods Mol Biol. 1994;31:371-87. doi: 10.1385 / 0-89603-258-2:371. PMID: 7921034).

[0081] The present disclosure employs the use of poly(a-olefin-co-maleic anhydride) copolymer for the synthesis of an amphiphilic polymer.CUB-PA12-PCT

[0082] The poly(a-olefin-co-maleic anhydride) copolymer used for the present disclosure can be purchased commercially with varying alkyl length of the a-olefin moiety. The molecular weight of the copolymer can be in the range of 2-20 kDa but is not limited to the described range.

[0083] The base copolymer of the anhydride can also be selected from the group consisting of the following: poly(maleic anhydride), poly(isobutylene-alt-maleic anhydride), poly(maleic anhydride-alt-l-octadecene), poly(maleic anhydride-alt-l-tetradecene), poly(ethylene-alt-maleic anhydride), polyethylene-graft-maleic anhydride, polyisoprene-graft-maleic anhydride, polypropylene-graft-maleic anhydride, poly(methylvinyl ether-alt-maleic anhydride).

[0084] Detailed Description

[0085] According to the present disclosure there is provided a copolymer comprising repeating units of the following formula (3) and / or the following formula (4)

[0086] It is explicitly pointed out that although the parts A and B are linked together, the formulas used herein are not to be understood that the copolymer according to the present disclosure is a block copolymer of the blocks A and B. The formulas provided herein are to be understood in the sense that the copolymer according to the present disclosure comprises the indicated repeating units. In particular, all possible forms of copolymers (random copolymer, gradient copolymer, alternating copolymer, block copolymer, graft polymer) are envisaged by the formulas used herein.CUB-PA12-PCT

[0087]

[0088] wherein

[0089] Ri, R2, R3, and R4 are bound to the back bone of the polymer as described above in particular via Xi, X2, X3. Ri, R2, R3, and R4can be independently selected from the group consisting of:

[0090] (C3-C10) cycloalkyls or (C3-C10) (hetero)cycloalkyls, unsubstituted or substituted by one or more radicals selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, and linear or branched (C1-C8) alkynyls;

[0091] (C3-C10) cycloalkenyls or (C3-C10) (hetero)cycloalkenyls, poly or monounsaturated, unsubstituted or substituted by one or more radicals selected from linear, cyclic or branched

[0092] (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, linear or branched (C1-C8) alkynyls;CUB-PA12-PCT

[0093] poly or monounsaturated polycycles, unsubstituted or substituted by one or more radicals selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, linear or branched (C1-C8) alkynyls;

[0094] further the pendant hydrophilic group can include one or more of the following: hydroxyl, amino, ether, carboxylic acid, carboxylate, phosphate, phosphonate, phosphocholine, carboxylic ether, carboxylic ester, phosphate ester, amide, phosphonamide, ammonium, or their respective salts. The pendant hydrophilic group can be positively charged, negatively charged, zwitterionic, or neutral.

[0095] positively charged hydrophilic groups may include, but are not limited to, ammonium cations (e.g., alkylammonium cations such as mono-, di-, tri-, or tetra-alkylammonium cations). Negatively charged hydrophilic groups may include, but are not limited to sulfates, carboxylate or phosphate groups;

[0096] linear alkyl primary or mono-, di- or trimethylated amines, wherein in case of the trimethylated amine a quaternary amine with a positive charge is present, in the embodiment of a zwitterionic hydrophilic side chain, the linear alkyl amine (e.g. dimethylaminopropylamine) can be further sulfonated by using exemplary 1,3 propane sultone, carboxylated by using exemplary a halo-(C2-C7)alkanoic acid (e.g. chloroacetic acid) or phosphorylated

[0097] molecules from the class of polyols like tris(hydroxymethyl)aminomethane, l-amino-2,3-propandiol, 2-amino-2-methyl-l,3-propandiol, l-amino-2,3-butanediol, 3-amino-l,2-propanediol, l-amino-1,2-ethanedio or 2-amino-2-deoxy-D-glucitol.

[0098] molecules from the class of amine functionalized linear or circular mono-, di- or polysaccharides like N-methyl-D-glucamine, 2-amino-2-deoxy-glucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxy-galactose, 2-amino-2-deoxy-mannose, 2-amino-2-deoxy-ribose, 2-amino-2-deoxy-arabinose, 2-amino-2-deoxy-xylose

[0099] molecules from the class of amine functionalized linear or branched polyethylene oxides like 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol.

[0100] molecules from the class of amine functionalized linear or branched alkylated quaternary trimethyl ammonium chlorides like (2-aminoethyl)trimethylammoniumchloridCUB-PA12-PCT

[0101] Ri, R2, and R3can be additionally to the above biological tags, fluorophores, peptides, biotin and functional groups for click chemistry

[0102] Ri,and R2can be additionally to the above H, OH, N, O, O~' S or an alkali metal ion and

[0103] R3can be additionally selected from the group consisting of:

[0104] 1. A linear alkane chain, optionally, containing or terminating with a cyclic carbon, 2. A linear chain alkoxy alkane of the formula -(CH2)qO(CH2)rCH3where q is 1 to 5 and r is 1 to 15,

[0105] 3. An alkoxy alkane containing or terminating with a cyclic carbon chain,

[0106] 4. A halogenated alkane,

[0107] 5. A halogenated cycloalkane

[0108] 6. A halogenated arene,

[0109] 7. A chain containing a repeating sequence of (CH2CH2O)tterminating with -OR6whereintequals a value of 1 to 50 and R6is hydrogen, a linear alkane, a cyclic alkane, trifluoromethyl, a halogenated alkane, a halogenated cycloalkane, or a halogenated arene,

[0110] 8. A hydrogen, and

[0111] 9. A mixture thereof.

[0112] A and B are greater than 0, that is in the copolymer according to the present disclosure, both monomer units are present.

[0113] The ratio of the hydrophobic and hydrophilic monomers can be in the range of 1:1 - 3:1, preferred in the range of 2:1 - 1.5:1 and most preferred in the range between 1.1:1 and 1.5:1.

[0114] The small letter "a" in all formulas as used herein is the number of carbon atoms of the alkyl chain of the hydrophobic a-olefin moiety of the polymer. The number of the carbon atoms can be in the range of 1-20. Preferably below 15 and most preferably below 10 carbon atoms.CUB-PA12-PCT

[0115] The sum of A and B can be 100 % of the copolymer. Furthermore, the copolymer according to the present disclosure can be a random copolymer or an alternating copolymer with no limitation with regard to the ratio of A and B.

[0116] The increment n indicates the total number of the monomers A and B in the polymer chain and determines the average molecular weight (Mw) of the polymer.

[0117] The negative charge of the carboxyl groups, if present, can be equalized with cations, if necessary, for example with sodium ion.

[0118] The copolymer according to the present disclosure is based on a poly(a-olefin maleic acid anhydride) copolymer which can be synthesized using polymerization techniques like RAFT (reversible-addition-fragmentation chain-transfer polymerization) or commercially obtained in various versions with respect to the a-olefine moiety and the needed molecular weight (e.g. poly(maleic anhydride-alt-l-octadecene). The maleic anhydride copolymer (the starting material) can be reacted with amines; this produces an acid amide (R-CO-NH-R1) with the amine on one side and the acid R-COOH (with R: polymer residue and R': hydrocarbon residue on the amine) on the other side.

[0119] If these compounds are heated, a ring closure to the maleimide can be achieved. However, the ring-closure can be achieved using different techniques based on condensation and water elimination like the DCC HOBt catalyzed ring-closure.

[0120] The copolymer according to the present disclosure can be a zwitterionic compound as exemplified in formula (3) and (4)CUB-PA12-PCT

[0121]

[0122] The amines moieties of R7-R10 are defined as follows:

[0123] R7can be a branched or linear C1-C7 alkyl;

[0124] can be a primary or secondary amine or tertiary amine alkylated with zero, one or two C1-C4 alkyl moieties as intermediate product before further functionalization with R9and Ri0; After alkylation or functionalization a quaternary amine may be present;

[0125] R9can be a branched or linear C1-C7 alkyl;

[0126] Rio can be a terminal functional group containing a negative charge such as carboxylic, phosphate or sulfonates moieties.

[0127] The copolymer according to the present disclosure exemplified in formula (5) as maleic acid amide can be turned in the maleimide form as shown in formula (6) for example by heating.

[0128] According to the present disclosure, the amide is formed on one of the two carbonyl carbons and a carboxylic acid on the other.

[0129] With respect of the stereochemistry of the a-olefin moieties and the ring-opening reaction during the amidation of the maleic acid anhydride, a random orientation of the hydrophobic chain and the amide with respect to the backbone and the carboxylic function of the maleic acid is possible.CUB-PA12-PCT

[0130] The copolymer can be according to the present disclosure extended to a terpolymer by addition of further molecular functionalization and can be represented by the following formula (7) or the following formula (8) (if required, the residues can be bound to the polymer back bone via the groups Xi, X2, and X3defined above)

[0131]

[0132] C denotes for the relative abundance of the introduced functionality Rn.

[0133] Rn denotes for the additional functionalities can be provided to the copolymers, such as fluorophores, tags (e.g., His, Rho, FLAG), biotin, functional groups for click chemistry (e.g. azides and alkynes) and others bearing a nucleophilic group (e.g. N, S, O). The functionality can be introduced using the amine reactivity of maleic acidCUB-PA12-PCT

[0134] anhydride or activation of the carboxylic acid groups of maleic acid to O-acylisourea intermediates which are reactive to nucleophilic attacks forming a stable amide bond. These compounds can be attached to the anhydrides as amine-functionalized molecules.

[0135] The copolymer according to the disclosure can be additionally to formula (7) and (8) converted in the ring-closed maleimide form before or after introducing Rn resulting in a copolymer bearing a randomized mixture of maleic acid amide as well as maleimide moieties.

[0136] In a further embodiment, block-copolymers of maleic acid anhydride and a-olefins as well as their functionalized derivatives according to this disclosure can be synthesized.

[0137] The letters A-C in the formulas denote for the relative number of statistical distributed units of the copolymer and give rise to the molecular weight of the copolymer which can be in the range of 2.000 to 24.000 Dalton. The sum of A-C can be 100% and is equal to the number of maleic acid anhydride units or a-olefin units or functionalities copolymer. The functionalization can be in the range from 0% - 100% to the respective maleic acid anhydride units and in general A + B + C = 100% of the total functionalized and non-functionalized maleic acid anhydride groups of the polymer.

[0138] The copolymer according to the present disclosure, Ri - R4can be independently selected from cycloalkyl residues, for example cyclooctyl, ethyl cyclohexyl, and methyl cyclohexyl. Surprisingly it has been found that cycloalkyl compounds solubilize membrane proteins. Thus, the copolymers according to the present disclosure are unexpectedly particularly useful for use in solubilization and stabilization of hydrophobic proteins, membrane proteins, and GPCRs.

[0139] According to the present disclosure there is also provided a method for preparing the above copolymer, wherein the anhydride of a maleic acid-a olefin copolymer can be reacted with the amines of Ri - Rn and to provide the copolymer of formulas (1) and (3) and (5), which subsequently can be reacted to the copolymers of formulas (2) and (4) and (6) by the elimination of water. The amines of Ri - Rn can be represented by RI-II-NH2. Ri- Rn are defined as above, wherein by these amines the atom N is introduced into the copolymers according to the present disclosure.CUB-PA12-PCT

[0140] There is further provided a complex comprising the copolymer as defined above and a hydrophobic protein, a membrane protein and / or a G protein coupled receptor (GPCR). The complex can further comprise a lipid. The membrane protein can be selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transporter proteins, such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases, and SLC transporters.

[0141] According to the present disclosure, there is further provided a method for obtaining the above complex comprising contacting the copolymer as defined above with the hydrophobic protein, the membrane protein, the G protein coupled receptor and optionally the lipid.

[0142] The copolymer according to the present disclosure can be used for solubilization, stabilization and / or purification of membrane proteins. In particular, the solubilization, stabilization and / or purification can be provided out of the native membrane surrounding. The membrane protein is selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transporter proteins, such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membranebased ATPases, and SLC transporters.

[0143] Furthermore, the copolymers according to the present disclosure can be used for cryo electron microscopy in particular with stabilization of the position and orientation of the membrane protein.

[0144] Furthermore, according to the present disclosure there is provided a complex comprising the copolymer according to the present disclosure, for example in the form of the above zwitterionic structure), a lipid and a biomolecule. Such a complex can be employed in the cell-free expression of proteins.

[0145] The present disclosure also provides a kit, comprising the copolymer according to the present disclosure and optional instructions for use in particular the uses as described above.

[0146] In the following, the present disclosure will be described more specifically.

[0147] According to the present disclosure, there is provided poly(a-olefine-co-maleic anhydride)-based copolymers.CUB-PA12-PCT

[0148] The monomers bearing the group Ri - Ru, respectively, are randomly distributed over the length of the polymer chain.

[0149] The polymer chains can bear further functionalities at the initial or terminal part of the copolymer chain. Either the initiator fragment derived by the radical starter molecule (e.g., azobis isobutyronitrile), the a-olefine monomer, a functional group (e.g., hydroxy, carboxylic acid, etc.) or a hydrogen atom as well as the used chain transfer agent.

[0150] The term functional group refers to any functional group known in the field of organic chemistry.

[0151] The letters A-C denote for the relative number of statistical distributed units of the polymer and give rise to the molecular weight of the polymer which can be in the range of 2.000 to 24.000 Dalton. The sum of A-C is 100% and is equal to the number of maleic acid anhydride units or a-olefine units of the initial polymer. The functionalization can be in the range from 0%- 100% to the respective maleic acid anhydride units and in general A + B + C = 100% of the total functionalized and non-functionalized maleic acid anhydride groups of the polymer.

[0152] Ri - Ru is the hydrophilic or the hydrophobic part of the polymer. Ri, R2, R4-R6 and Ru can be derived by the group of following amino-, thio- or hydroxy-bound substitutes:

[0153] (C3-C10) cycloalkyls or (C3-C10) (hetero)cycloalkyls, unsubstituted or substituted by one or more radicals selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1C8) alkenyls, and linear or branched (C1-C8) alkynyls; poly or monounsaturated, unsubstituted or substituted by one or more radicals selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, linear or branched (Cl-C8)alkynyls; poly or monounsaturated polycycles, unsubstituted or substituted by one or more radicals selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, linear or branched (C1-C8) alkynyls; phenyl, unsubstituted or substituted by one or more radicals selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, linear or branched (Cl-C8) alkynyls.

[0154] further the pendant hydrophilic group can include one or more of the following: hydroxyl, amino, ether, carboxylic acid, carboxylate, phosphate, phosphonate, phosphocholine, carboxylic ether, carboxylic ester,CUB-PA12-PCT

[0155] phosphate ester, amide, phosphonamide, ammonium, or their respective salts. The pendant hydrophilic group can be positively charged, negatively charged, zwitterionic, or neutral.

[0156] Positively charged hydrophilic groups may include, but are not limited to, ammonium cations (e.g., alkylammonium cations such as mono-, di-, tri-, or tetra-alkylammonium cations). Negatively charged hydrophilic groups may include, but are not limited to sulfates, carboxylate or phosphate groups;

[0157] Linear alkyl primary or mono-, di- or trimethylated amines. In case of the trimethylated amine a quaternary amine with a positive charge is present. In the embodiment of a zwitterionic hydrophilic side chain, the linear alkyl amine (e.g. Dimethylaminopropylamine) is further sulfonated (e.g., 1,3 propane sultone) but can also contain a carboxylic or phosphate moiety.

[0158] Molecules from the class of polyols like tris(hydroxymethyl)aminomethane, l-amino-2,3-propandiol, 2-amino-2-methyl-l,3-propandiol, l-amino-2,3-butanediol, 3-amino-l,2-propanediol, l-amino-1,2-ethanedio or 2-amino-2-deoxy-D-glucitol.

[0159] Molecules from the class of amine functionalized linear or circular mono-, di- or polysaccharides in the like N-methyl-D-glucamine, 2-amino-2-deoxy-glucose, N-methyl-D-glucamine, 2-amino-2-deoxy-glucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxy-galactose, 2-amino-2-deoxy-mannose, 2-amino-2-deoxy-ribose, 2-amino-2-deoxy-arabinose, 2-amino-2-deoxy-xylose.

[0160] Molecules from the class of amine functionalized linear or branched polytheylene oxides like 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol

[0161] Molecules from the class of amine functionalized linear or branched alkylated quaternary trimethyl ammonium chlorides like (2-aminoethyl)trimethylammoniumchlorid -hydrochlorid

[0162] Ri, 2, 3 and R4can be additionally to the above biological tags, fluorophores, peptides, biotin and functional groups for click chemistry.

[0163] Ri, R2and R4can be additionally to the above H, OH, N, O, S or an alkali metal ion andCUB-PA12-PCT

[0164] R3can be additionally selected from the group consisting of:

[0165] 1. A linear alkane chain, optionally, containing or terminating with a cyclic carbon, 2. A linear chain alkoxy alkane of the formula -(CH2)qO(CH2)rCH3 where q is 1 to 5 and r is 1 to 15,

[0166] 3. An alkoxy alkane containing or terminating with a cyclic carbon chain,

[0167] 4. A halogenated alkane,

[0168] 5. A halogenated cycloalkane

[0169] 6. A halogenated arene,

[0170] 7. A chain containing a repeating sequence of (CH2CH2O)tterminating with -OR6whereintequals a value of 1 to 50 and R6is hydrogen, a linear alkane, a cyclic alkane, trifluoromethyl, a halogenated alkane, a halogenated cycloalkane, or a halogenated arene, 8. A hydrogen, and

[0171] 9. A mixture thereof.

[0172] Amine containing biological tags (e.g., Biotin-PEG4-Amine) or fluorophores (e.g. Fluoresceine amine) or peptides (e.g., Rho tag).

[0173] The base copolymer can be synthesized via radical polymerization of the monomers with varying ration of the respective monomers and molecular weights ranging from 2-20 kDa.

[0174] The distribution of the monomers and functionalization can be randomly distributed or terminal using the terminal chain transfer agent (RAFT agent) for further modification.

[0175] The copolymer can be a co-block polymer or at least bear co-block elements additionally to copolymer parts where the monomers are randomly distributed.

[0176] The nature of the functionalization mechanism implies a random opening or functionalization reaction with respect to the ring-opening of the maleic acid anhydride while amide formation.CUB-PA12-PCT

[0177] For generation of the amphiphilic character of the copolymer according to the present disclosure, the copolymer can be grafted using different techniques inclusively but not exclusively amide formation of maleic acid anhydride moieties with a primary amine or thiol containing target via ring-opening reaction. The grafting percentage of the copolymer can be controlled via the amine excess. The ring-opened state is characterized by the formation of the amide as well as a carboxylic group. The carboxylic group can be used to form a maleimide by a heat induced ring closure reaction which can be beneficial to minimize negative charges in the copolymer.

[0178] The grafting of the base polymer with hydrophilic and hydrophobic molecules can be in the range of >1%, in particular between 40-100% and preferably between 60-100%. The grafting percentage is referred to the number of maleic acid anhydrides in the polymer backbone reacted with an amine-, thiol- or hydroxycontaining molecule forming an amide. After grafting reaction, if no ring-closure of the amide with the carboxylic group is done, a 100% grafting results in equal amounts of amides and carboxylic groups in the final copolymer. If a ring-closure of the amide and the carboxy group of the former maleic acid anhydride is done, a maleimide is formed by condensation reaction.

[0179] Polar molecules used for the grafting can be linear alkyl (primary or mono-, di- or trimethylated) amines.

[0180] In case of the trimethylated amine a quaternary amine with a positive charge is present.

[0181] In the case of polyols, tris(hydroxymethyl)aminomethane, l-amino-2,3-propandiol, 2-Amino-2-methyl-1,3-propandiol, l-Amino-2,3-butanediol, 3-Amino-l,2-propanediol, l-Amino-l,2-ethanedio or 2-Amino-2-deoxy-D-glucitol and similar substances can be used.

[0182] In the case of amine functionalized linear or circular mono-, di- or polysaccharides like N-methyl-D-glucamine, 2-Amino-2-deoxy-glucose, N-methyl-D-glucamine, 2-Amino-2-deoxy-glucose, N,N-Dimethylglucamine, N-Ethylglucamine, N-Methyl-D-mannosamine, N-Methyl-D-galactosamine, N-Methyl-D-glucosamine, 2-Amino-2-deoxy-galactose, 2-Amino-2-deoxy-mannose, 2-Amino-2-deoxy-ribose, 2-Amino-2-deoxy-arabinose, 2-Amino-2-deoxy-xylose can be used.

[0183] In the embodiment of a zwitterionic side chain, the linear alkyl amine (e.g. Dimethylaminopropylamine) can be further sulfonated (e.g., 1,3 propane sultone).CUB-PA12-PCT

[0184] Amine containing biological tags (e.g., Biotin-PEG4-Amine) or fluorophores (e.g. Fluoresceine amine) or peptides (e.g., Rho tag) can also grafted on the copolymer to implement a polymer intrinsic functionalization like target specificity, binding to affinity resins or labeling with chromophores for spectroscopic detection.

[0185] Molecules from the class of the polyole like tris(hydroxymethyl)aminomethane, l-amino-2,3-propandiol, 2-Amino-2-methyl-l,3-propandiol, l-Amino-2,3-butanediol, 3-Amino-l,2-propanediol, l-Amino-1,2-ethanedio or 2-Amino-2-deoxy-D-glucitol.

[0186] Molecules from the class of amine functionalized linear or circular mono-, di- or polysaccharides in the like N-methyl-D-glucamine, 2-Amino-2-deoxy-glucose, N-methyl-D-glucamine, 2-Amino-2-deoxy-glucose, N,N-Dimethylglucamine, N-Ethylglucamine, N-Methyl-D-mannosamine, N-Methyl-D-galactosamine, N-Methyl-D-glucosamine, 2-Amino-2-deoxy-galactose, 2-Amino-2-deoxy-mannose, 2-Amino-2-deoxy-ribose, 2-Amino-2-deoxy-arabinose, 2-Amino-2-deoxy-xylose.

[0187] Molecules from the class of amine functionalized linear or branched polytheylene oxides like 2-(2-(2-(2-Aminoethoxy)ethoxy)ethoxy)ethanol.

[0188] Molecules from the class of amine functionalized linear or branched alkylated quaternary trimethyl ammonium chlorides like (2-Aminoethyl)trimethylammoniumchlorid -hydrochlorid

[0189] Said functionalized and non-functionalized new amphiphilic based copolymers can be used for solubilization and stabilization of integral membrane proteins and membrane associated proteins.

[0190] The copolymers according to the present disclosure can be used for labelling of nanodisc complexes for their use in biomolecular research, diagnostic applications and medical product development.

[0191] Compared to the polymers and copolymers of the prior art, the copolymer according to the present disclosure has at least one of the following advantages:CUB-PA12-PCT

[0192] Enhanced solubilization efficiency of membrane proteins expressed in procaryotic and eucaryotic cells and / or native proteins expressed in cell membranes.

[0193] Enhanced stability of formed nanodisc assemblies of membrane proteins during solubilization, purification and during application of said new amphipols and cell membrane patches with respect to high ionic strength and high concentrations of cations compared to commercially available products.

[0194] Due to the enhanced solubilization efficiency, the polymers according to the present disclosure can be used in lower concentrations, leading to higher purity and functionality of the stabilized membrane proteins.

[0195] Possibility of functionalization of new copolymers with molecules (e.g., fluorophores, biotin derivatives, etc.) for protein purification and protein labelling.

[0196] The chemically modifiable backbone reveals a different molecular flexibility compared to other available copolymers (e.g. SMALPs) and enables site-specific modifications for placement of solubilized target proteins of cryo-TEM grids to enhance the orientation and thereof quality of the obtained protein structures via cryo-TEM.

[0197] The solubilization, stabilization and purification of membrane proteins out of the native membrane surrounding is dependent on a number of parameters. Most parameters can be optimized during the purification process to a higher efficiency. The parameters include buffer conditions (for example salt, pH), choice of polymer, protein-to-solubilization agent-ratio, temperature, and time. First, cell lysis and centrifugation are carried out by for example using the following parameters: Adding of protease inhibitors (PI) to buffer and readjust pH value then disrupting cells (e.g., Sonification, French Press), centrifugation at 9000 ref for 30 min at 4°C, discarding pellet (cell debris), collecting supernatant, centrifugation of the supernatant at 100 000 ref for 1 h at 4°C, discarding supernatant and homogenize pellet. Then the solubilization of membrane proteins is carried out: Polymers form synthetic nanodisc around the protein, thereby maintaining the native phospholipid environment and preserving the native and thus functional properties of the protein in a convenient one step manner (solubilization and stabilization). Detergents on the other hand form micelles around the hydrophobic belt, thus remove the lipids from the surrounding. For native conditions the unique lipid environment needs to be conserved.CUB-PA12-PCT

[0198] In one embodiment, the membrane protein is selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transporter proteins, such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases, and SLC transporters. That is, as a starting material for the method according to the present disclosure, a solution of the free polymer is used which stems from the solubilization, stabilization and purification of the above-mentioned membrane proteins out of their native surrounding by employing a polymer.

[0199] The amphiphilic copolymers according to the present disclosure can be used for solubilization and stabilization of membrane proteins for biotechnological and pharmaceutical applications. Thus, it is also possible to use the amphiphilic polymer according to the present disclosure as reagent, in reagent kits and diagnostic kits (e.g., lateral flow assays) comprising at least one part of the described disclosure. One application of the copolymer according to the present is the solubilization and stabilization of membrane proteins in solution with and without detergent pretreatment. This means, that they are capable of keeping fully functional or nonfunctional but still immunogenic membrane proteins in solution and prevent them from aggregation or precipitation upon solubilization and handling. Thus, the disclosure is also related to the formed water-soluble complex consisting of one or more amphiphilic polymer molecules, artificial or natural lipids derived from cell membranes as well as integral membrane proteins or membrane associated proteins.

[0200] The use of copolymers according to the present disclosure can be exemplified as follows:

[0201] One or more recombinant membrane or membrane associated proteins are expressed with high density in pro- or eucaryotic cells and are located either in or onto the cell membrane as well as potentially located in inclusion bodies.

[0202] The solubilizing step is carried out using either the whole cell suspension, the supernatant of cell lysate or the pellets of centrifuged supernatant derived from the cell lysate.

[0203] The protein solution or pellet is directly added to the polymer solution with a final polymer concentration up to 5% wt and incubated up to 24 h while stirring.CUB-PA12-PCT

[0204] Solubilization efficiency can be determined using standard biomolecular methods (e.g., SDS-PAGE, Western Blot).

[0205] By Centrifugation, insolubilized proteins and debris can be separated from the solubilized proteins located in the formed nanodisc complex.

[0206] In diagnostics, biological components such as DNA, RNA, proteins and metabolites are examined qualitatively and quantitatively. This provides information about diseases, genetic predispositions, or the state of health. Diagnostic tests can be performed by medical professionals, but also by private individuals.

[0207] The copolymers according to the present disclosure can be used to solubilize and stabilize membrane proteins, preferably in their native lipid environment, in order to maintain their activity. These stabilized membrane proteins can be used to detect interactions. The interaction of the copolymer stabilized membrane protein and its interaction partner can be detected inclusive but not exclusively via different analytical methods.

[0208] Examples for optical detections contain SPR (surface plasmon resonance), RM (resonant mirror), GCI (Grating-Coupled Interferometry), ELISA (enzyme-linked immunosorbent assay) as Direct ELISA, Sandwich ELISA, Competitive ELISA, or Reverse ELISA, and LFA (lateral flow assay).

[0209] The interaction of the copolymer stabilized membrane protein and its interaction partner can be detected inclusive but not exclusively via different analytical methods.

[0210] In addition to that, the copolymers can lyse eucaryotic cells and tissue in low concentration (0.01% to 5%) very quickly (in seconds to a few minutes) and without mechanical aids. Because of this capability for a mild lysis, it allows the user to obtain nucleic acids with a low level of fragmentation, and to get soluble and membrane proteins in their native state, the polymers of the disclosure are particularly suitable for diagnostic tests, especially basing on DNA, RNA, soluble proteins, and membrane proteins.

[0211] The present disclosure is described in the following by reference to the figures and the examples. It is explicitly pointed out that the examples and figures shall not be construed to limit the disclosure thereto,CUB-PA12-PCT

[0212] but they are intended only for illustrating the disclosure. In particular, specific combinations of the various residues R with Xi, X2and X3can be taken from the Figures and the Examples.

[0213] Fig. 1 shows the a-olefin-maleic acid base copolymer known from the prior art. In practice, the molecular weight is ~5-8 kDa. The ratio of olefimmaleic acid in this example is 1:1 - 1.2:1.

[0214] Fig. 2 shows a copolymer according to the disclosure consisting of the a-olefin maleic acid anhydride base copolymer from Fig. 2, in which 100% of all anhydrides are reacted with 3-amino-l,2-propanediol.

[0215] The special properties are:

[0216] - Very efficient in solubilising membrane proteins

[0217] - Well suited for cryo-TEM

[0218] - Reduced net negative charge of the polymer

[0219] Further alternative hydrophilic functionalisation via amide formation can be linear or branched alkyl chains (max C6, OH5). The variation of the degree of functionalisation with the hydrophilic residue is in practice between 50% and 100%. Remaining maleic acid anhydrides are hydrolysed to maleic acid

[0220] Fig. 3 shows another copolymer according to the disclosure, which is obtained by reacting up to 100% of all anhydrides of the base copolymer from Fig. 1 with 3-aminopropyldimethylamine. Further functionalisation with 1,3-propanesultone may have taken place.

[0221] The special properties are:

[0222] - Very efficient in solubilising membrane proteins

[0223] - Well suited for cryo-TEM

[0224] - Reduced net negative charge of the polymer to neutral

[0225] - Extremely resistant to Mg2+and Ca2+ions and pH stable (no precipitation <100 mM Mg2+ / Ca2+, pH >4)

[0226] Further alternative hydrophilic functionalisation via amide formation can be: instead of reaction with 1,3-propanesultone, reaction with 3-bromopropionic acid. In practice, the degree of functionalisation with the hydrophilic residue varies between 50% and 100%. Remaining maleic acid anhydrides are hydrolysed to maleic acidCUB-PA12-PCT

[0227] Fig. 4 shows another copolymer according to the disclosure, which is obtained by reacting 100% of the anhydride of the base copolymer from Fig. 1 with N-methyl-D-glucamine.

[0228] The special properties are:

[0229] - Very efficient in solubilising membrane proteins

[0230] - Well suited for cryo-TEM

[0231] - Reduced net negative charge of the polymer to neutral

[0232] - Reduced non-specific binding of proteins to nanodiscs

[0233] Further alternative hydrophilic functionalisation via amide formation can be carried out with linear or branched mono-, di-, tris- or max. tetrasaccharides. In practice, the degree of functionalisation with the hydrophilic residue varies between 50% and 100%. Remaining maleic acid anhydrides are hydrolysed to maleic acid.

[0234] Fig. 5 shows another copolymer according to the disclosure, which was obtained by reacting 100 % of all anhydrides of the base copolymer from Fig. 1 with amino-PEG-OH.

[0235] The special properties are:

[0236] - Very efficient in solubilising membrane proteins

[0237] - Well suited for cryo-TEM

[0238] - Reduced net negative charge of the polymer to neutral

[0239] - Reduced non-specific binding of proteins to nanodiscs

[0240] Further alternative hydrophilic functionalisation via amide formation:

[0241] - Linear or branched PEG chains

[0242] - Terminal functional group can vary

[0243] The variation of the degree of functionalisation with the hydrophilic residue is in practice between 50% and 100%. Remaining maleic acid anhydrides are hydrolysed to maleic acid

[0244] Fig. 6 shows another copolymer according to the disclosure, which was prepared by reacting 100% of all anhydrides with (2-aminoethyl)trimethylammonium chloride

[0245] The special properties are:

[0246] - Very efficient in solubilizing membrane proteins

[0247] - Well suited for cryo-TEM

[0248] - Neutral to positive net charge of the polymer

[0249] Further alternative hydrophilic functionalization via amide formation can beCUB-PA12-PCT

[0250] - Length and branching of the alkyl chain can vary

[0251] (aminoethyl-, propyl-...)

[0252] - Length and branching of the alkyl residues on the quaternary

[0253] ammonium ion can vary

[0254] The variation of the degree of functionalisation with the hydrophilic residue is in practice between 50% and 100%. Remaining maleic anhydrides are hydrolysed to maleic acid

[0255] Fig. 7 shows yet another copolymer according to the disclosure, in which 1 anhydride / polymer chain is reacted with amino fluoresceins. Ri and R2can be any of the substituents described in the present application; further alternative functionalisations via the amide formation are other fluorophores, chromophores or tags (e.g. Raman reporters).

[0256] Fig. 8 also shows a copolymer according to the disclosure, in which 1 anhydride / polymer chain is reacted with amine-PEG-biotin. Ri and R2can be any of the substituents described in the present application. Further alternative functionalisations via the amide formation:

[0257] - Other affinity tags, such as amino acid sequences (Rho tag) etc.

[0258] - Length of the PEG linker may vary.

[0259] Examples:

[0260] Synthesis of a-olefin maleic acid anhydride copolymer

[0261] Suspend 10.0 g maleic acid anhydride (102 mmol), 11.22 g 1-octene (102 mmol) 200 mg AIBN (1.2 mmol) and 800 mg S-butyl-S'-(l-phenyl ethyl) trithiocarbonat (BPT, synthesis described in literature, Postma, A et al. Synthesis of Well-Defined Polystyrene with Primary Amine End Groups through the Use of Phtalimido-Functional RAFT Agents. Macromolecules 39, 5293-5306 (2006) , in 80 ml MEK in a 200 ml round bottom flask while stirring vigorously. The solution is heated under argon atmosphere to 70°C and incubated for 24 h. The solution is precipitated in cold pentane and the polymer collected. If necessary, the terminal RAFT agent can be cleaved by dissolving in dioxane, cleavage with AIBN and reduction of the terminal radical by using TTMSS and purification via precipitation in pentane and diethyl ether. The solid polymer can be used in its anhydride form for further functionalization or can be hydrolyzed as follows.CUB-PA12-PCT

[0262] The solid polymer can be hydrolyzed by dissolution at 10 wt.% in 1 M NaOH followed by heating under reflux at 100°C. The hydrolyzed and ring-opened version of the a-olefin copolymer is precipitated with 1 M HCI and washed three times with double-distilled water. After freezing and lyophilization, the polymer is obtained.

[0263] The ratio of monomers as well as the RAFT agent to monomer ratio can be varied to obtain a copolymer with more hydrophilic or hydrophobic properties and to obtain polymers with different Mw.

[0264] Synthesis of sulfo-a -olefin maleic acid copolymer

[0265] To introduce the quaternary amine moieties 10.0 g of a-olefin maleic acid anhydride copolymer is dissolved in 60 ml DMF, 14.58 g (143 mmol) dimethylaminopropylamine dissolved in 40 ml DMF is added to the solution at room temperature with vigorous stirring. The flask is sealed with a Schlenk adapter and the suspension is placed in an oil bath and heated to 60°C for 24 hours. Then, to introduce the sulfonate moieties, 36.64 g (300 mmol) of 1,3-propane sultone dissolved in 50 ml of DMF is added to the solution at room temperature with vigorous stirring. The flask is sealed with a Schlenk adapter and the suspension is placed in an oil bath and heated to 60°C for 24 h. The DMF is then removed via precipitation in pentane and diethylether. The solid product is dissolved in 20 ml of NaOH (1 M) and dialyzed against H2O followed by lyophilization which gives the solid amphiphilic copolymer product

[0266] Synthesis of tagged or labelled a-olefin maleic acid anhydride precursor polymer

[0267] To introduce the fluorescent moieties 10.0 g of a-olefin maleic acid anhydride copolymer is dissolved in 60 ml DMF. A solution of 590 mg 5-aminofluorescein in 10 ml DMF is added dropwise. The flask is sealed with a Schlenk adapter and the suspension is placed in an oil bath and heated to 60°C for 24 h. Afterwards the DMF is removed under reduced pressure at 75°C using a rotary evaporator. The solid product is dissolved in 15 ml DMF and the procedure is repeated. The successful integration of one fluorescein per chain can be monitored using HPLC. For implementation of other hydrophilic moieties described in this patent, the resulting fluorescent a-olefin maleic acid anhydride copolymer can used for the synthesis described before.CUB-PA12-PCT

[0268] Synthesis of a-olefin maleic acid-Tris or a-olefin maleic acid-glycerol

[0269] To introduce the polyol moieties 10.0 g of a-olefin maleic acid anhydride copolymer is dissolved in 60 ml DMF. The reaction of the maleic acid anhydride monomers of the polymer backbone can be achieved by adding the threefold molar amount of polyol amine.

[0270] For a-olefin maleic acid-Tris in the ring opened version, 17.3 g tris(hydroxymethyl)aminomethane dissolved in 40 ml DMF is added dropwise.

[0271] For a-olefin maleic acid-glycerol, 13.0 g 2-aminol,3-propandiol dissolved in 40 ml DMF is added dropwise.

[0272] The solution is placed in an oil bath and heated to 60°C for 24 h. The DMF is then removed via precipitation in pentane and diethylether. The solid product is dissolved in 20 ml of NaOH (1 M) and dialyzed against H2O followed by lyophilization which gives the solid amphiphilic copolymer product.

[0273] Synthesis of glyco-a-olefin maleic acid

[0274] 10 g of the dried solid a-olefin maleic acid anhydride polymer is dissolved with 11.15 g N-methyl-D-glucamine in 100 ml of dry methanol at 65°C. After dissolution, a solution of 1.38 g sodium in 50 ml methanol was added dropwise under argon and stirring. After incubation for 24 h and addition of 100 ml ethanol the final glyco-a-olefin maleic acid can be isolated by rotational evaporation of the organic solvents. For purification, the crude product is dissolved in pure water and dialyzed against water. After freezing and lyophilization, the final polymer is obtained.

[0275] Synthesis of a-olefin maleic acid-PEG

[0276] 10 g of the dried solid a-olefin maleic acid anhydride polymer is dissolved in 60 ml of DMF at 65°C. After dissolution, a solution of 11 g PEG4-Amin in 40 ml DMF was added dropwise under argon and stirring. After incubation for 24 h the final a-olefin maleic acid-PEG can be isolated. For purification, the crudeCUB-PA12-PCT

[0277] product is dissolved in pure water and dialyzed against water. After freezing and lyophilization, the final polymer is obtained.

[0278] Synthesis of a-olefin maleic acid-amin

[0279] 10 g of the dried solid a-olefin maleic acid anhydride polymer is dissolved in 60 ml of DMF at 65°C. After dissolution, a solution of 10.0 g (2-aminoethyl)trimethylammoniumchlorid in 20 ml DMF and 10 ml H2O was added dropwise under argon and stirring. After incubation for 24 h the final a-olefin maleic acid-Amin can be isolated. For purification, the crude product is dissolved in pure water and dialyzed against water. After freezing and lyophilization, the final polymer is obtained.

[0280] Solubilization of his- or rho-tagged membrane protein G6PC and purification

[0281] The solubilization, stabilization and purification of membrane proteins out of the native membrane surrounding is dependent on a number of parameters. Most parameters can be optimized during the purification process to a higher efficiency. Parameters include: Buffer conditions (salt, pH etc.), choice polymer, protein-to-solubilization agent-ratio, temperature, time.

[0282] Cell lysis and centrifugation:

[0283] Add protease inhibitors (PI) to buffer and readjust pH value then disrupt cells (e.g., Bonification, French Press). Centrifuge at 9000 ref for 30 min at 4°C, discard pellet (cell debris), collect supernatant. Centrifuge supernatant at 100000 ref for 1 hat 4°C, discard supernatant and homogenize pellet.

[0284] Solubilization of membrane proteins:

[0285] Polymers form synthetic nanodisc around the protein, thereby maintaining the native phospholipid environment and preserving the native and thus functional properties of the protein in a convenient one step manner (solubilization and stabilization). Detergents on the other hand form micelles around theCUB-PA12-PCT

[0286] hydrophobic belt, thus remove the lipids from the surrounding. For native condition the unique lipid environment needs to be conserved.

[0287] If solubilization efficiency is low it is advised to screen variation of parameters to improve the yield of total solubilized protein. A standard protocol is described as follows:

[0288] Add solubilization agent (copolymer) to the protein solution Ideal concentrations may vary, good starting points are: 0.1 - 2.5 % copolymer (e.g. a-olefin maleic acid-based amphiphilic copolymers) Solubilize for 3 h to 24 hat 4 °C while stirring

[0289] Higher temperatures can be screened for optimization Centrifuge at 100000 ref for 1 h at 4 °C

[0290] Discard the pellet containing cell fragments, collect the supernatant

[0291] Use solubilized membrane protein in polymer nanodisc (supernatant) for affinity chromatography, separating the his- or rho-tagged membrane protein copolymer complex from the mixture by using commercially available agarose products.

[0292] Visualization of solubilized membrane proteins bySOS-PAGE, Western Blotand Rho Antibody / HRP staining for chemiluminescence detection.

[0293] In summary, the present disclosure describes a poly(a-olefine-co-maleic acid) copolymer comprising repeating units of formulas (1) and (2)

[0294]

[0295] CUB-PA12-PCT

[0296] formula (1)

[0297] or comprising repeating units of formulas (1) and (2a)

[0298]

[0299] formula (1) formula (2a)

[0300] wherein Xi and X2are independently NH, (Cl-C5-alkyl)N, in particular (CH3)N, O, O', S, S'; X3is N;

[0301] a is an integer of 1 to 20; Ri, R2, R3, and R4, can be independently derived from the following: a (C3-C10) cycloalkane or a (C3-C10) (hetero)cycloalkan, which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, and linear or branched (C1-C8) alkynyls; a (C3-C10) cycloalken or a (C3-C10) (hetero)cycloalken, which can be poly or monounsaturated, and which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, and linear or branched (C1-C8) alkynyls; a poly or monounsaturated polycycle, which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, and linear or branched (C1-C8) alkenyls, linear or branched (C1-C8) alkynyls; a linear C2 to CIO alkyl-amine, wherein one primary and / or mono-, di- or trimethylated amine-group is present , wherein in case of the trimethylated amine a quaternary amine with a positive charge is present, the linear alkyl amine e.g. dimethylaminopropylamine can be further sulfonated by using 1,3 propane sultone, carboxylated by using exemplary a halo-(C2- C7)alkanoic acid (e.g. chloroacetic acid) or phosphorylated an polyol, wherein a polyol is any compound having at least two hydroxyl groups; a linear or cyclic mono-, di- or polysaccharide; a linear or branched polyethylene oxide; an linear or branched alkylated quaternary trimethyl ammonium chlorides; a group selected from biological tags, fluorophores, peptides, biotin and functional groups for click chemistry; H, or an alkali metal ion for example Na+, and R3can be further selected, with the provisio that the above definitions are not included in this definition of R3, from the group consisting of a linear alkane chain,CUB-PA12-PCT

[0302] optionally, containing or terminating with a cyclic carbon; a linear chain alkoxy alkane of the formula -(CH2)qO(CH2)rCH3where q is 1 to 5 and r is 1 to 15; an alkoxy alkane containing or terminating with a cyclic carbon chain; a halogenated alkane; a halogenated cycloalkane; a halogenated arene; a chain containing a repeating sequence of (CH2CH2O)tterminating with -OR6whereintequals a value of 1 to 50 and R6is hydrogen, a linear alkane, a cyclic alkane, trifluoromethyl, a halogenated alkane, a halogenated cycloalkane, or a halogenated arene; a hydrogen, and a mixture thereof, and salts thereof, provided that R1 and R2 are not simultaneously O' or HO.

[0303] References:

[0304] 1. Rabilloud, T., Membrane proteins and proteomics: love is possible, but so difficult.

[0305] Electrophoresis, 2009. 30 Suppl 1: p. S174-80.

[0306] 2. Marconnet, A., et al., Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers. Biomacromolecules, 2020: p. 3459-3467.

[0307] 3. Smith, A.A.A., et al., Lipid Nanodiscs via Ordered Copolymers. Chem, 2020. 6(10): p. 2782-2795.

[0308] 4. Dorr, J.M., et al., The styrene-maleic acid copolymer: a versatile tool in membrane research. Eur Biophys J, 2016.45(1): p. 3-21.

[0309] 5. Marconnet, A., et al., Influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids SI. 2022.

[0310] 6. Zoonens, M. and J.L. Popot, Amphipols for each season. J Membr Biol, 2014.

[0311] 247(9-10): p. 759-96.

[0312] 7. Le Bon, C., et al., Folding and stabilizing membrane proteins in amphipol A8-35.

[0313] Methods, 2018. 147: p. 95-105.

[0314] 8. Smith, A. A. A., et al., Controlling Styrene Maleic Acid Lipid Particles through RAFT.

[0315] Biomacromolecules, 2017. 18(11): p. 3706-3713.

[0316] 9. Autzen, H.E., D. Julius, and Y. Cheng, Membrane mimetic systems in CryoEM:

[0317] keeping membrane proteins in their native environment. Curr Opin Struct Biol, 2019. 58: p. 259268.

[0318] 10. Timcenko, M., A.A.A. Autzen, and H.E. Autzen, Characterization of Divalent Cation Interactions with AASTY Nanodiscs. ACS Applied Polymer Materials, 2022. 4(2): p. 1071-1083.

[0319] 11. Glueck, D., et al., Electroneutral Polymer Nanodiscs Enable Interference-Free Probing of Membrane Proteins in a Lipid-Bilayer Environment. Small, 2022: p. e2202492.CUB-PA12-PCT

[0320] 12. Lin, C.A., et al., Design of an amphiphilic polymer for nanoparticle coating and functionalization. Small, 2008. 4(3): p. 334-41.

[0321] 13. Mueller, S., et al., The bigger picture: global analysis of solubilization performance of classical detergents versus new synthetic polymers utilizing shotgun proteomics. 2023.

[0322] 14. Yuan, Y., et al., Cryo-EM structure of human glucose transporter GLUT4. Nat Commun, 2022. 13(1): p.

[0323] 2671.

[0324] 15. Cookis, T., et al., Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation. J Vis Exp, 2023(202).

[0325] 16. Francisco, M., et al., Copolymerization of Maleic Anhydride with Styrene and a-Olefins. Molecular and Thermal Characterization. J. Macromol. Sci., Part A: Pure Appl. Chem., 2005(42).

Claims

1. CUB-PA12-PCTClaims1. A poly(a-olefine-co-maleic acid) copolymer comprising repeating units of formulas (1) and (2)or comprising repeating units of formulas (1) and (2a)formula (1) formula (2a)whereinXi and X2are independently NH, (Cl-C5-alkyl)N, in particular (CH3)N, O, O', S, S';X3is N;a is an integer of 1 to 20;Ri, R2, R3, and R4, can be independently derived from the following:CUB-PA12-PCTa (C3-C10) cycloalkane or a (C3-C10) (hetero)cycloalkan, which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, and linear or branched (C1-C8) alkynyls;a (C3-C10) cycloalken or a (C3-C10) (hetero)cycloalken, which can be poly or monounsaturated, and which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, linear or branched (C1-C8) alkenyls, and linear or branched (C1-C8) alkynyls;a poly or monounsaturated polycycle, which can be unsubstituted or substituted by one or more groups selected from linear, cyclic or branched (C1-C8) alkyls, and linear or branched (C1-C8) alkenyls, linear or branched (C1-C8) alkynyls;a linear C2 to CIO alkyl-amine, wherein one primary and / or mono-, di- or trimethylated aminegroup is present , wherein in case of the trimethylated amine a quaternary amine with a positive charge is present, the linear alkyl amine e.g. dimethylaminopropylamine can be further sulfonated by using 1,3 propane sultone, carboxylated by using exemplary a halo-(C2-C7)alkanoic acid (e.g. chloroacetic acid) or phosphorylateda polyol, wherein a polyol is any compound having at least two hydroxyl groups;a linear or cyclic mono-, di- or polysaccharide;a linear or branched polyethylene oxide;a linear or branched alkylated quaternary trimethyl ammonium chlorides;a group selected from biological tags, fluorophores, peptides, biotin and functional groups for click chemistry;H, or an alkali metal ion, andR3can be further selected, with the provisio that the above definitions are not included in this definition of R3, from the group consisting of a linear alkane chain, optionally, containing or terminating with a cyclic carbon; a linear chain alkoxy alkane of the formula -(CH2)qO(CH2)rCH3where q is 1 to 5 and r is 1 to 15; an alkoxy alkane containing or terminating with a cyclic carbon chain; a halogenated alkane; a halogenated cycloalkane; a halogenated arene; a chain containing a repeating sequence of (CH2CH2O)tterminating with -OR6whereintequals a value of 1 to 50 and R6is hydrogen, a linear alkane, a cyclic alkane, trifluoromethyl, a halogenated alkane, a halogenated cycloalkane, or a halogenated arene; a hydrogen, and a mixture thereofand salts thereof,provided that R1 and R2 are not simultaneously O' or HO.CUB-PA12-PCT2. Copolymer according to claim 1, wherein the polyol is selected from the group consisting of tris(hydroxymethyl)aminomethane, l-amino-2,3-propandiol, 2-amino-2-methyl-l,3-propandiol, 1-amino-2,3-butanediol, 3-amino-l,2-propanediol, l-amino-l,2-ethanedio and 2-amino-2-deoxy-D-glucitol.

3. Copolymer according to any of the preceding claims, wherein the linear or cyclic mono-, di- or polysaccharide is selected from the group consisting of N-methyl-D-glucamine, 2-amino-2-deoxy-glucose, N,N-dimethylglucamine, N-ethylglucamine, N-methyl-D-mannosamine, N-methyl-D-galactosamine, N-methyl-D-glucosamine, 2-amino-2-deoxy-galactose, 2-amino-2-deoxy-mannose, 2-amino-2-deoxy-ribose, 2-amino-2-deoxy-arabinose, 2-amino-2-deoxy-xylose4. Copolymer according to any of the preceding claims, wherein the linear or branched polyethylene oxide is 2-(2-(2-(2-aminoethoxy)ethoxy)ethoxy)ethanol.

5. Copolymer according to any of the preceding claims, wherein the linear or branched alkylated quaternary trimethyl ammonium chlorides is (2-aminoethyl)trimethylammoniumchlorid6. Copolymer according to any of the preceding claims, wherein the fluorophore is amino fluoresceine.

7. Copolymer according to any of the preceding claims, wherein the biotin is amino polyethylene glycol biotin.

8. Copolymer according to any of the preceding claims, wherein the copolymer has a number average molecular weight is 24000 g / mol or less.

9. A kit comprising the copolymer according to any of claims 1 to 8 and instructions for use in particular the uses as described above.

10. A method for preparing the copolymer according to any of claims 1 to 8, wherein a a-olefin-maleic acid copolymer having the group R3is reacted to provide the groups Ri, R2, and R4, optionally followed by a ring closure reaction.CUB-PA12-PCT11. Complex comprising the copolymer as defined in any of claims 1 to 8 and a hydrophobic protein, a membrane protein and / or a G protein coupled receptor (GPCR).

12. Complex according to claim 11 further comprising one or more lipids.

13. Complex according to claim 11 or 12, wherein the membrane protein is selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transporter proteins, such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membranebased ATPases, and SLC transporters.

14. Method for obtaining a complex according to any of claims 11 to 13 comprising contacting the copolymer as defined in any of claims 1 to 8 with the hydrophobic protein, the membrane protein, the G protein coupled receptor and optionally the lipid.

15. Use of the copolymer according to any of claims 1 to 8 for solubilization, stabilization and / or purification of membrane proteins.

16. Use according to claim 15, wherein the solubilization, stabilization and / or purification is provided out of a native membrane surrounding.

17. Use according to claim 15 or 16, wherein the membrane protein is selected from the group consisting of membrane receptor proteins, membrane enzymes, cell adhesion proteins, and transporter proteins, such as ABC transporters, ion channel proteins, water channel proteins (aquaporins), membrane-based ATPases, and SLC transporters.

18. Use of the copolymer according to any of claims 1 to 8 for cryo electron microscopy in particular with the stabilization of the position and orientation of the membrane protein.

19. Use of the copolymer according to any of claims 1 to 8 for labelling of nanodisc complexes.

20. Use of the copolymers according to any of claims 1 to 8 for the opening of cellular membranes.

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