Copolymers based on poly(isobutylene-ALT-maleic anhydride for the solubilization, isolation and molecular labelling of membrane proteins in aqueous media

Abstract

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

Description

COPOLYMERS BASED ON POLY(ISOBUTYLENE-ALT-MALEIC ANHYDRIDE) FOR THE SOLUBILIZATION, ISOLATION AND MOLECULAR LABELLING OF MEMBRANE PROTEINS IN AQUEOUS MEDIATECHNICAL FIELDThe present invention relates to specifically defined copolymers, a method for their preparation, a complex containing this copolymer, the use of the copolymer for solubilizing and stabilizing membrane proteins as well as a kit for this use containing the polymers according to the present invention.TECHNICAL BACKGROUNDMembrane 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.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.The commercially available detergents (e.g. SOS) 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 andinactivation. 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 function 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.[ODDS] 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 lass 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.A new class of amphiphilic polymers published and patented are SMAs (Styrene Maleic-Acid, WO2006129127, WO2011004158), 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],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. The solubilization efficiency as well as the stabilityof the formed membrane protein containing nanodisc strongly depends on the characteristics of the used polymer. Polymers, like SMAs, CyclApols and AASTYs 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 invention, 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.The length of the polymer as well as the polydispersity and the homogenous sequence of copolymerized 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. [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 hydrophilic and hydrophobic side chain combinations to enable protein structure analysis of membrane proteins using cryo-TEM.SUMMARY OF THE INVENTIONTo overcome the drawbacks of current copolymers for membrane solubilization, the object of the underlying invention is to provide a copolymer, which bears a new polymer backbone and is 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.This has been achieved by the subject-matter of the independent claims. Preferred embodiments are defined in the dependent claims.According to the present invention there is provided a copolymer comprising repeating units of the following formula (1) and (1 ') and / or the following formula (2) and (2')formula (2),formula (2') whereinRi and R3 are independently selected from the group consisting of (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;(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 (Cl- C8) alkyls, linear or branched (C1-C8) alkenyls, linear or branched (C1-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; 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; 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 phosphategroups; 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

[0018] molecules from the class of polyols like 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-1,2- ethanedio or 2-Amino-2-deoxy-D-glucitol; 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; molecules from the class of amine functionalized linear or branched polyethylene oxides like 2-(2- (2-(2-Aminoethoxy)ethoxy)ethoxy)ethanol, the number of ethoxy units may be varied between 1 and 15. The terminal functional group can be varied and may be a hydroxyl, methoxy, carboxyl, amino, thiol or others; molecules from the class of cholines like (2-Aminoethyl)trimethylammoniumchlorid, A-(3-Chlor- 2-hydroxypropyl)-A,A,A-trimethylammoniumchlorid,R.3 is additionally to the above H or an alkali metal ion;X is NH, 0 or S, and x and y are greater than 0, that is in the copolymer according to the present invention, both units bearing the index x and y respectively are present.The dashed lines in all formulas of the present application do not represent a chemical bond, but illustrate that the corresponding repeating units are in a relationship to each other, in particular that they arepresent together in a polymer of any kind, in particular as a random copolymer or as a block copolymer. It is common practice in the field in question here to describe such relationships within a polymer with dashed lines (see, for example, US 2022 / 0119558 Al and US 11,092,605 B2).The sum of x and y can be 100 % of the copolymer. Furthermore, the copolymer according to the present invention can be a random copolymer or an alternating copolymer.The negative charge of the carboxyl groups can be equalized with cations if necessary.The copolymer according to the present invention is based on a maleic acid-isobutylene copolymer. The starting maleic acid-isobutylene copolymer is commercially available as an anhydride. 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.If these compounds are heated, a ring closure to the maleimide is achieved. However, the ringclosure can be achieved using different techniques based on condensation and water elimination like the DCC HOBt catalyzed ring-closure.The copolymer according to the present invention can be a zwitterionic compound as exemplified in formula (5) and (5 ')formula (5')According to the present invention there is also provided a method for preparing the above copolymer, wherein the anhydride of a maleic acid-isobutylene copolymer is reacted with the amines of Re and the group Rs to provide the copolymer of formulas (5) and (5'). The amines of Re as well as the additional Rs functionality are defined as follows:Rs is a branched or linear C2-C7 alkyl;Re is a branched or linear C 1 -C6 alkyl with a terminal functional group containing a negative charge as carboxylic, phosphate or sulfonates moieties;R? and Rs are each independently a branched or linear C1-C4 alkyl;The copolymer according to the present invention exemplified in formula 5 and 5 ' as maleic acid amide can be turned in the maleimide form as shown in formula 6.According to the present disclosure, the amide is formed on one of the two carbonyl carbons and a carboxylic acid on the other.In one embodiment, the copolymer according to the present invention can be represented by thefollowing formula (3) and (3') or the following formula (4) and (4')formula (4)formula (4') whereinRi, R3 and X are defined as above. That is, the compound of formula (3) and formula (4) contain as one constituent the above compounds of formulas (1) and (2), respectively.R2 is derived from biological tags, fluorophores, peptides, biotin and functional groups for dick chemistry, and R4 is independently selected from the residues defined for R3.The letters A-D in formulas 3-4 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-D can be 100% and is equal to the number of maleic acid anhydride units or isobutylene units of the initial PIMA polymer. The functionalization can be in the range from 0% -100% to the respective maleic acid anhydride units and in general A + B + C + D= 100% of the total functionalized and non-functionalized maleic acid anhydride groups of the polymer.By the copolymers of formulas 3-4 additional functionalities can be provided to the copolymers, such as fluorophores, tags (His, Rho, FLAG), biotin, functional groups for dick chemistry (e.g. azides and alkynes) and others. These compounds can be attached to the anhydrides as amine- functionalized molecules. Theoretically, this can be done to the amide according to formula (1), but also via a ring closure according to formula (2).In one embodiment of the copolymer according to the present invention, Ri and R3 are independently selected from cycloalkyl residues, for example cyclooctyl, ethyl cyclohexyl, and methyl cyclohexyl. Surprisingly it has been found that cycloalkyl compounds solubilize membrane proteins, whereas linear alkyl compounds do not. Thus, the copolymers according to the presentinvention are unexpectedly particularly useful for use in solubilization and stabilization of hydrophobic proteins, membrane proteins, and GPCRs.According to the present invention there is also provided a method for preparing the above copolymer, wherein the anhydride of a maleic acid-isobutylene copolymer is reacted with the amines of Ri and R3 and the groups R2 and R4 are introduced to provide the copolymer of formulas (1), (1 ') and (3), (3 '), which subsequently can be reacted to the copolymers of formulas (2), (2') and (4), (4') elimination of water. The amines of Ri and R3 can be represented by R1-NH2 and R3- NH2, wherein Ri and R3 are defined as above, wherein by these amines the atom N is introduced into the copolymers according to the present invention.In a further embodiment, there is 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.According to the present invention, 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.The copolymer according to the present invention 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. 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.Furthermore, the copolymers according to the present invention can be used for cryo electron microscopy in particular with stabilization of the position and orientation of the membrane protein.Furthermore, according to the present invention there is provided a complex comprising thecopolymer according to the present invention, 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.The present invention also provides a kit, comprising the copolymer according to the present invention and optional instructions for use in particular the uses as described above.In the following, the present invention will be described more specifically.According to the present invention, there is provided poly(isobutylene-alt-maleic anhydride- based copolymers with the general formula (6):formula (6)The two segments bearing the group Ri and R2, respectively, are randomly distributed over the length of the polymer chain.In the above formula (6), X or Y is either the initiator fragment derived by the radical starter molecule, the isobutylene monomer, a functional group (e.g., hydroxy, carboxylic acid, etc.) or a hydrogen atom.The term functional group refers to any functional group known in the field of organic chemistry.The letters A-D 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-D is 100% and is equal to the number of maleic acid anhydride units or isobutylene units of the initial PIMA polymer. The functionalization can be in the range from 0% - 100% to the respective maleic acid anhydride units and in general A + B + C + D= 100%of the total functionalized and non-functionalized maleic acid anhydride groups of the polymer.Ri - R4 is the hydrophilic or the hydrophobic part of the polymer. R1-R4 can be derived by the group of following amino-, thio- or hydroxy-bound substitutes:(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.Further the pendant hydrophilic group can include one or more of the following: hydroxyl, amino, ether, carboxylic acid, carboxylate, phosphate, phosphonate, phosphocholine, , choline, 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.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 like cholines and their derivatives). 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. 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. Dimethyl aminopropyl amine) is further sulfonated (e.g., 1,3 propane sultone) but can also contain a carboxylic or phosphate moiety.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.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.Molecules from the class of amine functionalized linear or branched polyethylene oxides like 2-(2- (2-(2-Aminoethoxy)ethoxy)ethoxy)ethanol, the number of ethoxy units may be varied between 1 and 1 . The terminal functional group can be varied and may be a hydroxyl, methoxy, carboxyl, amino, thiol or others.Molecules from the class of cholines like (2-Aminoethyl)trimethylammoniumchlorid, A-(3-Chlor- 2-hydroxypropyl)-A,A,A-trimethylammoniumchlorid,Amine containing biological tags (e.g., Biotin-PEG4-Amine) or fluorophores (e.g. Fluoresceine amine) or peptides (e.g., Rho tag).Specifically, R3 and R4 can be a hydroxy group or an alkali metal (e.g., Li+, Na+, K+) ionically bound to an oxygen.DefinitionsThe term "amphipol" refers to vinyl polymers which are capable of solubilizing membrane proteins and keep them stable in solution in their native form.The term "nanodisc" refers to a small nanometer-sized complex formed by an amphiphilic polymer and lipids and is capable of stabilizing membrane proteins.The term "PIMA" comprises, with respect to the present invention, the polymer backbone based on polymerization of isobutylene and maleic acid anhydride monomers.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.The term "grafting" describes in the context of the present invention the functionalization of maleic acid anhydride units within the polymer chain with other amine, thio or hydroxy containing molecules.The prefix "sulfo-" refers to a 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.The prefix "glyco-" refers to a 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.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 apoly-his (for example his5 or his 10) or a TETSQVAPA amino acid sequence 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. and Corin et al..The present invention employs the use of poly(isobutylene-alt -maleic anhydride copolymer (PIMA) for the synthesis of an amphiphilic polymer.The PIMA used for the present invention can be purchased commercially (e.g., Thermo Scientific, Sigma Aldrich, 26426-80-2). The molecular weight of the PIMA can be in the range of 2-20 kDa but is not limited to the described range.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-1 -octadecene), Poly(maleic anhydride-alt-1 -tetradecene), Poly(ethylene-alt-maleic anhydride), Polyethylene-graft-maleic anhydride, Polyisoprene-graft-maleic anhydride, Polypropylene-graft- maleic anhydride, Poly(styrene-co- maleic anhydride), Poly(methyl vinyl ether-alt-maleic anhydride).For generation of the amphiphilic character of the copolymer according to the present invention, 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.The grafting of the base polymer with hydrophilic and hydrophobic molecules can be in the range of >1%, in particular between 1-100% and preferably between 40-60%. The grafting percentage is referred to the number of maleic acid anhydrides in the polymer backbone reacted with an amine-, thiol- or hydroxy- containing molecule forming an amide or ester. 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.Polar molecules used for the grafting can be 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 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, 1 -amino- 1,2-ethanedio or 2-amino-2- deoxy-D-glucitol and similar substances can be used.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.In the case of amine functionalized linear or branched polyethylene oxides like 2-(2-(2-(2- Aminoethoxy)ethoxy)ethoxy)ethanol can be used. The number of ethoxy units may be varied between 1 and 15. The terminal functional group can be varied and may be a hydroxyl, methoxy, carboxyl, amino, thiol group or others.In the case of amine functionalized cholines like (2-aminoethyl)trimethylammoniumchlorid, N-(3- Chlor-2-hydroxypropyl)-N,N,N-trimethylammoniumchlorid can be used.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). 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. Molecules from the class of the polyols like 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 or 2-Amino-2- deoxy-D-glucitol.Said functionalized and non-functionalized new amphiphilic based copolymers can be used for solubilization and stabilization of integral membrane proteins and membrane associated proteins.The copolymers according to the present invention can be used for labelling of nanodisc complexes for their use in biomolecular research, diagnostic applications and medical product development.Compared to the polymers and copolymers of the prior art, the copolymer according to the present invention has at least one of the following advantages:Enhanced solubilization efficiency of membrane proteins expressed in procaryotic and eucaryotic cells and / or native proteins expressed in cell membranes.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.Due to the enhanced solubilization efficiency, the polymers according to the present invention can be used in lower concentrations, leading to higher purity and functionality of the stabilized membrane proteins.Possibility of functionalization of new copolymers with molecules (e.g., fluorophores, biotin derivatives, etc.) for protein purification and protein labelling.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.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., Bonification, French Press), centrifugation at 9 000 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.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 asABC transporters, ion channel proteins, water channel proteins (aquaporins), membranebased ATPases, and SLC transporters. That is, as a starting material for the method according to the present invention, 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.The amphiphilic copolymers according to the present invention 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 invention as reagent, in reagent kits and diagnostic kits (e.g., lateral flow assays) comprising at least one part of the described invention. 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 invention 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.The use of copolymers according to the present invention can be exemplified as follows: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.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.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.Solubilization efficiency can be determined using standard biomolecular methods (e.g., SOS- PAGE, Western Blot).By Centrifugation, insolubilized proteins and debris can be separated from the solubilized proteins located in the formed nanodisc complex.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.The copolymers according to the present invention 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.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).The interaction of the copolymer stabilized membrane protein and its interaction partner can be detected inclusive but not exclusively via different analytical methods.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 invention are particularly suitable for diagnostic tests, especially basing on DNA, RNA, soluble proteins, and membrane proteins.The according to the present invention or the complex of the copolymer and the lipids, drugs or biomolecules can be used for diagnostic or therapeutic applications.ExamplesThe present invention is described in the following by examples. It is explicitly pointed out that the examples shall not be construed to limit the invention thereto.The figures show the following:Figure 1: ATR-FTIR spectra of PIMA and Cubipolnh (anhydride).Figure 2: ATR-FTIR spectra of Cubipolnh (anhydride) and the hydrolyzed Cubipol.Figure 3: ATR-FTIR spectra of Cubipolnh (anhydride) and the functionalized Cubipol Glycerol.Figure 4: ATR-FTIR spectra of Cubipolnh (anhydride) and the functionalized Glyco Cubipol.Figure 5: ATR-FTIR spectra of Cubipolnh (anhydride) and the functionalized Sulfo Cubipol.Figure 6: ATR-FTIR spectra of Cubipolnh (anhydride) and the functionalized Cubipol Amin.Figure 7: ATR-FTIR spectra of Cubipolnh (anhydride) and the functionalized Cubipol PEG.Figure 8: UV-Vis extinction spectra of Cubipol, Cubipol-Glycerol, Sulfo-Cubipol, Glyco-Cubipol, Cubipol PEG and Cubipol Amin. The polymers were measured at 25 mg / ml solution in 150 mM NaCl, 20 mM HEPES buffer, pH 7.5.Figure 9: Tolerance screening of Cubipol against divalent ions (top) and different pH values (bottom). Precipitation of the polymer as white gel indicates instability of the polymer.Figure 10: Tolerance screening of Cubipol Glycerol against divalent ions (top) and different pH values (bottom). Precipitation of the polymer as white gel indicates instability of the polymer.Figure 11: Tolerance screening of Glyco Cubipol against divalent ions (top) and different pH values (bottom). Precipitation of the polymer as white gel indicates instability of the polymer.Figure 12: Tolerance screening of Sulfo Cubipol against divalent ions (top) and different pH values (bottom). Precipitation of the polymer as white gel indicates instability of the polymer.Figure 13: Tolerance screening of Cubipol Amin against divalent ions (top) and different pH values (bottom). Precipitation of the polymer as white gel indicates instability of the polymer.Figure 14: Tolerance screening of Cubipol PEG against divalent ions (top) and different pH values (bottom). Precipitation of the polymer as white gel indicates instability of the polymer.Figure 15: HPLC chromatogram of Cubipol and Cubipol Fluoresceine. Excitation at 490 nm followed by fluorescence signal detection at 520 nm. The Cubipol base-polymer reveal no fluorescence while cubipol fluoresceine reveal a strong fluorescence. Agilent 1260 Infinity II system equipped with a Plgel 3 pM MIXED E column and DMF with 0.5 mg / ml LiBr as mobile phase was used at 60°C with 1 ml / min flow-rate. 50 pl (0. 1 mg / ml polymer) were injected.Figure 16: FPLC chromatogram of Cubipol Fluoresceine stabilized and affinity -resin (RholD4 MagBeads, Cube Biotech GmbH) purified human G6PC. Agilent 1260 Infinity II system equipped with a Superose 6 5 / 150 GL column and 150 mM NaCl, 20 mM HEPES, pH 7.5 as mobile phase at 0.5 ml / min flow-rate were used at room temperature. After elution from the purification resin, 50 pl protein eluate were injected. Excitation at 490 nm followed by fluorescence signal detection at 520 nm. The chromatogram reveals a void peak (5.3 min), the nanodisc (6-11 min) and excess fluorescent polymer (11-19 min).Figure 17: FPLC chromatogram of Cubipol Biotin (0.65 mg / ml), Streptavidin (1.5 mg / ml) and Cubipol Biotin (0.65 mg / ml)+Sfreptavidin (1.5 mg / ml) mixture. Agilent 1260 Infinity II system equipped with a Superdex 75 10 / 300 column and 150 mM NaCl, 20 mM HEPES, pH 7.5 as mobile phase at 0.5 ml / min flow -rate were used at room temperature. The change of the refractive index over time were monitored. The chromatogram reveals a Cubipol Biotin peak (22 min), the Streptavidin peak (17.8 min) and the shifted peak of Streptavidin upon binding of four Cubipol Biotin polymers (19.2 min).Figure 18: Coomassie stained SDS PAGE of the affinity resin purified and RholD4-tagged human membrane protein UCP 1 stabilized in Cubipol, Sulfo Cubipol, Glyco Cubipol, Cubipol PEG, Cubipol Amin, Cubipol Biotin or Cubipol Biotin. The expected size of UCP1 according to the sequence is 34. 17 kDa.Figure 19: Western blot of the SDS PAGE of the affinity resin purified and RholD4-tagged human membrane protein UCP 1 stabilized in Cubipol, Sulfo Cubipol, Glyco Cubipol, Cubipol PEG, Cubipol Amin, Cubipol Biotin or Cubipol Biotin. The expected size of UCP 1 according to the sequence is 34. 17 kDa.Figure 20: Hydrodynamic radii of copolymer DMPC nanodiscs determined via DLS.Synthesis of PIMA-C8C0Suspend 3.084 g PIMA (20.02 mmol MAnh monomers) in 50 ml THF in a 100 ml round bottom flask while stirring vigorously. The suspension is overlaid with argon and varying amounts of cyclooctylamine diluted in THF are added. For the synthesis of 40 % grafted PIMA-C8C0, in terms of the hydrolyzed form of the PIMA maleic anhydride and the total amount of available carboxyl groups, 2,037 g of cyclooctylamine (16,02 mmol) dissolved in 5 ml of THF is added slowly dropwise. The suspension is placed in an oil bath and heated to 60°C for 24 h. After the reaction, the suspended PIMA is grafted with the C8C0 monomer and undergoes dissolution followed by conversion to a clear solution. The THF is then evaporated under reduced pressure. The yellowish solid polymer is hydrolyzed by dissolution at 5 wt.% in 1 M NaOH followed by heating under reflux at 100°C. The hydrolyzed and ring -opened version of the amphiphilic PIMA-C8C0 copolymer is precipitated with 1 M HCI and washed three times with double-distilled water. After freezing and lyophilization, the polymer is obtained.To obtain the ring -closed version of the amphiphilic PIMA polymer, the THF is replaced by DMF as the solvent for the reaction. After reacting 1.019 g (8.008 mmol) C8C0 with the PIMA to obtain a 40% grafted ring-closed polymer version, the flask is prepared with an oil bubbler and heated to 120°C for 24 h. This converts the amide form of the PIMA-C8C0 to the maleimide form by eliminating H2O. Hydrolysis and purification are performed as described above.Synthesis of Sulfo-PIMA-C8C0Suspend 3.084 g PIMA (20.02 mmol MAnh monomers) in 50 ml THF in a 100 ml round bottom flask while stirring vigorously. The suspension is overlaid with argon and varying amounts of cyclooctylamine diluted in THF are added. For the synthesis of 40 % grafted PIMA-C8C0, in terms of the hydrolyzed form of the PIMA maleic anhydride and the total amount of available carboxyl groups, 2,037 g of cyclooctylamine (16,02 mmol) dissolved in 5 ml of THF is added slowly 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. After the reaction, the suspended PIMA is grafted with the C8C0 monomer and undergoes dissolution followed by conversion to a clear solution. To introduce the quaternary amine moieties, 0.613 g (6 mmol) dimethylaminopropylamine dissolved in 5 ml THF is added to the solution of PIMA-C8C0 in THF 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, 1 g (8 mmol) of 1,3 -propane sultone dissolved in 5 ml of THF is added to the solution of PIMA-C8C0 in THF 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 THF is then removed under reduced pressure and the solid product is purified by resuspension in 100 trichloromethane, filtration and drying in vacuo. 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 as a yellowish solid grafted with 40 mal% hydrophobic, 50 mal% carboxylic and 10 mal% zwitterionic moieties.Ring-opened Sulfo-PIMA-C8C0The ratio of the final hydrophobic, carboxylic and zwitterionic moieties in the polymer chain can be adjusted by varying the respective molar amounts. To obtain the ring-closed version of the described polymer, the THF in the reaction is replaced by DMF and, after introducing the dimethylaminopropylamine, the flask is sealed with an oil bubbler and the solution is heated to 120°C for 24 h, which yields the maleimide form under elimination of H2O.Synthesis of tagged or labelled PIMA precursor polymer3.084 g PIMA (20.02 mmol MAnh monomers) is dissolved under vigorous stirring in 10 ml DMF in a 100 ml round-flask under Argon atmosphere, at room temperature and protected from light. A solution of 178 mg 5 -Aminofluorescein in 5 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 usingHPLC. For implementation of the hydrophobic moieties, the PIMA-C80 synthesis described before can be applied.PIMA-Fluorescein as fluorescent precursor polymer for the synthesis of PIMA-C8C0-FluoresceinBy exchanging the molar amount and / or the described 5 -aminofluorescein with primary amine containing biomolecular tags, other fluorophores or molecules, a vast set of functionalities can be implemented in the final copolymer backbone.Synthesis of PIMA-C8C0-Tris or PIMA-C8C0-GlycerolSuspend 3.084 g PIMA (20.02 mmol MAnh monomers) in 50 ml THF in a 100 ml round bottom flask while stirring vigorously. The suspension is overlaid with argon and varying amounts of cyclooctylamine diluted in THF are added. For the synthesis of 40 % grafted PIMA-C8C0, in terms of the hydrolyzed form of the PIMA maleic anhydride and the total amount of available carboxyl groups, 1.273 g of cyclooctylamine (10.01 mmol) dissolved in 5 ml of THF is added slowly dropwise. The suspension is placed in an oil bath and heated to 60°C for 24 h. After the reaction, the suspended PIMA is grafted with the C8C0 monomer and undergoes dissolution followed by conversion to a clear solution. The reaction of the residual maleic acid anhydride monomers of the polymer backbone can be achieved by adding the threefold molar amount of polyol amine. For PIMA-C8C0-Tris in the ring opened version, 3.6 g Tris(hydroxymethyl)aminomethane dissolvedin 5 ml THF is added dropwise. For PIMA-C8C0-Glycerol, 4.6 g 2-aminol,3-propandiol dissolved in 5 ml THF is added dropwise. The solution is placed in an oil bath and heated to 60°C for 24 h. The THF is then evaporated under reduced pressure. The yellowish solid polymer is hydrolyzed by dissolution at 5 wt.% in 1 M NaOH followed by heating under reflux at 100°C. The hydrolyzed and ring-opened version of the amphiphilic PIMA-C8C0-Tris copolymer is dialyzed against double-distilled water. After freezing and lyophilization, the polymer is obtained.PIMA-C8C0-Tris in the ring-open statePIMA-C8C0-Glycerol in the ring-open stateSynthesis of Glyco-PIMA-C8C0Suspend 8.0 g PIMA (51.93 mmol MAnh monomers) in 40 ml DMF in a 100 ml round bottom flask while stirring vigorously. The suspension is overlaid with argon and varying amounts of cyclooctylamine diluted in THF are added. For the synthesis of 40 % grafted PIMA-C8C0, in terms of the hydrolyzed form of the PIMA maleic anhydride and the total amount of available carboxyl groups, 5.28 g of cyclooctylamine (41.5 mmol) dissolved in 10 ml of DMF is added slowly dropwise. The suspension is placed in an oil bath and heated to 60°C for 24 h. After the reaction, the suspended PIMA is grafted with the C8C0 monomer and undergoes dissolution followed by conversion to a clear solution. The anhydride form of the grafted polymer is precipitated in 400 ml diethyl ether and dried in vacuo. 10 g of the dried solid polymer is dissolved with 6.75 g N-methyl-D-glucamine in 100 ml of dry methanol at 65°C. After dissolution, a solution of 0.651 mg sodium in 25 ml methanol was added dropwise under argon and stirring. After incubation for 24 h and addition of 100 ml ethanol the final glycol-PIMA-C8C0 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.Glyco-PIMA-C8C0 in the ring-open stateSynthesis of PIMA-C8C0-PEGSuspend 8.0 g PIMA (51.93 mmol MAnh monomers) in 40 ml DMF in a 100 ml round bottom flask while stirring vigorously. The suspension is overlaid with argon and varying amounts of cyclooctylamine diluted in THF are added. For the synthesis of 40 % grafted PIMA-C8C0, in terms of the hydrolyzed form of the PIMA maleic anhydride and the total amount of available carboxyl groups, 5.28 g of cyclooctylamine (41.5 mmol) dissolved in 10 ml of DMF is added slowly dropwise. The suspension is placed in an oil bath and heated to 60°C for 24 h. After the reaction, the suspended PIMA is grafted with the C8C0 monomer and undergoes dissolution followed by conversion to a clear solution. The anhydride form of the grafted polymer is precipitated in 400 ml diethyl ether and dried in vacuo. 10 g of the dried solid polymer is dissolved in 20 ml of DMF at 65°C. After dissolution, a solution of 5.34 g PEG4-Amin (27.6 mmol) in 20 ml DMF was added dropwise under argon and stirring. After incubation for 24 h the final PIMA-C8C0- PEG 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. A ring-closed derivative can be synthesized by condensation of ring-opened version as described.Ring-open PIMA-C8C0-PEGSynthesis of PIMA-C8C0-AminSuspend 8.0 g PIMA (51.93 mmol MAnh monomers) in 40 ml DMF in a 100 ml round bottom flask while stirring vigorously. The suspension is overlaid with argon and varying amounts of cyclooctylamine diluted in THF are added. For the synthesis of 40 % grafted PIMA-C8C0, in terms of the hydrolyzed form of the PIMA maleic anhydride and the total amount of available carboxyl groups, 5.28 g of cyclooctylamine (41.5 mmol) dissolved in 10 ml of DMF is added slowly dropwise. The suspension is placed in an oil bath and heated to 60°C for 24 h. After the reaction, the suspended PIMA is grafted with the C8C0 monomer and undergoes dissolution followed by conversion to a clear solution. The anhydride form of the grafted polymer is precipitated in 400 ml diethyl ether and dried in vacuo. 10 g of the dried solid polymer is dissolved in 20 ml of DMF at 65°C. After dissolution, a solution of 5.0 g (2-Aminoethyl)trimethylammoniumchlorid -hydrochi orid (5.71 mmol) in 10 ml DMF and 5 ml H2O was added dropwise under argon and stirring. After incubation for 24 h the final PIMA-C8C0-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. A ring-closed derivative can be synthesized by condensation of ring-opened version as described.Ring-open PIMA-C8C0-AminSolubilization of his- or rho-tagged membrane protein G6PC and purificationDynamic Light ScatteringDynamic light scattering experiments were performed using a Nanotemper Prometheus Panter with the included software. Each sample was loaded in a 10 pl glass capillary. Each measurement consisted of 10 scans of 5 s carried out at 25°C. The attenuator position and laser power automatically optimized for size determination.DMPC nanodisc preparationDMPC was suspended with 1.25 mg / ml in the respective 5 w / v% copolymer solutions in 150 mM NaCl, 20 mM HEPES buffer (pH 7.5) and were stirred or 4 h at room temperature. Prior to analysis the solutions were centrifuged for 30 min at 10.000 ref. pH and divalent ion stability assayCopolymer solutions (2.5 w / v%) in 150 mM NaCl, 20 mM HEPES with 0-150 mM MgCL orCaCH or pH ranging from 3-10 were prepared.Expression of rho-tagged membrane protein G6PC and UCP1The production, expression and purification of G6PC and UCP1 (M. Jastroch, V. Hirschberg and M. Klingenspor, 2012, Functional characterization of UCP1 in mammalian HEK293 cells excludes mitochondrial uncoupling artefacts and reveals no contribution to basal proton leak, BBA, 1817 (9), 1660-1670, doi.org / 10.1016 / j.bbabio.2012.05.014) is described in literature.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.Cell lysis and centrifugation:Add protease inhibitors (PI) to buffer and readjust pH value then disrupt cells (e.g., Sonification, French Press). Centrifuge at 9 000 ref for 30 min at 4°C, discard pellet (cell debris), collect supernatant. Centrifuge supernatant at 100 000 ref for 1 hat 4°C, discard supernatant and homogenize pellet.Solubilization of membrane proteins: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 condition the unique lipid environment needs to be conserved.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:Add solubilization agent (copolymer) to the protein solution Ideal concentrations may vary, good starting points are:0.1 - 2.5 w / v% copolymer (e.g. PIMA based amphiphilic copolymers) Solubilize for 3 h to 24 hat 4 °C while stirring.Higher temperatures can be screened for optimization Centrifuge at 100 000 ref for 1 h at 4 °C Discard the pellet containing cell fragments, collect the supernatantUse 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.Visualization of solubilized membrane proteins by SDS-PAGE, Western Blot and Rho Antibody / HRP staining for chemiluminescence detection.Results:The functionalization of the base-polymer PIMA with the modifications described within this invention can be monitored using standard analytical techniques as electronic absorption spectroscopy (UV-Vis), high performance liquid chromatography (HPLC) with different online detectors, infrared spectroscopy (ATR-FTIR) and dynamic light scattering (DLS) to characterize the intrinsic physicochemical properties of the copolymers and their ability to solubilize lipids and proteins.Figure 1 shows the conversion of the unfunctionalized polyisobutylene-maleic acid anhydride (PIMA) backbone polymer with cyclooctylamine to the Cubipol polymer in the anhydride state. For all FTIR spectra (Figure 1-7) characteristic vibrational modes can be detected. Upon functionalization of the maleic acid anhydride, the characteristic C=O carbonyl stretching band at —1770 cm'1and —1850 cm'1are weakend or completely diminished upon hydrolyzation while the carboxylic acid C=O stretching at -1720 cm'1appears together with the amide C=O stretching at 1660 cm'1and the amide N-H vibration at 1550 cm1. Additionally, for Sulfo Cubipol characteristic S=O stretching bands appear between 1100-1250 cm'1which indicates a successful functionalization. For Cubipol PEG, the C-O-C deformation can be detected at 1100 cm1.The different chemical properties of the copolymers described in this invention can be related to two characteristics. First the hydrophobicity which is derived by the aliphatic backbone and the hydrophobic modification of the backbone. In this example, the hydrophobic modification is cyclooctylamide. Both hydrophobic elements, the backbone and the modification are responsiblefor the solubilization efficiency and can be varied through the modification rate of the backbone. Second, the hydrophilicity is derived by the hydrophilic modification of the backbone. This hydrophilic modification is responsible for the stability of the polymer in solution as well as the stability of formed nanodiscs with or without stabilized protein. The modifications are numerous and exemplary the modifications and results of PIMA-C8C0 (Cubipol), PIMA-C8C0-Glycerol (Cubipol Glycerol), Glyco-PIMA-C8C0 (Glyco Cubipol), PIMA-C8C0-PEG (Cubipol PEG), PIMA-C8 CO-Amin (Cubipol Amin) and Sulfo- PIMA-C8C0 (Sulfo Cubipol) are described. Most important, the resistance against mono- and divalent ions (e.g. H+, Mg2+. Ca2+) which was determined by precipitation experiments. The results are shown in Table 1 as well as Figure 9-14. Upon adjusting of the pH from 3-10 or addition of the respective divalent ion amount for the target concentration, precipitation of the polymer indicated the instability under the chosen conditions.Table 1:Overview of the properties of Cubipol, Cubipol-Glycerol, Sulfo-Cubipol, Glyco-Cubipol, Cubipol PEG and Cubipol Amin. Tested was the stability of the respective copolymer in aqueous solution with 25 mg / ml polymer in 150 mM NaCl, 20 mM HEPES buffer with varying calcium chloride and magnesium chloride concentrations at pH 7.5. For pH stability assays, the pH of the polymer solution was adjustet accordingly.Copolymers used for the stabilization of membrane proteins should reveal minimal absorption above 260 nm to enable the determination of the protein concentration by UV-Vis absorption. Specifically, quantifying the amino acid tryptophane which absorbs light at 280 nm. Figure 8 shows UV-Vis absorption spectra of exemplary copolymers described in this invention. The absorption of a 25 mg / ml copolymer solution is <0.1 a.u. which enables the quantification of protein yield after protein purification and removal of excess copolymer.The copolymer technology described in this invention allows the functionalization of the different copolymers with biomolecular tags and fluorophores as described before. Figure 15 shows the HPLC chromatogram of Cubipol and Cubipol Fluoresceine. The fluorescence signal at 520 nm was detected after excitation at 490 nm. The chromatogram reveals no unspecific fluorescence of the Cubipol polymer in contrast to the strong fluorescence signal of Cubipol Fluoresceine with a peak maximum at ~7 min. Figure 16 shows the FPLC chromatogram of the human G6PC membrane protein stabilized in Cubipol Fluoresceine and purified with RholD4 Magbeads. The chromatogram reveals a fluorescent void peak at 5.3 min of not sufficiently stabilized protein, the fluorescent nanodisc peak containing the membrane protein G6PC between 6-11 min as well as the excess copolymer from 11-18 min.The attachment of biomolecular tags at the copolymer backbone was exemplary shown via FPLC with Cubipol Biotin in Figure 17. Figure 17 shows the refractive index signal change after size exclusion chromatography of Cubipol Biotin, Streptavidin and a sample of a mixture of both. The fourfold molar amount of streptavidin was incubated with the onefold molar amount of Cubipol Biotin to ensure the saturation of streptavidin. The successful binding of Cubipol Biotin can be seen upon the peak shifting from ~22 min (Cubipol Biotin) to 19.5 min (Cubipol Biotin + Streptavidin). Streptavidin itself reveals a weak refractive index signal at ~18 min.To assess the ability of the polymers described in this invention to solubilize lipids, the capability was evaluated using l,2-dimyristoyl-sn-glycero-3 -phosphocholine (DMPC) as exemplary lipid. Using dynamic light scattering as analytical technique allows the characterization of the formed synthetic copolymer nanodiscs with respect to their hydrodynamic size. The formed lipid copolymer nanodiscs have a hydrodynamic radius between 2.5 nm and 12 nm. However, it has to be noted that the DLS data does not reveal the physical size of the formed copolymer nanodiscs but the hydrodynamic size including bound ions. Thus, the size correlates with the actual charge of the copolymer backbone. Sulfo Cubipol has the highest number of charges per copolymer chain leading to the largest number of bound ions resulting in a large hydrodynamic radius.The ability to solubilize membrane proteins was shown with the human membrane protein UCP1 tagged with a RholD4 tag and overexpressed in HEK cell culture. The solubilization and purification was achieved following a protocol previously described. After affinity resin purification and elution, samples of the eluate were analyzed using SDS-PAGE, Western blot and DLS. Figure 18 shows the Coomassie stained gel with the target protein UCP1 at around 35 kDa which fits the expected size of 34.2 kDa. The identity of the bands at around 35 kDa was furtherconfirmed by Western blot with anti-RholD4 antibody labeling followed by a secondary HRP antibody labeling and detection of the chemiluminescence. Furthermore, the cumulant radius of the UCP1 containing copolymer nanodiscs were determined via DLS indicating stable copolymer nanodiscs. The low PDI indicates no aggregation of the stabilized membrane protein in the different copolymers.Table 2.: Cumulant radius and cumulant PDI of the protein UCP1 stabilized in different Cubipol derivatives analyzed via dynamic light scattering.Polymer Cumulant Radius [nm] Cumulant PDI [nm]Cubipol 12.57 0.31Sulfo-Cubipol 22.59 0.30Glyco-Cubipol 18.24 0.50Glycerol- Cubipol 15.16 0.40Cubipol PEG 20.71 0.43Cubipol Amin 10.55 0.32Cubipol Biotin 14.30 0.39The invention of this multimodal copolymer platform described in this invention enables the efficient stabilization of different membrane proteins resulting in copolymer nanodiscs with tunable properties regarding the chemical properties of the copolymer belt which is a unique feature. Furthermore, the functionalization of the copolymers described in this invention with biomolecular tags, fluorophores or in the embodiment with lipids or bioactive molecules enable their use in life sciences or for biomedical applications.The above description is provided for illustrative purposes and is not intended to be exhaustive or to restrict the invention to the specific forms disclosed. Those skilled in the relevant field will recognize that various modifications and adaptations can be made based on the provided disclosure.The terminology used in this specification is primarily chosen for clarity and instructional purposes and is not necessarily intended to define the precise scope of the invention. Therefore, the scope should be determined not by this detailed description but by the claims issued in connection with this application.Accordingly, the embodiments described herein serve as examples rather than limitations of the invention, as defined by the following claims.Furthermore, throughout this specification and the accompanying claims, unless explicitly stated otherwise, the term "comprise" and its variations (such as "comprises" or "comprising") should be understood to indicate the inclusion of the specified element or elements without excluding others.References:1. Rabilloud, T Membrane proteins and proteomics: love is possible, but so difficult.Electrophoresis, 2009. 30 Suppl 1: p. S 174-80.2. Marconnet, A., et al., Solubilization and stabilization of membrane proteins by cycloalkane- modified amphiphilic polymers. Biomacromolecules, 2020: p. 3459-3467.3. Smith, A.A.A., et al., Lipid Nanodiscs via Ordered Copolymers. Chem, 2020. 6(10): p. 2782- 2795.

[0099] 4. Darr, J.M., et al., The styrene-maleic acid copolymer: a versatile tool in membrane research. Eur Biophys J, 2016. 45(1): p. 3-21.5. Marconnet, A., et al., influence of Hydrophobic Groups Attached to Amphipathic Polymers on the Solubilization of Membrane Proteins along with Their Lipids SI. 2022.6. Zoonens, M. and J.L. Popot, Amphipols for each season. J Membr Biol, 2014, 247(9-10): p. 759- 96.7. Le Bon, C., et al., Folding and stabilizing membrane proteins in amphipol AB-35. Methods, 2018. 147: p. 95-105.8. Smith, A. A. A., et al., Controlling Styrene Maleic Acid Lipid Particles through RAFT. Biomacromolecules, 2017. 18(11): p. 3706-3713.9. Autzen, H.E., D. Julius, and Y. Cheng, Membrane mimetic systems in CryoEM: keeping membrane proteins in their native environment. Curr Opin Struct Biol, 2019. 58: p. 259268.10. 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. 1071-1083.11. Glueck, D., et al., Electroneutral Polymer Nanodiscs Enable Interference-Free Probing of Membrane Proteins in a Lipid-Bilayer Environment. Small, 2022: p. e2202492.12. Lin, C.A., et al., Design of an amphiphilic polymer for nanoparticle coating and functionalization. Small, 2008. 4(3): p. 334-41.13. Mueller, S., et al., The bigger picture: global analysis of solubilization performance of classical detergents versus new synthetic polymers utilizing shotgun proteomics. 2023.14. Yuan, Y., et al., Cryo-EM structure of human glucose transporter GLUT4. Nat Commun, 2022. 13(1): p. 2671.15. Cookis, T., et al., Streptavidin-Affinity Grid Fabrication for Cryo-Electron Microscopy Sample Preparation. J Vis Exp, 2023(202).

Claims

Claims1. A copolymer comprising repeating units of the following formula (1) and / or the following formula (2)and / or ofRi and R3 are independently selected from the group consisting of(C3-C10) cycloalkyls or (C3-C10) (hetero)cycloalkyls, unsubstituted or substituted by one or more radicals selected from linear (C1-C8), cyclic or branched (C3-C8) alkyls, linear (C1-C8) or branched (C3-C8) alkenyls, and linear (C 1-C8) or branched (C3-C8) alkynyls,(C3-C10) cycloalkenyls or (C3-C10) (hetero)cycloalkenyls, poly or monounsaturated, unsubstituted or substituted by one or more radicals selected from linear (C1-C8), cyclic or branched (C3-C8) alkyls, linear (C1-C8) or branched (C3-C8) alkenyls, linear (C1-C8) or branched (C3-C8) alkenyls, and linear (C1-C8) or branched (C3-C8) alkynyls, poly ormonounsaturated polycycles, unsubstituted or substituted by one or more radicals selected from linear (C1-C8), cyclic or branched (C3-C8) alkyls, linear (C1-C8) or branched (C3-C8) alkenyls, and linear (C1-C8) or branched (C3-C8) alkynyls, phenyl, unsubstituted or substituted by one or more radicals selected from linear (C1-C8), cyclic or branched (C3-C8) alkyls, linear (C 1-C8) or branched (C3-C8) alkenyls, and linear (C1-C8) or branched (C3-C8) alkynyls, 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 case of a zwitterionic hydrophilic side chain, the linear alkyl amine can be further sulfonated, residues of polyols liketris(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, residues 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, residues of amine functionalized linear or branched polyethylene oxides like 2-(2-(2-(2- aminoethoxy)ethoxy)ethoxy)ethanol, residues of cholines like (2-aminoethyl)trimethylammoniumchlorid, JV-(3-chlor-2-hydroxypropyl)- 2V,JV,JV-trimethylammoniumchlorid,R.3 additionally to the above is H or an alkali metal ion, and X is NH, 0 or S, and y and x are greater than 02. Copolymer according to claim 1 having the following formula (3) or the following formula (4)Formula (4) and / or of formulas (3’) and (4’)Formula (4') wherein Ri, R3 and X are defined as in claim 1R2 is derived from biological tags, fluorophores, peptides, biotin and functional groups for dickchemistry, andR.4 is independently selected from the residues defined for R3.

3. Copolymer according to claim 1 or 2, wherein Ri and R3 are independently selected from cycloalkyl residues, in particular a cyclooctyl residue.

4. The copolymer according to any of the preceding claims wherein the copolymer is a zwitterionic compound as exemplified in formulas (5) or (5’) or the maleimide compound obtained by a ring closure of the compound of formulas (5) or (5’)Formula (5)Formula (5') whereinRs is a branched C3-C7 or linear C1-C7 alkyl;Re is a branched C3-C6 or linear C1-C6 alkyl with a terminal functional group containing a negative charge as carboxylic, phosphate or sulfonates moieties.R7 and Rs are each independently a branched C3-C4 or linear C1-C4 alkyl.

5. The copolymer according to any of the preceding claims wherein the copolymer is a compound grafted with aminated linear or cyclic and mono-, di- or polysaccharides like 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 as exemplified in formulas (6) or (6’) or the maleimide compound obtained by a ring closure of the compound of formulas (6) or (6’)Formula (6')6. The copolymer according to any of the preceding claims, wherein the copolymer is a compound grafted with an aminated polyol like tris(hydroxymethyl)aminomethane, 1-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- ethanediol or 2-amino-2-deoxy-D-glucitol and exemplified in formulas (7) (7’) or the maleimide compound obtained by a ring closure of the compound of formulas (7) or (7’)Formula (7')7. Method for preparing the copolymer according to any of claims 1 to 6, wherein theanhydride of a maleic acid-isobutylene copolymer is reacted with the amines of Ri and R3 and the groups R2 and R4 are introduced to provide the copolymer of formulas (1), (1 ’), (3), and (3 ’), which subsequently can be reacted to the copolymers of formulas (2), (2’), (4), and (4’).

8. Complex comprising the copolymer as defined in any of claims 1 to 6 and a hydrophobic protein, a membrane protein and / or a G protein coupled receptor (GPCR).

9. Complex according to claim 8 further comprising a lipid.

10. Complex according to claim 8 or 9, 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.

11. Method for obtaining a complex according to any of claims 8 to 10 comprising contacting the copolymer as defined in any of claims 1 to 6 with the hydrophobic protein, the membrane protein, the G protein coupled receptor and optionally the lipid.

12. Use of the copolymer according to any of claims 1 to 6 for solubilization, stabilization and / or purification of membrane proteins.

13. Use according to claim 12, wherein the solubilization, stabilization and / or purification is provided out of a native membrane surrounding.

14. Use according to claim 12, wherein the solubilization, stabilization and / or purification is provided using a cell-free expression system.

15. Use according to claim 12 or 13 or 14, 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.

16. Use of the copolymers according to any of claims 1 to 6 for cryo-electron microscopy.

17. Complex comprising the copolymer according to any of claims 1 to 6, a lipid and optionally a biomolecule.

18. Kit, comprising the polymer according to any of claims 1 to 6 and optional instructions for use.

19. Use ofthe copolymers of any claims 1 to 6 or a complex of the copolymers and lipids, drugs or biomolecules for diagnostic or therapeutic applications.

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