Functionalized and non-functionalized amphiphilic polymers based on polyacrylic acid for solubilization, isolation, and molecular labeling of membrane proteins in aqueous media

Abstract

The present application relates to water-soluble membrane protein-solubilizing polymers having the general formula (1), where X is either an initiator fragment derived from a radical starter molecule, or a functional group or a hydrogen atom; Y is a functional group, hydrogen atom, terminal RAFT agent, or conjugated molecule available for further functionalization; a and b represent the relative number of repeat units of the polymer, resulting in the molecular weight of the polymer; R1 is the hydrophilic portion of the polymer, based on grafted proteinogenic and non-proteinogenic amino acids and amino acid derivatives; and R2 is the hydrophobic portion of the polymer. Additionally, methods for preparing the polymers and their uses are described. [Formula 1] JPEG2026503553000012.jpg44159

Description

[Technical Field]

[0001] The present invention relates to specifically defined polymers, methods for their preparation, their use for solubilizing and stabilizing membrane proteins, and kits for this use containing the polymers according to the invention. [Background technology]

[0002] Membrane proteins are highly relevant in biomedical research, as approximately one-third of all proteins are membrane proteins, which are strongly involved in cellular communication, transmembrane transport of molecules, and enzymatic reactions, making them potential drug targets. Their natural interactions with the alkyl chains of cell membrane lipids and their structure, with hydrophobic membrane-inserted helices and sheets, make membrane protein isolation and stabilization challenging, and amphiphilic detergents are required for membrane disruption. Generally, membrane protein surfaces are characterized by the localization of hydrophobic amino acids within the membrane-inserted region and hydrophilic amino acids within the peripheral portion of the membrane protein. This Janus-like structure is responsible for the reduced solubility and, therefore, stability of membrane proteins in aqueous media.

[0003] However, the functional activity of cell membrane proteins often depends on their native lipid environment, which is disrupted by solubilization using detergents. Furthermore, membrane proteins tend to aggregate, especially during large-scale expression and purification, and the structural stability of membrane proteins is disrupted after solubilization using common detergents, such as sodium dodecyl sulfate (SDS) or n-dodecyl-beta-maltoside (DDM).

[0004] Commercially available detergents (e.g., SDS) enable membrane dissolution and solubilization of target membrane proteins with high yields. [1] However, membrane protein solubilization is often accompanied by membrane protein denaturation and inactivation and is limited by detergent concentrations above the critical micelle concentration (CMC). Detergent concentrations at the CMC can adversely affect membrane protein function because they poorly mimic the lipids of cell membranes. In addition, the presence of detergents in membrane protein solutions can adversely affect further analysis or applications of membrane proteins (e.g., cryo-transmission electron spectroscopy or crystallization). Below the CMC, membrane protein solubility cannot be ensured due to hydrophobic patches in the protein's transmembrane region that are not completely covered by the detergent.

[0005] Traditional amphiphilic polymers, called amphiphiles, have been used to overcome these challenges by stabilizing solubilized membrane proteins. These amphiphilic polymers directly bind to similar hydrophobic portions of protein transmembrane domains. This allows for membrane protein solubilization and stabilization in a unique one-step process, eliminating the need for detergents. The amphiphile prototype A8-35, described in International Publication No. 1998027434, consists of a polyacrylic acid backbone grafted with octylamine (approximately 25%), isopropylamine (approximately 40%), and approximately 35% remaining carboxylic acid groups. However, the use of A8-35 generally requires detergent-based membrane protein solubilization, which can result in loss of protein function, because the amphiphilic polymer surrounding the membrane protein does not mimic the native environment of the cellular membrane surrounding the membrane protein.

[0006] New classes of amphiphilic polymers that have been published and patented include SMA (styrene maleic acid, WO 2006129127, WO 2011004158), CyclApol (US Patent No. 20220119558), and AASTY (poly(acrylic acid-co-styrene)).[2,3] The stabilization of these new polymers is based on their ability to mimic the natural environment of membrane proteins through the formation of small complexes called nanodiscs.[3]

[0007] Ring-shaped nanometer-sized nanodisc complexes consist of one or more amphiphilic polymer molecules that surround patches of cell membrane containing membrane proteins. [4] Furthermore, SMA, CyclApol, and AASTY allow for the solubilization and stabilization of membrane proteins without the use of detergents. However, the described polymers are limited in terms of control over polymer length, polydispersity, monomer sequence, flexibility to vary the hydrophobic or hydrophilic side chains of the polymer, and the ability to end-functionalize the polymer chains without affecting the polymerization efficiency itself.

[0008] The solubilization efficiency and stability of the membrane protein-containing Nanodiscs formed depend strongly on the characteristics of the polymer used. Polymers such as SMA, CyclApol, and AASTY are stabilized by electrostatic repulsion of the negatively charged polymer chains. In the case of SMA and AASTY, the charge is generated by copolymerization of styrene with carboxyl groups, which are realized in the polymer by using monomers such as acrylic acid or maleic acid. These Nanodisc-forming polymers are sensitive to the presence of divalent ions and are at risk of aggregation at low pH values ​​(<6.5) and high ionic strength due to the negative charge of the stabilizing carboxylic acid groups. [5] In contrast, polymers with highly charged polar groups such as phosphate or sulfate [6] and flexible nonpolar side chains, as described in this invention, enhance both solubilization efficiency and Nanodisc stability.

[0009] The length of the polymer, as well as the polydispersity and uniform sequence of comonomers such as acrylic acid and styrene, are important factors for solubilization itself or for applications such as cryo-transmission electron microscopy. [7] Therefore, further control over the polymerization is crucial. Previously used techniques, such as radical batch polymerization, result in polymers with high polydispersity (PDI > 2) and a non-uniform distribution of the monomers within the polymer depending on their chemical properties.

[0010] In recent years, the use of reversible addition-fragmentation chain transfer polymerization (RAFT) has enabled the synthesis of monodisperse polymers (polydispersities <1.5), as demonstrated by AASTY.[8] RAFT polymerization was first described by Rizzardo et al. in 1998 and is defined as "a degenerative transfer radical polymerization involving a degenerative chain transfer process in which chain activation and chain deactivation occur via a two-step addition-fragmentation mechanism" (IUPAC definition).[9] A typical RAFT polymerization consists of one or more monomers (e.g., vinyl derivatives), a radical source (e.g., a thermochemical initiator), and a RAFT agent (thiocarbonylthio compound). Furthermore, RAFT-synthesized polymers offer the opportunity for terminal functionalization due to terminal thiocarbonylthio moieties that can be chemically substituted using various techniques, such as maleimide conjugation chemistry.

[10] Terminal modifications of polymers can include, among other things, fluorophores or biomolecular tags, such as biotin, for further analysis and purification of synthetic nanodiscs containing membrane proteins. [Prior art documents] [Patent documents]

[0011] [Patent Document 1] International Publication No. 1998027434 [Patent Document 2] International Publication No. 2006129127 [Patent Document 3] International Publication No. 2011004158 [Patent Document 4] U.S. Patent No. 20220119558 Summary of the Invention [Problem to be solved by the invention]

[0012] To overcome the shortcomings of current polymers for membrane solubilization, the underlying objective of the present invention is to develop polymers that are adaptive in terms of the hydrophilic and hydrophobic side chains used, stable at high ionic strength, and resistant to divalent ions (e.g., Ca). 2+ The objective of this study is to provide a polymer that is tolerant to high concentrations (>5 mM) and efficiently solubilizes and stabilizes membrane proteins. Furthermore, the newly invented polymer possesses the important feature of selective solubility. Polymer not incorporated into Nanodiscs can be removed by high-speed centrifugation, which reduces the excess amount of polymer in the lysate, enabling highly efficient purification using affinity resins and advanced downstream applications (e.g., cryo-electron microscopy) that would be adversely affected by excess copolymer not incorporated into Nanodiscs.

[0013] In another embodiment, the polymer is monodisperse (PDI<1.25), more preferably has a PDI<1.2, and even more preferably a PDI<1.1, and is preferably end-functionalizable.

[0014] This is achieved by the subject matter of the independent claims. Preferred embodiments are defined in the dependent claims. [Means for solving the problem]

[0015] According to the present invention, there is provided an amphiphilic vinyl-derived polymer having the general formula (1): [ka]

[0016] The two segments, each carrying the groups R1 and R2, are randomly distributed along the length of the polymer chain.

[0017] In formula (1) above, X can be an initiator fragment derived from radical starter molecules including, but not exclusively, 4'-azobis(4-cyanopentanoic acid) (ACPA), 2,2'-azobis(2-methylpropionitrile), 2-(azo(1-cyano-1-methylethyl))-2-methylpropanenitrile (AIBN), or can be a functional group (e.g., hydroxy, carboxylic acid, etc.) or a hydrogen atom.

[0018] Y can be a terminal RAFT agent used for further functionalization, including, but not exclusively, a functional group (e.g., hydroxy, carboxylic acid, thiol, etc.), a hydrogen atom, or 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CTCTA), which can be chemically substituted or modified with, for example, but not exclusively, a fluorophore bearing either a primary amine (e.g., fluoresceinamine) or a maleimide (e.g., 5-maleimido-fluorescein), or can be substituted with a biomolecular tag such as biotin or a Rho1D4 Tag (e.g., biotin-maleimide, biotin-polyoxyethylene-maleimide, biotin-propargylamide).

[0019] The term functional group refers to any functional group known in the art of organic chemistry.

[0020] The letters a and b indicate the relative number of statistically distributed units in the polymer, resulting in the molecular weight of the polymer, which can range from 2,000 to 24,000 daltons. a and b can range from 0% to 90% for each unit, and generally a + b = 100% of the total functionalized and non-functionalized carboxylic acid groups in the polymer.

[0021] R1 is the hydrophilic part of the polymer based on grafted proteinogenic and non-proteinogenic amino acids and amino acid derivatives, and is ionically bonded to the hydroxyl groups or oxygens (e.g., Li + , Na+ , K. + ), but can also be chemically modified and exchanged via amide formation with phosphate- or sulfate-containing molecules, including, but not exclusively, phosphorylethanolamine (2-aminoethyl dihydrogen phosphate) or homotaurine (3-aminopropane-1-sulfonic acid). Additionally, R1 can be polar amino acids with charged side chains, including, but not exclusively, aspartic acid, glutamic acid, and amino acids with uncharged side chains, including, but not exclusively, serine or threonine linked by amide formation. Grafting of acrylic acid can be achieved using organic or aqueous conjugation reactions including, but not exclusively, N-hydroxysuccinimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, (benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate), (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate), and similar compounds and derivatives of such reagents.

[0022] R2 is the hydrophobic part of the polymer and is derived from amino acid.The example of the non-polar molecule described can be, for example, but not exclusively, phenylalanine benzyl ester (for example, Sigma Aldrich, CAS 2462-32-0) or tryptophan benzyl ester (for example, Sigma Aldrich, CAS 141595-98-4), whose carboxylic acid group can be chemically protected by methyl-, ethyl-, tert-butyl-, benzyl- or benzhydryl-ester.In addition, hydrophobic amino acids such as phenylalanine, tryptophan and tyrosine can be used without protective groups.

[0023] definition 1. The term "amphipol" refers to a vinyl polymer that is able to solubilize membrane proteins and keeps them stable in solution in their native form. 2. The term "nanodisc" refers to small nanometer-sized complexes formed by amphiphilic polymers and lipids, which are capable of stabilizing membrane proteins. 3. The term "vinyl" in the context of the present invention includes polymer backbones based on the polymerization of ethenyl groups. 4. The term "membrane protein" includes proteins that contain a hydrophobic transmembrane domain or are associated with a membrane by at least one hydrophobic domain. Membrane proteins can be monomeric or oligomeric and can be associated with cofactors. 5. The term "grafting" in the context of the present invention denotes the functionalization of functional groups within a polymer chain by other molecules. 6. The term "dispersity" is expressed as the polydispersity index (PDI), which can be calculated as the ratio of the weight average by number to the average molecular weight, as determined by aqueous size exclusion chromatography (SEC). 7. The term "Aminopol" refers to a polyacrylic acid backbone grafted with proteinogenic and non-proteinogenic amino acids in varying proportions. 8. The term "Phenylopol" refers to a polyacrylic acid backbone onto which various proportions of L-phenylalanine benzyl ester molecules have been grafted. 9. The prefixes "phospho-" and "sulfo-" refer to polymers grafted with varying proportions of phosphate- or sulfate-containing molecules to increase the solubility of the polymer and the efficiency of solubilization and stabilization obtained during the solubilization and handling of membrane proteins. 10. The term "affinity chromatography" refers to a method for separating biomolecules from a mixture based on highly specific macromolecular binding interactions between the biomolecule and another substance. Affinity chromatography is useful due to its high selectivity and resolution of separation compared to other chromatographic methods. An example of affinity chromatography is the purification of his-tagged or rho-tagged proteins. These proteins contain poly-his (e.g., his6 or his7) at the C- or N-terminus. 10) or TETSQVAPA amino acid sequence, allowing purification from mixtures via selective binding to Ni-NTA agarose (for his-tagged proteins) or anti-Rho-1D4 antibody agarose (for rho-tagged proteins). Protocols for these purification procedures can be found on the Cube Biotech website and in Hochuli et al. and Corin et al.

[0024] The present invention employs the use of polyacrylic acid (PAA) for the synthesis of amphiphilic polymers. PAA contains an alkyl backbone and carboxylic acid side chains.

[0025] The PAAs used in the present invention can be purchased commercially (e.g., Thermo Scientific, Sigma Aldrich, CAS 79-10-7) or synthesized using radical polymerization, such as ATRP polymerization

[11] or RAFT polymerization

[12] , resulting in polymers with low polydispersity (PDI<2), more preferably pdi<1.2, and even more preferably pdi<1.1, and the ability to be end-functionalized. The PAAs described are not limited to the described sources, synthetic procedures, or PDIs.

[0026] In the case of ATRP polymerization of polyacrylic acid, the synthesis is carried out using methyl acrylate monomer, initiator, copper bromide, and a catalytic agent. After polymerization, the resulting polymethyl acrylate polymer is hydrolyzed to yield the desired PAA. ATRP-PAA is not limited to the synthesis procedure described.

[0027] In the case of RAFT polymerized polyacrylic acid, the synthesis is carried out using a thiocarbonylthio compound, an initiator (e.g., a thermochemical or photochemical initiator), and the acrylic acid monomer to be polymerized.

[0028] The molecular weight of the polymerized PAA can range from 2 to 20 kDa, but is not limited to the ranges described.

[0029] To generate the amphiphilic properties of the polymers according to the present invention, the polymers can be grafted using a variety of techniques including, but not exclusively, amide formation of the carboxyl groups of PAA with primary amine-containing target molecules via carbodiimide-, isothiocyanate-, isocyanate-, acyl azide-, NHS ester-, anhydride, imidoester-, aldehyde-, or epoxide-crosslinker chemistries.

[0030] PAA grafting involves activation of carboxyl groups and chemical modification using either a crosslinking molecule or a zero-length crosslinker, as well as chemically active target molecules (e.g., ester formation, acid halide formation).

[0031] The polymer class of PAAs grafted with polar or nonpolar amino acids and proteinogenic and nonproteinogenic amino acids and their derivatives is called "Aminopol" or named using amino acid-related prefixes, as in the case of L-phenylalanine-grafted PAA (Phenylopol).

[0032] Grafting of PAA with polar molecules is in the range of >10%, preferably 20-75%, most preferably 25-50%, but may be in the range of 60-90%.

[0033] The polar molecules used for grafting can be phosphate- or sulfate-containing molecules, including, but not limited to, phosphorylethanolamine (2-aminoethyl dihydrogen phosphate, e.g., Sigma Aldrich, CAS 1071-23-4) or homotaurine (3-aminopropane-1-sulfonic acid, e.g., Sigma Aldrich, CAS 3687-18-1). In addition, polar proteinogenic and non-proteinogenic amino acids with charged side chains, including, but not limited to, aspartic acid (e.g., Sigma Aldrich, CAS 56-84-8) and glutamic acid (e.g., Sigma Aldrich, CAS 56-86-0), and amino acids with uncharged side chains, including, but not limited to, serine (Sigma Aldrich, CAS 56-45-1) or threonine (e.g., Sigma Aldrich, CAS 80-68-2), as well as derivatives of these molecules, can be used.

[0034] Grafting of PAA with non-polar molecules is in the range of >10%, preferably 20-75%, most preferably 25-50%, but may be in the range of 60-90%.

[0035] The non-polar molecules used for grafting can be proteinogenic and non-proteinogenic amino acids, and derivatives thereof, whose carboxylic acid groups are chemically protected, for example, but not exclusively, by using methyl-, ethyl-, tert-butyl-, benzyl-, or benzhydryl-esters. Examples of non-polar molecules described can be phenylalanine benzyl ester (e.g., Sigma Aldrich, CAS 2462-32-0) or tryptophan benzyl ester (e.g., Sigma Aldrich, CAS 141595-98-4). In addition, hydrophobic amino acids without protecting groups, such as phenylalanine, tryptophan, and tyrosine, can be used. The grafting process can be achieved here by sequential removal of the protected amino acid and protecting group, or by direct coupling with activated acrylic acid.

[0036] In the case of RAFT-polymerized PAAs, the terminal thiocarbonylthio moiety allows for functionalization of the polymer with target molecules, including, but not limited to, chromophores (e.g., fluorophores such as fluorescein or Alexa, or Raman-active dyes such as CY3 or CY5) or biomolecular tags (e.g., biotin, Rho1D4, peptides). Substitution of the thiocarbonylthio moiety with target molecules can be achieved using, for example, RAFT or ATRP polymerization, thermolysis, radical-induced reduction, addition-fragmentation coupling, radical-induced oxidation, or hetero-Diels-Alder reactions, to name a few, but not exclusively. A preferred modification method is the reaction of the polymer's thiol functional groups with maleimide-functionalized molecules, functionalized alkenes, or functionalized alkynes.

[0037] In one embodiment, functional polymers can also be prepared by grafting functionalized amines, such as 5-aminofluorescein or aminobiotin, onto carboxyl functional groups. PAA grafting involves activation of the carboxyl groups and chemical modification using either a crosslinking molecule or a zero-length crosslinker, as well as chemically active target molecules (e.g., ester formation, acid halide formation).

[0038] There are at least two methods of preparation: Reaction of polyacrylic acid with one or more mixtures of amino acids, such as L-phenylalanine, with, for example, aminofluorescein, for example, by EDC and HNS. EDC / NHS activation, e.g., reaction of aminofluorescein with phenylopol polymers, phospho-phenylopol polymers or taurine phenylopol polymers.

[0039] The novel end-functionalized and end-non-functionalized amphiphilic polymers can be used to solubilize and stabilize integral membrane proteins and membrane-associated proteins.

[0040] Amphipols according to the present invention can be used to label Nanodisc complexes for use in biomolecular research, diagnostic applications and medical product development.

[0041] To conclude the described invention, a new class of amphipols is based on: 1. Polyacrylic acid (PAA) synthesized via radical polymerization (e.g., commercially available atom transfer radical polymerization (ATRP) with variable molecular weight, e.g., Acros, Sigma-Aldrich) or via reversible addition-fragmentation chain transfer (RAFT) polymerization. Notably, the PAA synthesized using RAFT and used for functionalization exhibits a polydispersity index of <1.1. 2. Grafting of the PAA with polar and non-polar proteinogenic and non-proteinogenic amino acids and their derivatives, and their combinations with or without derivatives bearing chemically protected functional groups. 3. Optionally, grafting the new amphipols with or without molecules containing strongly charged or polar functional groups such as sulfate and phosphate.

[0042] Compared to the polymers of the prior art, the polymers according to the invention have at least one of the following advantages: 1. Improved solubilization efficiency of membrane proteins expressed in prokaryotic and eukaryotic cells and / or native proteins expressed within the cell membrane. 2. Improved stability of the formed nanodisc assemblies of membrane proteins during solubilization, purification and application of the new amphipol and cell membrane patch with respect to high ionic strength and high concentration of cations compared to commercially available products. 3. Due to the increased solubilization efficiency, the polymers according to the invention can be used at lower concentrations, increasing the purity and functionality of the stabilized membrane proteins. 4. Removal of excess polymer after solubilization by simple high-speed centrifugation to enable efficient binding of tagged membrane proteins or tagged copolymers to affinity resins and advanced downstream applications. 5. Possibility of terminal functionalization of new amphipols synthesized using RAFT with molecules (e.g., fluorophores, biotin derivatives, etc.) using conjugation chemistries (e.g., maleimide, thiol-ene, thiol-yne click chemistry) for protein purification and protein labeling. 6. High polymer monodispersity, achieved through RAFT or ATRP polymerization, results in extremely narrow particle distributions with pD values ​​of less than 1.25, 1.2, or even 1.1, leading to more uniform complexes with membrane proteins and superior results in analyses such as X-ray structural analysis or cryo-electron microscopy. These superior results can be attributed to higher resolution or faster computational times.

[0043] The solubilization, stabilization, and purification of membrane proteins from their native membrane surroundings depend on several parameters. Most parameters can be optimized for greater efficiency during the purification process. These parameters include buffer conditions (e.g., salt, pH), polymer selection, protein-to-solubilizer ratio, temperature, and time. First, cell lysis and centrifugation are performed, for example, using the following parameters: adding a protease inhibitor (PI) to the buffer and readjusting the pH value. Then, cell disruption (e.g., sonication, French press), centrifugation at 9,000 rcf for 30 minutes at 4°C, discarding the pellet (cell debris, excess copolymer), collecting the supernatant, centrifugation of the supernatant at 100,000 rcf for 1 hour at 4°C, discarding the supernatant, and homogenizing the pellet. Next, membrane protein solubilization is performed: the polymer forms synthetic nanodiscs around the protein, thereby maintaining the native phospholipid environment and preserving the protein's native and therefore functional properties in a convenient one-step process (solubilization and stabilization). On the other hand, surfactants form micelles around the hydrophobic belts, thus removing lipids from the surroundings, which is necessary to preserve the native lipid environment for the native state.

[0044] 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, SLC transporters. That is, as starting material for the method according to the invention, a solution of free polymer is used, which solution is derived from the solubilization, stabilization and purification of the above-mentioned membrane proteins from their natural surroundings by employing polymers.

[0045] The amphiphilic polymers of the present invention can be used to solubilize and stabilize membrane proteins for biotechnological and pharmaceutical applications. Therefore, reagent kits and diagnostic kits (e.g., lateral flow assays) incorporating at least some of the described inventions can also use the amphiphilic polymers of the present invention as reagents. One use of the amphipols of the present invention is the solubilization and stabilization of membrane proteins in solution, with or without detergent pretreatment. This means that they can keep fully functional or non-functional but still immunogenic membrane proteins in solution, preventing them from aggregating or precipitating during solubilization and handling. Therefore, the present invention also relates to the formation of water-soluble complexes consisting of one or more amphiphilic polymer molecules, artificial or natural lipids derived from cell membranes, and integral membrane proteins or membrane-associated proteins.

[0046] The use of the polymers according to the invention can be exemplified as follows: 1. One or more recombinant membrane or membrane-associated proteins are expressed at high density in prokaryotic or eukaryotic cells and located either in or on the cell membrane, potentially in inclusion bodies. 2. The solubilization step is performed using either a whole cell suspension, the supernatant of a cell lysate, or the pellet of the centrifuged supernatant derived from the cell lysate. 3. The protein solution or pellet is added directly to the polymer solution with a final polymer concentration of up to 5% by weight and incubated with stirring for up to 24 hours. 4. Solubilization efficiency can be determined using standard biomolecular methods (e.g., SDS-PAGE, Western blot). 5. By centrifugation, insolubilized proteins, debris and excess copolymers can be separated from the solubilized proteins located in the formed Nanodisc complexes.

[0047] Diagnostics involves qualitative and quantitative testing of biological components such as DNA, RNA, proteins, and metabolites, which can provide information about disease, genetic predisposition, or health conditions. Diagnostic tests can be performed by medical professionals or by individuals.

[0048] The polymers according to the present invention can be used to solubilize and stabilize membrane proteins, preferably in their natural lipid environment, to maintain their activity. These stabilized membrane proteins can be used to detect interactions. The interaction between the copolymer-stabilized membrane protein and its interaction partner can be detected, for example, but not exclusively, through various analytical methods.

[0049] Examples of optical detection include 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).

[0050] The interaction of the copolymer-stabilized membrane protein with its interaction partner can be detected, for example, but not exclusively, through a variety of analytical methods.

[0051] Examples of optical detection include 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).

[0052] Additionally, the copolymers can lyse eukaryotic cells and tissues at low concentrations (0.01%-5%) very quickly (within seconds to minutes) and without mechanical assistance. This ability for gentle lysis allows users to obtain nucleic acids with low levels of cleavage, as well as soluble and membrane proteins in their native state, making the polymers of the present invention particularly suitable for diagnostic tests based on DNA, RNA, soluble proteins, and membrane proteins.

[0053] The present invention is further illustrated by the following examples and figures, which should be understood as being merely illustrative of the present invention and not limiting thereof. [Brief explanation of the drawings]

[0054] [Figure 1] Figure 1 shows size exclusion chromatograms of 0.5 wt% polyacrylic acid aqueous solutions with various molecular weights and low PDI (<1.1) synthesized using RAFT polymerization. [Figure 2] UV-Vis absorbance spectrum of a 0.7 mg / ml solution of phenylopol in methanol. [Figure 3] Figure 1 shows a size-exclusion chromatogram of 0.5 wt% phenyllopol fluorescently labeled with end-conjugated fluorescein in DMF. The refractive index signal and the fluorescence intensity signal are overlaid. For better visualization, the fluorescence intensity was multiplied by 50. [Figure 4] 1 shows solubilization blots of model solubilization of a membrane protein (G6PC) using various copolymers. [Figure 5]Photographs of a Phenylopol sample after solubilization of the membrane protein G6PC from cell lysates before 1) and after 2) high-speed centrifugation at 60,000 g for 1 hour. Excess copolymer and cell debris are sedimented. 3) [Figure 6] UV-Vis spectra of cell lysates with and without Phenylopol for membrane protein solubilization, and before and after centrifugation to remove cell debris and excess copolymer are shown. [Figure 7] Shown are isolated blots of a model purification of a membrane protein (G6PC) using various copolymers and Rho affinity resin purification. [Example]

[0055] The present invention will now be illustrated by examples, which it is expressly pointed out should not be construed as limiting the present invention thereto.

[0056] 1. Synthesis of monodisperse polyacrylic acid (PAA) using RAFT polymerization: [ka]

[0057] Polyacrylic acid (PAA) was synthesized by RAFT polymerization according to a modified protocol derived by Chaduc et al. (2013).

[12] The target molecular weight of the PAA polymer was varied between 2 kDa and 15 kDa by varying the amount of acrylic acid used in the synthesis. The synthesis of 5 kDa PAA was achieved by mixing 1.87 g (26 mmol) of acrylic acid with 20 mg of 4,4'-azobis(4-cyanopentanoic acid) (ACPA), 149 mg (0.49 mmol) of CTCTA, and 8 mL of deionized water in a 50 mL round-bottom flask with a ground glass stopper. For the synthesis of 2, 3, 4, 7, 9, 11, and 15 kDa PAA, 0.75, 1.15, 1.5, 1.87, 2.8, 3, 4.3, and 5.9 g of acrylic acid were added, respectively. After dissolution of the solid extract, the resulting yellow solution was deoxygenated by argon bubbling for 30 minutes. The flask was then sealed and incubated at 70 °C for 24 hours. The crude PAA product was purified by precipitation of PAA in 10 mL of diethyl ether prior to functionalization with the ligand. The molecular weight of the polymer was determined using aqueous size-exclusion chromatography (Agilent Infinity 1260, 2x aquagel-OH 20 columns, 200 mM NaNO3, 20 mM Na2HPO4, pH 7.5).

[0058] The goal of synthesizing polyacrylic acid by RAFT polymerization is to obtain a polymer with a low polydispersity index and the ability to precisely functionalize the terminals. The molecular weight of the polyacrylic acid synthesized using RAFT polymerization was determined by size exclusion chromatography. The results show highly monodisperse polymers with PDIs of less than 1.1.

[0059] Furthermore, the size of PAA can be precisely varied across a wide range of molecular weights relevant for solubilizing membrane proteins.

[0060] Figure 1 shows size-exclusion chromatograms of polyacrylic acids with various molecular weights and low PDIs (<1.1) synthesized using RAFT polymerization. Size characterization was performed using an Agilent Infinity II 1260 system, including an isocratic pump, column oven, and refractive index detector. Two Agilent Aquagel-OH 20 columns were used for separation, with a mobile phase containing sodium nitrate (200 mM) and sodium phosphate (20 mM, pH 7.5). A polyacrylic acid calibration kit (Agilent) was used as a molecular weight standard.

[0061] 2. Synthesis of polyacrylic acid using ATRP polymerization

[11] In a 50 ml round-bottom flask with a ground glass stopper, 54.1 mg (0.38 mmol) of CuBr, 2 mL (22.2 mmol) of methyl acrylate, 0.08 mL (0.38 mmol) of N,N,N',N",N"-pentamethyldiethylenetriamine (PMDETA), and 0.0 mL (0.45 mmol) of methyl 2-bromopropionate (2-MBP) were mixed and deoxygenated by argon bubbling for 30 min, followed by incubation at 50 °C for 4.5 h. The crude PAA product was purified by precipitation of the PAA in 10 mL of diethyl ether prior to functionalization with the ligand. Hydrolysis of the PMA backbone was achieved by dissolving 0.2 g of PMA in 20 mL of THF, adding 25 mL of sodium hydroxide solution (1.37 g), and incubating at 60 °C for 10 h. Aqueous size exclusion chromatography (Agilent Infinity 1260, 2x aquagel-OH 20 columns, 200 mM NaNO3, 20 mM Na2HPO4, pH 7.5) was used to determine the molecular weight of the polymer.

[0062] 3. Functionalization of PAA with L-phenylalanine benzyl ester Briefly, 1 g of synthetic PAA (approximately 6 kDa) prepared as described above or purchased commercially was mixed with 2 g (6.6 mmol) of L-phenylalanine benzyl ester hydrochloride and 1.3 g (14.18 mmol) of N-hydroxysuccinimide in 50 mL of phosphate buffer (100 mM, pH 7.2) and 50 mL of methanol. While stirring, 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added, and the resulting mixture was stirred for 30 min. This last step was repeated three times, and the reaction mixture was incubated for 24 h. The crude reaction mixture was then purified by dialysis against distilled water using 1 kDa dialysis tubing until the water conductivity reached <10 μS. The solid polymer, hereafter referred to as "Phenylopol," was isolated by vacuum drying. The grafting rate was determined using titration with hydrochloric acid to obtain the amount of remaining free carboxylic acid groups. Size exclusion chromatography (Agilent Infinity 1260, 1× PlGel mixed column, DMF, 0.05% LiBr) was used to determine the functionalization of the polymer. [ka]

[0063] 4. Deprotection of Phenylopol Using Base-Catalyzed Hydrolysis of Benzyl Ester Deprotection was carried out using 1 g (0.17 mmol) of 50% grafted phenylol and a 5-fold molar excess (1.4 g, 35 mmol) of NaOH relative to the benzyl ester moiety dissolved in 30 mL of deionized water. The resulting solution was heated to 100 °C under reflux for 10 min and purified using dialysis. [ka]

[0064] 5. Synthesis of Phospho-Phenylopol by Functionalization of Phenylopol with Phosphorylethanolamine Using the example of phosphorylethanolamine, functionalization of phenyllopol with strongly charged functional groups, such as phosphate or sulfate, was carried out as described. Briefly, 1 g of phenyllopol was mixed with 3.25 g (20 mmol) of phosphorylethanolamine and 1.1 g (17.7 mmol) of N-hydroxysuccinimide in 50 ml of phosphate buffer (100 mM, pH 7.2). While stirring, 0.5 g of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was added, and the resulting mixture was stirred for 30 minutes. This last step was repeated three times, and the reaction mixture was incubated for 24 hours. The crude reaction mixture was then purified using dialysis against distilled water using 1 kDa dialysis tubing until the water conductivity reached <10 μS. The solid polymer, designated phospho-phenylopol, was isolated by vacuum drying. Titration with hydrochloric acid was used to determine the grafting rate and to obtain the amount of remaining free carboxylic acid groups (<10%). [ka]

[0065] End-functionalization of RAFT polymers was achieved via three different mechanisms: a) Decomposition of the RAFT agent thiocarbonate using butylamine and generation of a reducing end thiol group via covalent attachment of the reduced thiol to a biological tag or a fluorophore containing a maleimide moiety. b) Conjugation of an amine-containing fluorophore or biological tag with a heterobifunctional linker molecule (e.g., 1-[(4-([(2,5-dioxo-1-pyrrolidinyl)oxy]carbonyl)cyclohexyl)methyl]-1H-pyrrole-2,5-dione (SMCC)), followed by conjugation of the degraded thiocarbonate described in 1 to the reducing-end thiol. c) The third mechanism is the application of thiol-yne or thiol-ene click chemistry.

[13]

[0066] 6. Conjugation of Phenylopol Synthesized via RAFT Polymerization to Fluorescein-Maleimide Briefly, 1 g of phospho-phenylopol, 74 mg of tris(2-carboxyethyl)phosphine (TCEP), and 125 mg of fluorescein-maleimide were dissolved in 20 mL of phosphate buffer (100 mM, pH 7.0) with stirring, followed by the addition of 210 mg of butylamine. The resulting reaction mixture was incubated for 48 h. The dissolved polymer was then purified and isolated using dialysis and vacuum drying as previously described. Size exclusion chromatography (Agilent Infinity 1260, 1x PlGel mixed column, DMF, 0.05% LiBr) was used to determine the functionalization of the polymer. [ka]

[0067] 7. Conjugation of Phenylopol Synthesized via RAFT Polymerization with Amino-Fluorescein Using Heterobifunctional Crosslinkers Briefly, 396 mg (1.14 mmol) of aminofluorescein and 191 mg (0.57 mmol) of SMCC were dissolved in 40 mL of dimethylformamide and 10 mL of methanol and incubated at room temperature for 3 h. Then, 1 g of phospho-phenylolol was added and dissolved with stirring. Subsequently, 166 mg (2.29 mmol) of butylamine was added and incubated on an end-over-end shaker for 24 h. The dissolved polymer was purified and isolated using dialysis and vacuum drying as previously described. [ka]

[0068] 8. Conjugation of phospho-phenylopol, synthesized via RAFT polymerization, to biotin-propargylamide via thiol-ene click chemistry [10, 14] Briefly, in a 50 ml round-bottom flask with a ground glass stopper, 1 g (77 μmol) of phospho-phenylol, 2.2 mg of 4,4'-azobis(4-cyanopentanoic acid) (ACPA) (7.7 μmol), and 109 mg (388 μmol) of biotin-propargylamide were dissolved in 10 ml of deionized water. The reaction mixture was deoxygenated by bubbling argon through it for 30 min, heated to 70 °C, and incubated for 24 h. The crude product was then purified using dialysis as previously described and dried under vacuum. [ka]

[0069] 9. Solubilization and purification of his-tagged or rho-tagged membrane protein G6PC The solubilization, stabilization, and purification of membrane proteins from their native membrane periphery depend on several parameters, most of which can be optimized for greater efficiency during the purification process. These parameters include buffer conditions (salt, pH, etc.), the selected polymer, the protein-to-solubilizing agent ratio, temperature, and time.

[0070] Cell lysis and centrifugation: Add protease inhibitors (PI) to buffer and readjust the pH value, then disrupt the cells (e.g., sonicate, French press). Centrifuge at 9,000 rcf at 4°C for 30 minutes, discard the pellet (cell debris), and collect the supernatant. Centrifuge the supernatant at 100,000 rcf at 4°C for 1 hour, discard the supernatant, and homogenize the pellet.

[0071] Membrane protein solubilization: The polymer forms synthetic nanodiscs around the protein, thereby maintaining the natural phospholipid environment and preserving the natural and therefore functional properties of the protein in a convenient one-step manner (solubilization and stabilization). In contrast, surfactants form micelles around the hydrophobic belt, thus removing lipids from the surroundings. For the natural state, it is necessary to preserve the unique lipid environment.

[0072] If the solubilization efficiency is low, it is recommended to screen parameter variations to improve the yield of total solubilized protein. A standard protocol is described as follows: Adding a solubilizer (copolymer) to the protein solution The ideal concentration can vary, but a good starting point is: 0.1-2.5% copolymers (e.g., Aminopol, Phenylopol-Fluorescein) Solubilize for 3 to 24 hours at 4°C with stirring. Higher temperatures can be screened for optimization. Centrifuge at 100,000 rcf for 1 hour at 4°C. Discard the pellet containing cell fragments and excess polymer and collect the supernatant. For affinity chromatography, solubilized membrane proteins are used in polymer nanodiscs (supernatant) to separate his-tagged or rho-tagged membrane protein copolymer complexes from the mixture by using commercially available agarose products. Visualize solubilized membrane proteins by SDS-PAGE, Western blot, and Rho antibody / HRP staining for chemiluminescence detection

[0073] Further standard protocols are described as follows: Adding a solubilizer (copolymer) to the protein solution The ideal concentration can vary, but a good starting point is: 0.1~2.5% Phenylopol Solubilize for 3 to 24 hours at 4°C with stirring. Higher temperatures can be screened for optimization. Centrifuge at 100,000 rcf for 1 hour at 4°C. Discard the pellet and collect the supernatant · For affinity chromatography, solubilized membrane proteins are used in polymer nanodiscs (supernatant) to separate his-tagged or rho-tagged membrane protein copolymer complexes from the mixture.

[0074] result: The first step in generating well-defined copolymers grafted with amino acids is the synthesis of well-defined acrylic acid polymers with varying molecular weights, as previously described. Figure 1 shows size-exclusion chromatograms of polyacrylic acid polymers with varying molecular lengths. To ensure low polydispersity, RAFT polymerization was used in this example. The molecular weight of the resulting polyacrylic acid can be varied, illustratively between 2 kDa and 15 kDa with a polydispersity of less than 1.2, by varying the amount of acrylic acid added to the polymerization solution. As evident from Figure 1, as the amount of monomer present in the polymerization solution increases, the molecular weight of the final polymer increases. As the molecular weight decreases, the retention time of the polymer during size-exclusion chromatography is extended as the polymer further interacts with the size-exclusion column. A polyacrylic acid calibration kit (Agilent) was used to determine molecular weights.

[0075] The synthesis of Phenylopol was achieved by grafting 40 mol% of polyacrylic acid with L-phenylalanine benzyl ester. As can be seen in Figure 2 (solid line), after purification and drying, the resulting copolymer exhibits a strong absorption mode with a peak absorption at 258 nm. As a control, polyacrylic acid, which does not exhibit a clear absorption mode at 260 nm, was measured (dashed line). The increased absorption of Phenylopol at 220 nm is attributed to the polyacrylic acid-amide backbone of the polymer.

[0076] Further functionalization of the previously synthesized Phenylopol is demonstrated by end-modification with aminofluorescein and SMCC. The resulting fluorescently labeled polymer was analyzed using organic phase size-exclusion chromatography. Figure 3 shows the chromatograms of refractive index detection and correlated fluorescence detection (excitation 489 nm, emission 521 nm). Due to the highly hydrophobic nature of the polymer after functionalization with fluorescein and L-phenylalanine benzyl ester, the polymer was analyzed using DMF as the mobile phase containing 0.05 wt% LiBr. For better visualization, the fluorescence signal was multiplied by 50. The Phenylopol polymer peak located at approximately 7 min showed a fluorescent signal, confirming successful functionalization. The second peak located at approximately 9 min corresponds to trace amounts of unconjugated SMCC-fluorescein.

[0077] As an example, 40% grafted phenyllopol was used to solubilize the model membrane protein G6PC induced by HEK cells. G6PC was overexpressed in a cell line by transformation. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze and size-separate the cell-expressed proteins for molecular weight. Figure 4 shows the results of membrane solubilization on SDS-PAGE using Western blot, primary labeling with a primary anti-Rho tag antibody for chemiluminescence detection, and secondary HRP-conjugated antibody. Successful solubilization of G6PC results in a band with a molecular weight of approximately 80 kDa, as evidenced by a broad range of markers. To compare solubilization efficiency, crude cell lysates and various commercially available prior art copolymers were used as controls. For each polymer, the crude solubilized lysate (T) and the supernatant after centrifugation (100,000 rcf, 1 hour) were analyzed in the gel. The performance of Phenylopol can be compared to the most efficient commercially available copolymers (e.g., Ultrasolute 17 and 18), with a dark, distinct band at approximately 80 kDa indicating high purity and high yield of solubilization.

[0078] Stable and efficient isolation of solubilized target proteins using affinity resin purification may be the most critical step for membrane proteins. Because binding of affinity tags to affinity resins (e.g., His-tags to NTA resins) can be strongly affected and inhibited by excess copolymers, removing excess copolymers present in the lysate prior to affinity purification can dramatically improve the quality of the isolated protein. Therefore, removal of excess polymer is crucial. As shown in Figure 5, phenylopol has unique properties, allowing the excess polymer to be removed by simple centrifugation before binding of tagged proteins to affinity resins. Figure 5 shows cell lysates solubilized with 0.1, 1.0, and 2.5 wt% phenylopol and centrifuge tubes containing cell lysates (L). The turbidity of the cell lysate decreased with the addition of 0.1 wt% phenylopol, indicating membrane solubilization. As the weight percent of polymer increases, turbidity increases as excess Phenylopol, which does not form nanodiscs, is suspended in the cell lysate (A). Centrifugation can be used to sediment the excess polymer and unsolubilized cell membranes, which can then be removed from the supernatant prior to affinity resin binding (B, C, D). Figure 5B shows the sedimented unsolubilized cell membranes (yellowish) and excess polymer (white) after centrifugation. The importance of this feature is exemplified by comparing the blot intensities of the S Phenylopol 0.1 wt% (Figure 4) and S Ultrasolute 18 samples. G6PC blot intensity is stronger in the S Ultrasolute 18 sample. However, isolation of G6PC using Rho affinity purification shows comparable blot intensities with less carryover of off-target proteins, despite initially using 25-fold less copolymer (Figure 7).

[0079] Analysis of cell lysate and cell lysate / Phenylopol samples using UV-Vis absorbance spectroscopy demonstrates efficient removal of unsolubilized cell membranes, proteins, and excess copolymers (Figure 6). To compensate for the contribution of cell membranes and non-soluble proteins, each sample was blanked using the respective cell lysate and cell lysate 60,000 rcf controls. The decrease in L-phenylalanine benzyl ester absorbance at 260 nm indicates removal of excess polymer. Furthermore, analysis of the supernatant allows identification of the polymer concentration for the most efficient solubilization of cell membranes (Figure 6, Phenylopol, cell lysate supernatant 60,000 rcf).

[0080] More efficient solubilization with respect to isolation efficiency and removal of excess copolymer prior to affinity resin purification are key features of the new inventive polymer series, exemplified by the use of Phenylopol for membrane protein isolation. Advantageously, the physicochemical properties of the Aminopol polymer series can be varied by using different protected and unprotected amino acids for polyacrylic acid grafting, making the Aminopol series suitable for a variety of membrane proteins and downstream applications.

[0081] Furthermore, the use of polymerization techniques such as RAFT polymerization allows for the synthesis of well-defined polymers with extremely narrow polydispersities and chain-end functionalization as previously described and exemplified in the Results, features not offered by currently commercially available polymers (e.g., DIBMA, SMA, Ultrasolute).

[0082] The foregoing description is for purposes of illustration and is not intended to be exhaustive or to limit the invention to the precise form disclosed.

[0083] In summary, the present invention relates to polymers of general formula (1) based on acrylic acid functionalized with polar and nonpolar chemically protected proteinogenic and nonproteinogenic amino acids and charged molecules such as phosphorylethanolamine or homotaurine to enhance solubilization efficiency, stability of the formed complexes, and resistance to high ionic strength. The amphiphilic polymers according to the present invention are used for the solubilization and stabilization of membrane-bound proteins in aqueous media and for the use of membrane protein-polymer complexes in biotechnological and medical applications. Furthermore, the newly developed amphiphilic polymers have the advantage of being further chemically labeled with molecular tags and dyes for protein labeling. [ka]

[0084] References: 1. Rabilloud, T., Membrane proteins and proteomics: love is possible, but so difficult. Electrophoresis, 2009.30 Suppl 1:p.S174-80. 2.Marconnet, A., et al., Solubilization and stabilization of membrane proteins by cycloalkane-modified amphiphilic polymers.Biomacromolecules,2020:p.3459-3467. 3.Smith,AAA,et al.,Lipid Nanodiscs via Ordered Copolymers.Chem,2020.6(10):p.2782-2795. 4.Zoonens, M. and JLPopot, Amphipols for each season.J Membr Biol,2014.247(9-10):p.759-96. 5.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. 6.Dahmane,T.,et al.,Sulfonated amphipols:synthesis,properties,and applications.Biopolymers,2011.95(12):p.811-23. 7.Zampieri,V.,et al.,CryoEM reconstructions of membrane proteins solved in several amphipathic solvents,nanodisc,amphipol and detergents,yield amphipathic belts of similar sizes corresponding to a common ordered solvent layer.Biochim Biophys Acta Biomembr,2021.1863(11):p.183693. 8.Smith,A.A.A.,et al.,Controlling Styrene Maleic Acid Lipid Particles through RAFT.Biomacromolecules,2017.18(11):p.3706-3713. 9.Chiefari,J.,et al.,Living Free-Radical Polymerization by Reversible Addition-Fragmentation Chain Transfer:The RAFT Process.Macromolecules,1998.31:p.5559-5562. 10.Moad,G.,E.Rizzardo,and S.H.Thang,End-functional polymers,thiocarbonylthio group removal / transformation and reversible addition-fragmentation-chain transfer(RAFT)polymerization.Polymer International,2011.60(1):p.9-25. 11.Peng,D.,et al.,Synthesis of poly(acrylic acid)-g-polystyrene copolymer by successive ATRP.Polymer Bulletin,2010.65(7):p.657-667. 12.Chaduc,I.,et al.,Effect of the pH on the RAFT Polymerization of Acrylic Acid in Water.Application to the Synthesis of Poly(acrylic acid)-Stabilized Polystyrene Particles by RAFT Emulsion Polymerization.Macromolecules,2013.46(15):p.6013-6023. 13.Fairbanks,B.D.,D.M.Love,and C.N.Bowman,Efficient Polymer-Polymer Conjugation via Thiol-ene Click Reaction.Macromolecular Chemistry and Physics,2017.218(18). 14.Oishi,E.,M.Takamura,and T.Takahashi,Removal of Trithiocarbonyl End Group of RAFT-Polymerized Poly(stearyl acrylate)and Effect of the End Group on Thermal and Structural Properties.Polymers(Basel),2021.13(23). 15.His affinity purification:Hochuli,E.,Bannwarth,W.,Dobeli,H.et al.Genetic Approach to Facilitate Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent.Nat Biotechnol 6,1321-1325(1988) 16.Rho affinity purification:Corin,K.,Baaske,P.,Geissler,S.et al.Structure and function analyses of the purified GPCR human vomeronasal type 1 receptor 1.Sci Rep 1,172(2011).https: / / doi.org / 10.1038 / srep00172

Claims

1. A water-soluble membrane protein-solubilizing polymer having the general formula (1) 【Chemistry 1】 (In the formula, X is either an initiator fragment derived from the radical starter molecule used to prepare the polymer, or a functional group or a hydrogen atom; Y is a functional group, a hydrogen atom or a terminal RAFT agent available for further functionalization and can also be conjugated with other molecules such as fluorophores and biomolecular tags; a and b represent the relative numbers of statistically distributed units of the polymer, resulting in the molecular weight of the polymer; R １ is the hydrophilic part of the polymer based on grafted proteinogenic and non-proteinogenic amino acids and amino acid derivatives, and polar functional groups such as phosphoethanolamine and derivatives, R ２ is the hydrophobic part of the polymer, based on grafted proteinogenic and non-proteinogenic amino acids and amino acid derivatives).

2. 2. The polymer of claim 1, wherein the initiator fragment is selected from the group consisting of 4'-azobis(4-cyanopentanoic acid), 2,2'-azobis(2-methylpropionitrile), and 2-(azo(1-cyano-1-methylethyl))-2-methylpropanenitrile.

3. 2. The polymer of claim 1, wherein the RAFT agent is selected from the group consisting of 4-((((2-carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid, and may be chemically substituted or modified with, for example, but not exclusively, a fluorophore bearing either a primary amine or a maleimide, or may be substituted with a biomolecular tag.

4. 2. The polymer of claim 1, wherein the molecular weight is from 2,000 to 24,000 daltons.

5. 2. The polymer of claim 1, wherein a and b range from 0% to 90% for each of said units, and generally a+b=100% of the total functionalized and non-functionalized carboxylic acid groups of said polymer.

6. 2. The polymer of claim 1, wherein the hydrophilic moiety is selected from the group consisting of a hydroxy group, an alkali metal ionically bonded to oxygen (the hydrophilic moiety can also be chemically modified and exchanged via amide formation with a phosphate- or sulfate-containing molecule), a mono- or polyamino acid with a charged side chain, or an amino acid with an uncharged side chain, or a combination.

7. The polymer of claim 1 , wherein the hydrophobic moiety is a proteinogenic or non-proteinogenic amino acid whose carboxylic acid group is chemically protected.

8. 8. The polymer of claim 7, wherein the protected amino acids are selected from the group consisting of phenylalanine and tryptophan protected by groups such as benzyl esters and tertbutyl esters.

9. The polymer of claim 1 , wherein the hydrophobic moiety is an amino acid.

10. 10. The polymer of claim 9, wherein the amino acid is selected from the group consisting of phenylalanine and tryptophan.

11. 10. The polymer of claim 1, wherein the polydispersity index of the polymer, defined as the ratio of the weight average by number to the average molecular weight as determined by aqueous size exclusion chromatography (SEC), is 1.25 or less.

12. 12. The polymer of claim 11, wherein the polydispersity index of the polymer is 1.1 or less.

13. 2. The polymer of claim 1, wherein the functional group introduced at position Y is biotin, a peptide, a tag, a fluorophore, or a dye.

14. A polyacrylic acid polymer is prepared, and the substituent R １ and / or R ２ 14. A method for preparing a polymer as defined in any one of claims 1 to 13, which is functionalized to introduce

15. Use of a polymer according to any one of claims 1 to 13 for the solubilisation and stabilisation of membrane proteins.

16. A water-soluble membrane protein amphiphilic vinyl polymer conjugate comprising a polymer of Formula 1.

17. 17. The complex of claim 16, further comprising a lipid compound.

18. A method for preparing a water-soluble membrane protein amphiphilic vinyl polymer conjugate according to claim 16, comprising a solution process in which a protein fraction from a biological or synthetic membrane containing the membrane protein, or a mixture of membrane proteins, is contacted with a polymer of formula 1.

19. Use of the polymer according to any one of claims 1 to 18 in diagnostics in which biological components such as DNA, RNA, proteins and metabolites are examined qualitatively and quantitatively.

20. A kit for solubilizing and stabilizing membrane proteins, comprising a polymer as defined in claims 1 to 13.

21. A kit for the lysis of eukaryotic cells or tissues, comprising one or more polymers according to any one of claims 1 to 20.

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