Mimetic structures

Abstract

A membrane assembly comprising: (a) a porous support; and (b) a plurality of membrane mimetic structures on a surface of the support; wherein the membrane mimetic structures comprise: (i) a channel protein which comprises a transmembrane domain having a hydrophobic surface, and (ii) one or more amphiphilic molecules; and wherein the one or more amphiphilic molecules form a ring structure around the hydrophobic surface of said transmembrane domain of the channel protein.

Description

[0001] Mimetic structures

[0002] Field of the Invention

[0003] The present invention concerns mimetic structures. More particularly, but not exclusively, this invention concerns a membrane assembly comprising: (i) a porous support and (ii) a plurality of membrane mimetic structures on a surface of the support, wherein at least a portion of the membrane mimetic structures each comprise a membrane protein. The invention also concerns methods of making such a membrane assembly, uses of the membrane assembly (e.g. in filtration applications) and filtration methods involving the membrane assembly.

[0004] Background of the Invention

[0005] Conventional nanofiltration or reverse osmosis water filtration membranes are known in the art. Typically, they are made by casting a support membrane (e.g. poly sulfone or poly ether sulfone); immersing the resulting cast in an aqueous solution of a diamine; removing excess from the surface of the membrane; immersing the membrane in an organic solution of a trifunctional acyl halide; and curing the resulting product to produce a polyamide layer. Washing and secondary coating are then carried out as necessary.

[0006] Alternative approaches which mimic the filtration properties of biological membranes (so-called “biomimetic” membranes) have also been proposed. Membrane proteins in biological cell membranes have unique and desirable characteristics of ultra- high water permeability and near-perfect solute selectivity. Membrane proteins are functional and maintain their activity when embedded in the native lipid bilayer environment of cell membranes. There has been considerable interest in extracting these membrane proteins as isolated particles and reconstituting them in lipid bilayers (proteoliposomes) or lipid bilayer-like environments (e.g. proteopolymersomes). Such lipid bilayers or lipid bilayer-like environments can be immobilized on a porous support membrane to fabricate a semipermeable biomimetic membrane assembly for filtration applications (Shen et al., Journal of Membrane Science (2014), vol. 454, p. 359-381).

[0007] Proteopolymersomes are dense assemblies of amphiphilic block copolymers into which proteins (e.g. membrane proteins) are reconstituted. The amphiphilic block copolymers self-assemble to form vesicles that mimic lipid bilayers but provide a more chemically and structurally robust host environment to the reconstituted proteins than when using lipids. Amphiphilic block copolymers also support cross-linking (either within the vesicle wall or between vesicles) and more generally functionalisation of the vesicle surface for various applications.

[0008] Biomimetic membranes can be used for high-resolution selective separation of any application that involves the use of a semipermeable membrane for filtration applications. Such applications include, but are not limited to, microfiltration, ultrafiltration, nanofiltration, reverse osmosis and engineered osmosis applications.

[0009] To fabricate a synthetic biomimetic membrane, the protein must be immobilized on a stable, porous support. Coating protein on porous substrates (e.g. via the use of proteoliposomes or proteopolymersomes) is not trivial and several factors govern a robust coating.

[0010] Current biomimetic membranes suffer from two major disadvantages: 1) low density of protein (e.g. membrane protein, for example transmembrane protein) on the surface of the support, i.e., the number of molecules of protein per unit area of the membrane surface is low; and 2) the orientation of the protein on the surface of the support may not be optimal. The present invention seeks to mitigate the above- mentioned problems.

[0011] Summary of the Invention

[0012] The present invention provides, according to a first aspect, a membrane assembly comprising: a. a porous support; and b. a plurality of membrane mimetic structures on a surface of the support, wherein at least a portion of said membrane mimetic structures each comprise a membrane protein; wherein the membrane mimetic structures are selected from the group consisting of: (i) nanodiscs; (ii) membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein, wherein said amphiphilic polymer is not a lipid or an amphiphilic block copolymer and (iii) a mixture of (i) and (ii). According to a second aspect of the invention there is also provided a method of making a membrane assembly according to the first aspect of the invention, which comprises the following steps: a. providing a porous support; b. applying an aqueous solution of the membrane mimetic structures as defined herein to a surface of the porous support; thereby forming the membrane assembly.

[0013] According to a third aspect of the invention there is also provided a membrane assembly comprising: a. a porous support; and b. a plurality of membrane mimetic structures on a surface of the support; wherein the membrane mimetic structures comprise: (i) a channel protein which comprises a transmembrane domain having a hydrophobic surface, and (ii) one or more amphiphilic molecules; and wherein the one or more amphiphilic molecules form a ring structure around the hydrophobic surface of said transmembrane domain of the channel protein.

[0014] According to a fourth aspect of the invention there is also provided a method of making a membrane assembly according to the third aspect of the invention, which comprises the following steps: a. providing a porous support; b. applying an aqueous solution of the membrane mimetic structures as defined herein to a surface of the porous support; thereby forming the membrane assembly.

[0015] According to a fifth aspect of the invention there is also provided a membrane assembly preparable by a method according to the second or fourth aspects of the invention.

[0016] According to a sixth aspect of the invention there is also provided a use of a membrane assembly according to the first, third or fifth aspects of the invention in a filtration application. According to a seventh aspect of the invention there is also provided a filtration method comprising the following steps: a. providing a membrane assembly according to the first, third or fifth aspects of the invention; b. filtering a liquid using the membrane assembly.

[0017] It will of course be appreciated that features described in relation to one aspect of the present invention may be incorporated into other aspects of the present invention. For example, the methods and uses of the invention may incorporate any of the features described with reference to the membrane assemblies of the invention and vice versa.

[0018] Embodiments of the present invention will now be described by way of example only with reference to the accompanying figures of which:

[0019] Figure 1 shows: (A) a schematic of RsAqpZ and membrane scaffold protein (MSP) expression constructs; (B) affinity and size exclusion (SEC) chromatography based purification of wild type RsAqpZ (WT) and RsAqpZ mutant (R188A); (C) SDS- PAGE gels showing the purification of wild type (WT) RsAqpZ and RsAqpZ mutant (R188A). The R188A mutant shown in the Figure was used for the remainder of the experiments described herein where a transport-dead RsAqpZ mutant was required. Figure 2 is a schematic showing the structure and construction of a biomimetic membrane assembly according to embodiments of the first aspect of the invention.

[0020] Figure 3 shows stopped flow light scattering data for wild-type RsAqpZ and RsAqpZ mutant (R188A), showing normal permeability (wild-type) and decreased permeability (mutant) under osmotic stress. Control: Empty polymersomes (i.e. without aquaporin) in buffer. Top panel: Overlaid graph of normalised traces, normalised to a minimum voltage of 0 dV and a maximum voltage of 1.0 dV. Bottom panel: Fold- increase in permeability compared to control. The R188A mutant shown in the Figure was used for the remainder of the experiments described herein where a transport-dead RsAqpZ mutant was required. Figure 4 shows schematics of aquaporin Z incorporated into (A) polymer vesicles and (B) nanodiscs; and negative stain cryo-TEM images of aquaporin-incorporated vesicles (C) and nanodiscs (D).

[0021] Figure 5 shows a comparison of (A) intact (folded) RsAqpZ protein coverage (assessed using the signal intensity of GFP fluorescence) and (B) total RsAqpZ protein coverage (assessed using signal intensity of conjugated dye) on biomimetic membrane assemblies using nanodiscs or vesicles. (C) is a graph illustrating the percentage coverage of protein on the membrane assemblies based on the signal intensities.

[0022] Figure 6 shows (A) initial scans of biomimetic membrane assemblies using nanodiscs prior to denaturation treatments; (B) the same membrane assemblies following heat denaturation and rehydration and (C) the same membrane assemblies following treatment with 99% ethanol and rehydration. Top panels, total protein (detected using the conjugated dye); bottom panels, intact (folded) protein (detected using GFP fluorescence).

[0023] Figure 7 shows the surface topology of biomimetic membrane assemblies, imaged using tapping atomic force microscopy (AFM). (A), negative control (piperazine- based coating, no vesicles or nanodiscs), (B) and (D) vesicles containing RsAqpZ, (C) and (E) nanodiscs containing RsAqpZ. The top half of panels (A), (B) and (C) show the scanning AFM images and the bottom half of panels (A), (B) and (C) show plots measuring the surface characteristics (waviness, roughness and texture) of representative biomimetic membrane assemblies. Note that the scale of the y-axis of the plot in bottom panel of (C) (nanodiscs) is in nm, compared to the y-axes in the bottom panels of (A) and (B) which are in microns. The AFM images in (B) and (C) are of a 2 micron x 2 micron area while the AFM images in (D) and (E) are of a smaller 1 micron x 1 micron area of the same membrane assemblies.

[0024] Figure 8 shows SEM images of cross-sections (top row) and surfaces (bottom row) of control (A), vesicle-incorporating (B) and nanodisc-incorporating (C) membrane assemblies.

[0025] Figure 9 shows top view schematics of (A) aquaporin Z and (B) OmpG incorporated into individual nanodiscs; and a top view schematic of contiguous OmpG nanodiscs (C) as would be arranged on the surface of a support of an embodiment of a membrane assembly as described herein. Figure 10 shows the performance of membrane assemblies containing different loading densities of aquaporin Z-incorporated nanodiscs (permeability and NaCl rejection), compared to control membrane assemblies (‘CTRL’, without any membrane mimetic structures; ‘Empty’, membrane assembly loaded with ‘empty’ nanodiscs without AqpZ protein incorporated). The membrane assemblies were tested at 25 °C in a cross-flow setup at 225 psi, with a salt concentration of 2000 ppm and a feed flow rate of 1 L / min.

[0026] Figure 11 shows the performance of membrane assemblies containing OmpG- incorporated nanodiscs (permeability and NaCl rejection), compared to control membrane assemblies (‘CTRL’, without any membrane mimetic structures; ‘EMPTY’, loaded with empty nanodiscs without OmpG protein incorporated). Performance was tested at 25 °C in a cross-flow setup at 225 psi, with a salt concentration of 2000 ppm and a feed flow rate of 1 L / min.

[0027] Figure 12 shows the performance of membrane assemblies containing detergent- solubilised OmpG (permeability and NaCl rejection) compared to a commercially available control (BW30 membrane with no membrane mimetic structures). Performance was tested at 25 °C in a cross-flow setup at 225 psi, with a salt concentration of 2000 ppm and a feed flow rate of 1 L / min.

[0028] Detailed Description

[0029] According to an embodiment of a first aspect of the invention, there is provided a membrane assembly comprising: a. a porous support; and b. a plurality of membrane mimetic structures on a surface of the support, wherein at least a portion of said membrane mimetic structures each comprise a membrane protein; wherein the membrane mimetic structures are selected from the group consisting of: (i) nanodiscs; (ii) membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein, wherein said amphiphilic polymer is not a lipid or an amphiphilic block copolymer and (iii) a mixture of (i) and (ii).

[0030] Figure 2 (not drawn to scale) shows a schematic of the structure and construction of a membrane assembly according to an embodiment of the first aspect of the invention. The membrane assembly comprises a porous support 1, optionally comprising a backing 2 such as a non-woven layer, with a plurality of the membrane mimetic structures 3 on a surface of the support 1 (Figure 2A). The backing 2 (when present) is on the opposite surface of the porous support 1 from the plurality of membrane mimetic structures 3, i.e. the porous support 1 is located between the plurality of membrane mimetic structures 3 and the backing 2. The membrane assembly may also optionally comprise a coating 4 on the surface of the support 1 comprising the membrane mimetic structures 3 (Figure 2B). The above reference signs are not to be construed as limiting the extent of the matter protected by the claims.

[0031] The present invention also provides a membrane assembly comprising: a. a porous support; and b. a plurality of membrane mimetic structures on a surface of the support, wherein at least a portion of said membrane mimetic structures each comprise a membrane protein; wherein the membrane mimetic structures are selected from the group consisting of: (i) nanodiscs; (ii) membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein and (iii) a mixture of (i) and (ii).

[0032] The present invention also provides a membrane assembly comprising: a. a porous support; and b. a plurality of membrane mimetic structures on a surface of the support; wherein the membrane mimetic structures comprise: (i) a channel protein which comprises a transmembrane domain having a hydrophobic surface, and (ii) one or more amphiphilic molecules; and wherein the one or more amphiphilic molecules form a ring structure around the hydrophobic surface of said transmembrane domain of the channel protein.

[0033] In embodiment, the one or more amphiphilic molecules form a ring structure around the external hydrophobic surface of the transmembrane domain of the channel protein. A side view and a top view of an example of a ring structure as referred to herein is shown in Figure 4B.

[0034] The present inventors have surprisingly found that membrane assemblies (which comprise a porous support and a plurality of membrane mimetic structures on a surface of the support as described herein) have a much higher density of protein (e.g. membrane protein, for example a channel protein, a membrane channel protein or a transmembrane protein) on the surface of the porous support when formed using membrane mimetic structures as described herein, when compared with membrane assemblies which are formed using membrane mimetic structures such as proteoliposomes or proteopolymersomes. Furthermore, use of membrane mimetic structures of the present invention permits optimisation of the orientation of the protein (e.g. channel protein) with respect to the support, i.e. a portion (e.g. a majority, substantially all or all) of the proteins are oriented orthogonally or substantially orthogonally to the plane of the support. Such membrane assemblies, when used for filtration of liquids, show surprising enhancements of both flux (permeability) and selectivity (rejection), effectively overcoming the trade-off between permeability and selectivity that may be associated with currently available filtration membrane assemblies. Finally, membrane assemblies made according to the invention have good robustness when exposed to denaturing conditions (e.g. heat), which should be beneficial in real- world filtration applications.

[0035] In an embodiment of the invention, (in respect of the membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein wherein said amphiphilic polymer is not a lipid or an amphiphilic block copolymer), the amphiphilic polymer which contacts said membrane protein is capable of stabilising said membrane protein, e.g. in particular in aqueous solution. Thus, when the surface of the support comprising the membrane mimetic structures is exposed to an aqueous environment, the amphiphilic polymer stabilises the membrane protein that it contacts. As will be appreciated, it is the hydrophobic moieties of the amphiphilic polymer which contact the hydrophobic surfaces of the membrane protein and the hydrophilic moieties of the amphiphilic polymer which are directed away from the hydrophobic surfaces of the membrane protein (towards the external aqueous environment when such an environment is present). Orienting the amphiphilic polymer in this manner thus shields the hydrophobic surfaces of the membrane protein from a surrounding aqueous environment and also prevents aggregation and / or denaturation of the membrane protein.

[0036] In an embodiment of the invention, said membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein (wherein said amphiphilic polymer is not a lipid or an amphiphilic block copolymer) are essentially, e.g. completely, free of lipids or amphiphilic block copolymers. In other words, these membrane mimetic structures are not nanodiscs (the membrane protein is not surrounded by a disc of lipid as is the case in nanodiscs) and instead the amphiphilic polymer directly contacts the membrane protein and is capable of stabilising the membrane protein in an aqueous solution. Thus, such membrane structures may comprise essentially (e.g. consist of) the membrane protein and the amphiphilic polymer. In an embodiment of the invention, said membrane mimetic structures (which comprise an amphiphilic polymer which contacts said membrane protein) are not proteoliposomes or proteopolymersomes. In an embodiment of the invention, the amphiphilic polymer (in the membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein) is an amphipol polymer, for example an amphipol polymer as defined herein. Accordingly, in an embodiment, the present invention is directed to a membrane assembly comprising: a. a porous support; and b. a plurality of membrane mimetic structures on a surface of the support, wherein at least a portion of said membrane mimetic structures each comprise a membrane protein; wherein the membrane mimetic structures are selected from the group consisting of: (i) nanodiscs; (ii) membrane mimetic structures which comprise an amphipol polymer which contacts said membrane protein and (iii) a mixture of (i) and (ii). In this embodiment, as is explained above, it is the hydrophobic moieties of the amphipol polymer which contact the hydrophobic surfaces of the membrane protein and the hydrophilic moieties of the amphipol polymer which are directed away from the hydrophobic surfaces of the membrane protein (towards the external aqueous environment when such an environment is present).

[0037] In embodiments of the third aspect of the invention, wherein the membrane mimetic structures comprise: (i) a channel protein which comprises a transmembrane domain having a hydrophobic surface, and (ii) one or more amphiphilic molecules, the ring structure formed by the one or more amphiphilic molecules around the hydrophobic surface of said transmembrane domain of the channel protein is a ringshaped structure. In an embodiment, the membrane mimetic structures are soluble when exposed to an aqueous environment.

[0038] The ring structure may also be referred to as an annular structure or an annularshaped structure. In another embodiment, such a ring structure is capable of shielding the hydrophobic surface of the transmembrane domain of the channel protein from an aqueous environment (for example, an aqueous medium) such that the membrane mimetic structures are soluble when exposed to an aqueous environment (for example, an aqueous medium). Vesicles, including but not limited to liposomes, polymersomes and hybrid polymer / lipid vesicles, do not form a ring structure around the channel protein embedded therein (e.g. around a hydrophobic surface of a transmembrane domain of such an embedded channel protein), but rather form a spherical or substantially spherical structure.

[0039] In further embodiments of the third aspect of the invention, the structure of the transmembrane domain of said channel protein is selected from the group consisting of one or more alpha helices or a beta barrel. For example, the transmembrane domain of said channel protein may be a beta barrel comprising from 8 to 24 beta strands, for example from 10 to 24 beta strands or from 12 to 24 beta strands, for example 10, 12, 14, 16, 19, 22, or 24 beta strands, for example a beta barrel comprising 14 beta strands or for example a beta barrel having 14 beta strands. In an embodiment, the structure of the transmembrane domain of said channel protein is selected from the group consisting of one or more alpha helices or a beta barrel having from 10 to 24 beta strands, for example from 12 to 24 beta strands. When the transmembrane domain of said channel protein is formed of one or more alpha helices, i.e. the channel protein is an alphahelical channel protein. Such an alpha-helical channel protein may be monotopic, bitopic (also termed ‘single-pass’ or ‘single-spanning’) or polytopic.

[0040] In further embodiments of the third aspect of the invention, at least a portion (for example, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% or more) of the hydrophobic moieties of said one or more amphiphilic molecules are oriented towards said channel protein and at least a portion (for example, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 96%, 97%, 98%, 99% or more) of the hydrophilic moieties of said amphiphilic molecule are oriented away from said channel protein. In an embodiment, substantially all, e.g. all of the hydrophobic moieties of said one or more amphiphilic molecules are oriented towards said channel protein and substantially all, e.g. all of the hydrophilic moieties of said amphiphilic molecule are oriented away from said channel protein.

[0041] In an embodiment of the third aspect, the membrane mimetic structures comprising (i) a channel protein which comprises a transmembrane domain having a hydrophobic surface, and (ii) one or more amphiphilic molecules as described herein are soluble when exposed to an aqueous medium. For example, an aqueous solution of the membrane mimetic structures can be made in which (at least a portion of) the membrane mimetic structures remain soluble. Similarly, when the membrane mimetic structures are in situ on the porous support and the assembly is exposed to an aqueous medium, the membrane mimetic structure remain soluble.

[0042] In an embodiment of the third aspect, the membrane mimetic structures each comprise a single channel protein.

[0043] The ring structure formed by the one or more amphiphilic molecules may be planar or essentially planar, i.e. may have a flat or level surface extending in a plane outwards from the embedded channel protein. Therefore, in an embodiment of the third aspect, the one or more amphiphilic molecules form an essentially planar ring structure around the hydrophobic surface of the transmembrane domain of the channel protein. In another embodiment of the third aspect, the one or more amphiphilic molecules form a planar ring structure around the hydrophobic surface of the transmembrane domain of the channel protein. As will be appreciated by the skilled person, since vesicles have a spherical or substantially spherical shape, the amphiphilic molecules which surround a membrane or channel protein which is embedded in said vesicles do not extend outwards in a plane from the embedded protein.

[0044] In an embodiment of the third aspect, the ring structure is planar or essentially planar, and the plane of the ring structure is orthogonal or essentially orthogonal to the axis of the channel protein. The axis of a channel protein in a membrane assembly according to the third aspect of the invention refers to the axis of the central pathway (or pore) through which water, ions and / or other substances such as small molecules pass across the membrane where the channel protein is embedded. This axis is perpendicular or approximately perpendicular to the plane of the membrane in the native state.

[0045] In another embodiment of the third aspect, the membrane mimetic structures further comprise lipids or lipid-mimic polymers. Suitable lipids and lipid-mimic polymers which can be used in said membrane mimetic structures according to the third aspect of the invention are described elsewhere herein. In particular, ‘lipid-mimic polymers’ refer to polymers, such as are described elsewhere herein, which mimic the amphiphilic nature of lipid molecules, but which are not lipids, and which serve as a structural and functional replacement for lipids. Suitable lipids may include phosopholipids, such as are described elsewhere herein. Suitable lipid-mimic polymers may for example include amphiphilic block copolymers as described herein. In another embodiment of the third aspect, said lipids or lipid-polymers (as referred to above and as described elsewhere herein) are located between the hydrophobic surface of the transmembrane domain of the channel protein and the one or more amphiphilic molecules.

[0046] In another embodiment of the third aspect, at least a portion of the one or more amphiphilic molecules contacts the hydrophobic surface of the transmembrane domain of the channel protein, for example at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% of the one or more amphiphilic molecules contacts the hydrophobic surface of the transmembrane domain of the channel protein. For example, at least a portion of the one or more amphiphilic molecules directly contacts the hydrophobic surface of the transmembrane domain of the channel protein.

[0047] In another embodiment of the third aspect, the membrane mimetic structures are selected from the group consisting of: nanodiscs (as described herein), membrane mimetic structures stabilised using nanodisc scaffold peptide (NSP) or reversed nanodisc scaffold peptide (NSPr) (as described elsewhere herein), detergent- stabilised membrane mimetic structures (as described elsewhere herein), saposin-stabilised membrane mimetic structures (as described elsewhere herein) and amphipol-stabilised membrane mimetic structures (as described elsewhere herein). The sequences of NSP and NSPr are shown in SEQ ID NO: 2 and SEQ ID NO: 3 respectively. In an embodiment, the membrane mimetic structures are selected from the group consisting of: nanodiscs, detergent- stabilised membrane mimetic structures, and amphipol- stabilised membrane mimetic structures. In an embodiment, the membrane mimetic structures are selected from the group consisting of: nanodiscs and detergent- stabilised membrane mimetic structures. In an embodiment, the membrane mimetic structures are nanodiscs as described elsewhere herein.

[0048] In another embodiment, the membrane mimetic structures are detergent- stabilised membrane mimetic structures, for example detergent-stabilised membrane mimetic structures comprising a non-denaturing detergent. Non-denaturing detergents are particularly suitable for preparing membrane assemblies comprising a channel protein, since they are formulated to maintain the native structure and function of proteins during solubilization. This facilitates downstream applications of the solubilised channel proteins (e.g. filtration applications) which rely on the preserved state of the proteins. Suitable detergents that may be used in membrane mimetic structures in a membrane assembly according to the invention may also include nonionic and zwitterionic detergents, which are known to be less denaturing than ionic detergents, and are often used to solubilize membrane proteins and channel proteins where it is important to retain native protein function. In an embodiment, the detergent in a detergent-stabilised membrane mimetic structure is CHAPS. In another embodiment, the detergent in a detergent-stabilised membrane mimetic structure is a Triton-X non-ionic detergent. In another embodiment, the detergent in a detergent- stabilised membrane mimetic structure is selected from the group consisting of: DDM (n-Dodecyl-P-D-Maltopyranoside), OG (n-Octyl-P-D-Glucopyranoside), LDAO (n- Dodecyl-N,N-Dimethylamine-N-Oxide), n-Octylpolyoxyethylene, Lauryl Maltose Neopentyl Glycol, and Decyl Maltose Neopentyl Glycol. For example, the detergent in a detergent-stabilised membrane mimetic structure is DDM.

[0049] In an embodiment of the third aspect, the membrane mimetic structure is an amphipol- stabilised membrane mimetic structure, and the amphiphilic molecule is an amphipol polymer as described elsewhere herein. In a further embodiment, the amphipol polymer contacts (e.g. directly contacts) the channel protein. In an embodiment, the amphipol polymer is a polymer according to Formula IV :

[0050] Formula IV wherein,

[0051] Ri is carboxylate, or V-phosphorylcholinc-V’- ethylenedioxybis(ethyl)acrylamide;

[0052] R2 is a Ci-Ce alkylene, or is a bond;

[0053] R3 is a Ci-Ce alkyl, a C3-C9 cycloalkyl, a C6-C14 aryl which is optionally substituted with a C1-C4 alkyl;

[0054] R4 is a Ci-Ce alkyl, or ethylsulfonate; wherein x, y and z are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85, y is from about 10 to about 85 and z is from 0 to about 60 and wherein the sum of x, y and z equals 100.

[0055] In another embodiment of the third aspect, the channel protein is a channel protein as described elsewhere herein. For example, the channel protein is selected from the group consisting of: an aquaporin (as described elsewhere herein), an aquaglyceroporin, OmpX (Escherichia coli), PagP (Escherichia coli), OmpW (Escherichia coli), OmpT (Escherichia coli), EspP autotransporter beta domain (Escherichia coli), OmpLA (Escherichia coli), OmpG (Escherichia coli), OmpF (Escherichia coli), FhuA (Escherichia coli), T7 DNA ejectosome periplasmic tunnel (Escherichia phage T7), Aerolysin (Aeromonas hydrophila), Main porin MspA (Mycolicibacterium smegmatis), and Alpha-hemolysin (Staphylococcus aureus). For example, the channel protein is selected from the group consisting of: Aquaporin AqpZ (Escherichia coli; GenBank ID U38664), Aquaporin AQPO (Ovis aries), Aquaporin AQP1 (red blood cell; Homo sapiens), Aquaporin AQP2 (kidney; Homo sapiens (expressed in Pichia pastoris)), Aquaporin AQP4 (glial cell; Rattus norvegicus), Aquaporin AQP4 (Homo sapiens (expressed in Pichia pastoris)), Aquaporin AQP5 (HsAQP5) (Homo sapiens), Aquaglyceroporin AQP7 (Homo sapiens (expressed in Komagataella pastoris), Aquaglyceroporin AQP10 (Homo sapiens (expressed in S. cerevisiae), Aquaporin AqpM (Methanothermobacter marburgensis), GlpF glycerol facilitator channel (Escherichia coli), Aquaglyceroporin AQP (Plasmodium falciparum), OmpX (Escherichia coli), OmpW (Escherichia coli), OmpT (Escherichia coli), OmpLA (Escherichia coli), OmpG (Escherichia coli; Gen Bank ID P76045), OmpF (Escherichia coli). For example, the channel protein is selected from Aquaporin AqpZ or OmpG

[0056] In another embodiment of the third aspect, the channel protein is selected from the group consisting of: aquaporins (as described herein) and glyceroporins (as described herein); beta-barrel channel proteins (for example beta-barrel channel proteins having from 10 to 24 beta strands); and alpha-helical channel proteins. In another embodiment, the channel protein is an aquaporin, for example Aquaporin Z. In another embodiment, substantially all the channel proteins present in the membrane assembly are oriented orthogonally to the surface of the porous support. In another embodiment, a portion or substantially all the channel proteins present in the membrane assembly are oriented orthogonally to the surface of the porous support. For example, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the channel proteins present in the membrane assembly are oriented orthogonally to the surface of the porous support. In an embodiment of the invention, all (100%) of the channel proteins present in the membrane assembly are oriented orthogonally to the surface of the porous support. The orientation of the channel proteins in the membrane assembly relative to the surface of the porous support can be determined by the use of known microscopy techniques, for example atomic force microscopy (AFM)

[0057] In the membrane assemblies according to the invention, there may be a proportion of the membrane mimetic structures (e.g. nanodiscs) on the surface of the support which do not comprise a membrane protein (e.g. which do not comprise a channel protein). Such nanodiscs may be referred to as “empty nanodiscs”. What is essential is that the membrane assemblies according to the invention comprise or include a plurality of membrane mimetic structures on a surface of the support, wherein at least a portion of said membrane mimetic structures each comprise a membrane protein (e.g. a channel protein). There may therefore be some membrane mimetic structures (e.g. nanodiscs) on the surface of the support which do not comprise a membrane protein (e.g. a channel protein). Advantageously, the number of membrane mimetic structures (e.g. nanodiscs) on the surface of the support which do not comprise a membrane protein (e.g. a channel protein) is minimised. For example, the percentage of membrane mimetic structures (e.g. nanodiscs) on the surface of the support which do not comprise a membrane protein (for example which do not contain a channel protein) is <50%, <40%, <30%, <25%, <20%, <15%, <10%, <5% or <1%. Expressed alternatively, the portion of the membrane mimetic structures on the surface of the support which each comprise a membrane protein (for example which each comprise a channel protein) is >50%, >60%, >70%, >75%, >80%, >85%, >90%, >95% or >99%.

[0058] In an embodiment of the invention, the density of the membrane proteins (e.g. channel proteins) on the surface of the support is at least 1000 proteins / pm2, for example at least 1000 proteins / pm2, at least 2000 proteins / pm2, at least 3000 proteins / pm2, at least 5000 proteins / pm2, at least 7500 proteins / pm2, at least 10,000 proteins / pm2, at least 12,000 proteins / pm2, at least 15,000 proteins / pm2, at least 18,000 protcins / p m2, at least 20,000 protcins / p m2or at least 25,000 protcins / p m2. For example, in an embodiment, the density of the membrane proteins on the surface of the support is from about 500 proteins / pm2to about 50,000 proteins / pm2, for example from about 1000 proteins / pm2to about 50,000 proteins / pm2, for example from about 2000 proteins / pm2to about 50,000 proteins / pm2, for example from about 3000 proteins / pm2to about 50,000 proteins / pm2, for example from about 5000 proteins / pm2to about 50,000 proteins / pm2, from about 7500 proteins / pm2to about 50,000 proteins / pm2, from about 10,000 proteins / pm2to about 50,000 proteins / pm2, from about 12,000 proteins / pm2to about 50,000 proteins / pm2, from about 15,000 proteins / pm2to about 50,000 proteins / pm2, from about 18,000 proteins / pm2to about 50,000 proteins / pm2, from about 20,000 proteins / pm2to about 50,000 proteins / pm2, from about 25,000 proteins / pm2to about 50,000 proteins / pm2, from about 500 proteins / pm2to about 25,000 proteins / pm2, from about 500 proteins / pm2to about 20,000 proteins / pm2, from about 500 proteins / pm2to about 18,000 proteins / pm2, from about 500 proteins / pm2to about 15,000 proteins / pm2, from about 500 proteins / pm2to about 10,000 proteins / pm2, from about 500 proteins / pm2to about 5000 proteins / pm2, from about 500 proteins / pm2to about 2500 proteins / pm2, from about 1000 proteins / pm2to about 2000 proteins / pm2, for example about 1500 proteins / pm2. For example, in an embodiment, the density of the membrane proteins on the surface of the support is from about 1000 proteins / pm2to about 25,000 proteins / pm2, from about 1000 proteins / pm2to about 20,000 proteins / pm2, from about 1000 proteins / pm2to about 18,000 proteins / pm2, from about 1000 proteins / pm2to about 15,000 proteins / pm2, from about 2000 proteins / pm2to about 20,000 proteins / pm2, from about 2000 proteins / pm2to about 15,000 proteins / pm2, from about 5000 proteins / pm2to about 15,000 proteins / pm2, from about 5000 proteins / pm2to about 20,000 proteins / pm2, from about 8000 proteins / pm2to about 15,000 proteins / pm2or from about 10,000 proteins / pm2to about 15,000 proteins / pm2, for example about 12,000 proteins / pm2.

[0059] The density of the membrane proteins (e.g. channel proteins) on the surface of the support as described herein may be measured using atomic force microscopy (AFM).

[0060] In an embodiment of the various aspects of the invention, the membrane mimetic structures as described herein have a diameter of from about 0.2 nm to about 90 nm, from about 0.2 nm to about 50 nm, from about 0.2 nm to about 20 nm, from about 0.2 nm to about 10 nm, from about 0.2 nm to about 5 nm, from about 0.2 nm to about 1 nm, for example about 0.5 nm. In an embodiment of the invention, the membrane mimetic structures as described herein have a diameter of from about 1 nm to about 90 nm, from about 1 nm to about 80 nm, from about 1 nm to about 60 nm, from about 1 nm to about 50 nm, from about 1 nm to about 40 nm or from about 1 nm to about 20 nm. In an embodiment of the invention, the membrane mimetic structures as described herein have a diameter of from about 2 nm to about 20 nm, from about 2 nm to about 15 nm, for example from about 5 nm to about 15 nm. In an embodiment of the invention, the membrane mimetic structures (e.g. nanodiscs) have a diameter of from about 5 nm to about 90 nm, for example from about 7 nm to about 90 nm, from about 5 nm to about 50 nm, from about 5 nm to about 20 nm, from about 7 nm to about 50 nm, from about 7 nm to about 20 nm, from about 8 nm to about 18 nm, from about 8 nm to about 15 nm, from about 10 nm to about 15 nm, from about 10 nm to about 12 nm or from 8 nm to about 11 nm, for example about 10 nm.

[0061] Nanodiscs

[0062] In an embodiment of the invention, the membrane mimetic structures are nanodiscs. A nanodisc is a small, disc-shaped structure that finds use in proteomics

[0063] (Sligar & Denisov, Protein Sci., 2021 Feb; v. 30(2):297-315 - https : / / doi g / 10 A nanodisc consists of two main components lipids

[0064] (which may be of artificial origin or from a cell membrane) or lipid- like molecules and a stabilising belt molecule that holds the lipids / lipid-like molecules together and which provides a hydrophobic surface facing the hydrophobic portion of the lipids or lipid- like molecules, and a hydrophilic surface on the outside facing the aqueous medium (i.e. the stabilising belt molecule is amphipathic / amphiphilic). A representation of a nanodisc comprising a membrane protein (which is the channel protein AqpZ) is shown in Figure 4B . The ring structure formed by the membrane scaffold protein and the lipids, which surrounds the external hydrophobic surface of the transmembrane domain of AqpZ is clearly shown in that figure. Moreover, it can be seen that the ring structure shields the external hydrophobic surface of the transmembrane domain of AqpZ from an aqueous environment. This composition makes nanodiscs highly soluble in aqueous solutions. The purpose of a nanodisc is to mimic the native phospholipid bilayer of cells for target molecules which are unstable in an aqueous environment. Such target molecules are usually membrane proteins such as transmembrane proteins (e.g. transmembrane proteins such as channel proteins as described herein) which comprise hydrophobic surfaces which are embedded in the native lipid bilayer, as well as hydrophilic surfaces which are exposed to the aqueous environment outside of the native lipid bilayer. Due to the presence of both hydrophobic and hydrophilic surfaces in a transmembrane protein, such transmembrane proteins are not soluble in standard aqueous buffers without a solubilising agent, e.g. a detergent. However, when embedded in a nanodisc, the hydrophobic surfaces of a target molecule, e.g. a membrane protein, for example a transmembrane protein such as a channel protein, are shielded within the lipid / lipid-like environment of the nanodisc and the hydrophilic surfaces of the target molecule, e.g. a membrane protein, for example a transmembrane protein such as a channel protein, are exposed to the aqueous environment outside of the nanodisc (with the whole assembly being held together by the stabilising belt molecule). The target molecule, e.g. a membrane protein, for example a transmembrane protein such as a channel protein, is thus stabilised in a native membrane-like environment via the nanodisc.

[0065] In an embodiment of the invention, the nanodiscs are selected from the group consisting of lipid-based nanodiscs, polymer-based nanodiscs, hybrid lipid / polymer- based nanodiscs and a combination thereof. Lipid-based nanodiscs comprise lipids, e.g. phospholipids, such as are described elsewhere herein. Polymer-based nanodiscs comprise polymers, such as are described elsewhere herein, which mimic the amphiphilic nature of lipid molecules, but which are not lipids, and which structurally and functionally replace the lipids which are found in a conventional lipid-based nanodisc. Such polymers may be referred to herein as “lipid-mimic polymers”. Examples of such lipid-mimic polymers include amphiphilic block copolymers as described herein. Hybrid lipid / polymer-based nanodiscs comprise both lipids and lipidmimic polymers.

[0066] In an embodiment of the invention, the stabilising belt molecule is a membrane scaffold protein (MSP), i.e. the nanodisc is an MSP nanodisc. In an embodiment of the invention, the stabilising belt molecule is a membrane scaffold protein (MSP) and the nanodisc comprises one or more lipids selected from the group consisting of POPC and DMPC. In an embodiment of the invention, the stabilising belt molecule is a synthetic polymer, i.e. a non-peptide-based or a non-protein-based polymer, which may be referred to herein as a “synthetic belt polymer”. Such a nanodisc may be referred to herein as a “synthetic nanodisc”. In an embodiment of the invention, the stabilising belt molecule is an amphipathic peptide, which may be referred to herein as a “peptide nanodisc”. Examples of peptide nanodiscs are provided in Larsen et al., Soft Matter, 2016, v. 12, p. 5937 (DOI: 10.1039 / c6sm00495d). The amphipathic peptide in such peptide nanodiscs may be based on, e.g. comprise, an apo Al mimetic sequence 18A originally designed by Anantharamaiah et al., Journal of Biological Chemistry, v. 260(18), p. 10248 (1985) (DOI: https: / / doi.org / 10.1016 / S0021-9258(17)39238-4). In an embodiment of the invention, the stabilising belt molecule is selected from the group consisting of a membrane scaffold protein (MSP) or a derivative thereof, and a synthetic polymer, i.e. a non-peptide-based or a non-protein-based polymer.

[0067] MSP nanodiscs, peptide nanodiscs and synthetic nanodiscs are three broad categories of nanodisc which differ essentially in the identity of the stabilising belt molecule and (in the case where the nanodisc is a lipid-based nanodisc), the type of lipids found within the nanodisc. In MSP and peptide nanodiscs, the lipid composition is artificial and chosen by the maker of the nanodisc, i.e. it can be completely controlled. In contrast, in synthetic nanodiscs, the synthetic belt polymer can insert into a cell membrane surrounding a membrane protein of interest and, like a cookie cutter, the membrane protein is dissolved from the membrane and the synthetic belt polymer keeps the membrane protein stable in the newly formed nanodisc. Examples of stabilising belt molecules which are synthetic belt polymers are poly(diisobutylene-a / t-maleic acid) (DIBMA), poly(styrene-co-maleic acid) (SMA), poly(acrylic acid- co -styrene) (AASTY) or an amphipol, for example an amphipol copolymer of formula V or VI as described elsewhere herein. It should be noted that nanodiscs comprising all combinations of lipids, lipid-mimic polymers and stabilising belt molecules are contemplated by the present invention. Thus, for example, lipid-based nanodiscs comprising an MSP or amphipathic peptide, lipid-based nanodiscs comprising a synthetic belt polymer, polymer-based nanodiscs comprising an MSP or amphipathic peptide, polymer-based nanodiscs comprising a synthetic belt polymer and mixed lipid / polymer-based nanodisc comprising an MSP, an amphipathic peptide or a synthetic belt polymer are all contemplated by the present invention.

[0068] In an embodiment of the invention, the membrane mimetic structures, such as the lipid-based or hybrid lipid / polymer-based nanodiscs, comprise one or more lipids selected from the group consisting of branched lipids, cationic lipids, cholesterol, fluorescent lipids, glycerolipids, helper lipids, ionisable lipids, isotope-labelled lipids, native lipids, PEGylated lipids, phospholipids, sphingolipids and stearic lipids. In an embodiment of the invention, the membrane mimetic structures, such as the lipid-based or hybrid lipid / polymer-based nanodiscs, comprise one or more lipids selected from the group consisting of fluorescent lipids, isotope-labelled lipids, native lipids (e.g. native lipids from E. coli) and phospholipids. In an embodiment of the invention, the membrane mimetic structures, such as the lipid-based or hybrid lipid / polymer-based nanodiscs, comprise one or more lipids selected from the group consisting of a phospholipid and native lipids (e.g. native lipids from E. coli). In an embodiment of the invention, the membrane mimetic structures, such as the lipid-based or hybrid lipid / polymer-based nanodiscs, comprise one or more lipids selected from the group consisting of l,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC); 1,2-dimyristoyl- sn-glycero-3-phospho-( 1 '-rac-glycerol) (DMPG) ; 1 ,2-dioleoyl-5n-glycero-3- phosphocholine (DOPC); l-myristoyl-2-hydroxy-5n-glycero-3-[phospho-rac-(l- glycerol)] (LMPG); I -palmitoyl-2-hydroxy-.s / 7-glyccro-3-|phospho-rac-( I -glycerol) | (LPPG); l-palmitoyl-2-oleoyl-5n-glycero-3-phosphocholine (POPC); l-palmitoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (POPE); palmitoyl-oleoyl- phosphatidylglycerol (POPG); l-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS); phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipins, cholesterols and native lipids (e.g. native lipids from E. coli). In an embodiment of the invention, the membrane mimetic structures, such as the lipid-based or hybrid lipid / polymer-based nanodiscs, comprise one or more lipids selected from the group consisting of POPC and DMPC and native lipids (e.g. native lipids from E. coli). As will be understood by the skilled person, when using a stabilising belt molecule which is a synthetic polymer, the lipid composition of the nanodisc is determined by the lipid composition of the native cell membrane from which the nanodisc is made.

[0069] Polymer-based nanodiscs

[0070] In an embodiment of the invention, the nanodiscs are polymer-based nanodiscs (see, for example Fiori et al., Scientific Reports, 2017, 7: 15227, Polymer-based nanodiscs comprise polymers, which are not lipids, but which mimic the amphiphilic nature of lipid molecules, and wherein the polymers functionally and structurally replace the lipids in a conventional lipid-based nanodisc. Such polymers may be referred to herein as “lipidmimic polymers”. In an embodiment of the invention, the polymer-based nanodiscs comprise one or more lipid- mimic polymers which are amphiphilic block copolymers. In an embodiment of the invention, the amphiphilic block copolymer comprises at least one hydrophilic block and at least one hydrophobic block. The stabilising belt molecule in a polymer-based nanodisc may be an MSP as described elsewhere herein, a synthetic polymer as described elsewhere herein, or an amphipathic peptide as described elsewhere herein.

[0071] In an embodiment of the invention, the nanodiscs are formed from a lipid-mimic polymer which is an amphiphilic block copolymer, for example which is a diblock copolymer AB or BA, or a triblock copolymer ABA or ABC. In a further embodiment, the nanodiscs are formed from an amphiphilic diblock copolymer AB or BA, or a triblock copolymer ABA. In a further embodiment of this aspect, the nanodiscs are formed from an amphiphilic diblock copolymer AB or BA. In such a di- or triblock copolymer, the B block represents the hydrophobic block and the A block (and C block, when present) represent the hydrophilic block(s).

[0072] A very wide range of hydrophilic polymers and hydrophobic polymers may form the blocks A and B (and C if present). Suitable hydrophobic polymers may include for example polysiloxanes, for example polydimethylsiloxane or polydiphenylsiloxane, perfluoropolyether, polystyrene, polyoxypropylene, polyvinylacetate, polyoxybutylene, polyisoprene, polybutadiene, polyvinylchloride, polyalkylacrylates, polyalkylmethacrylates, polyacrylonitrile, polypropylene, polytetrahyrofuran, polymethacrylates, polyacrylates, polysulfones, polyvinylethers, and polypropylene oxide), and copolymers thereof. The hydrophobic segment of a block copolymer as described herein contains a predominant amount of hydrophobic monomers. A hydrophobic monomer is a monomer that typically gives a homopolymer that is insoluble in water and can absorb less than 10% by weight of water.

[0073] Suitable hydrophobic monomers may include for example dimethylsiloxanes, Cl -Cl 8 alkyl and C3-C18 cycloalkyl acrylates and methacrylates, C3-C18 alkylacrylamides and -methacrylamides, acrylonitrile, methacrylonitrile, vinyl C1-C18 alkanoates, C2-C18 alkenes, C2-C18 haloalkenes, styrene, (lower alkyl) styrene, C4 C12 alkyl vinyl ethers, C2-C10 perfluoro-alkyl acrylates and methacrylates and correspondingly partially fluorinated acrylates and methacrylates, C3-C12 perfluoroalkylethylthiocarbonylaminoethyl acrylates and methacrylates, acryloxy- and methacryloxyalkylsiloxanes, N-vinylcarbazole, C1-C12 alkyl esters of maleic acid, fumaric acid, itaconic acid, mesaconic acid, vinyl acetate, vinyl propionate, vinyl - l - butyrate, vinyl valerate, chloroprene, vinyl chloride, vinylidene chloride, vinyltoluene, vinyl ethyl ether, perfluorohexyl ethylthiocarbonylaminoethyl methacrylate, isobornyl methacrylate, trifluoroethyl methacrylate, hexa-fluoroisopropyl methacrylate, hexafluorobutyl methacrylate, tristrimethylsilyloxysilylpropyl methacrylate, and 3- methacryloxypropylpentamethyldisiloxane.

[0074] The hydrophobic portion of the block copolymer may include a single type of polymer or more than one type of polymer, such as two or more of those mentioned above. Typically, the hydrophobic portion of a block copolymer of an embodiment of the present invention includes only a single type of polymer.

[0075] The mean molecular weight (g / mol) of one hydrophobic segment B within a block copolymer as described herein may range from about 400 to about 50,000, for example from about 400 to about 15,000, for example from about 500 to 5,000, for example from about 500 to about 3,000, for example from about 500 to about 1000. In an embodiment of this aspect, the B block is formed from a polysiloxane (for example polydimethylsiloxane or poly diphenylsiloxane), perfluoropoly ether, polystyrene, polyoxypropylene, polyvinylacetate, polyoxybutylene, polyisoprene, polybutadiene, polyvinylchloride, a polyalkylacrylate, a polyalkylmethacrylate, polyacrylonitrile, polypropylene, polytetrahyrofuran, a polymethacrylate, a polyacrylate, a polysulfone, a poly vinylether, or poly(propylene oxide). In an embodiment of this aspect, the B block is formed from a polysiloxane (for example polydimethylsiloxane or polydiphenylsiloxane) or polybutadiene, for example polydimethylsiloxane (PDMS) or polybutadiene. In an embodiment of this aspect, the B block is formed from polybutadiene.

[0076] In addition to the hydrophobic segment B, the amphiphilic copolymer includes at least 1, for example 2, segments A (or an A segment and a C segment) which include at least one hydrophilic polymer, for example a hydrophilic polymer selected from the group consisting of a polyoxazoline (for example a (poly)2-Ci-3alkyl-2-oxazoline (PAOXA)), polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, poly (meth) acrylic acid, polyethylene oxide-co- polypropyleneoxide block copolymers, poly (vinylether), poly(N,N- dimethylacrylamide), polyacrylic acid, polyacyl alkylene imine, polyhydroxyalkylacrylates such as hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, and polyols, and copolymers thereof. The hydrophilic segment of a block copolymer as described herein contains a predominant amount of hydrophilic monomers. A hydrophilic monomer is a monomer that typically gives a homopolymer that is soluble in water or can absorb at least 10% by weight of water.

[0077] Suitable hydrophilic monomers include hydroxy 1-substituted lower alkyl acrylates and methacrylates, acrylamide, methacrylamide, (lower alkyl) acrylamides and methacrylamides, N,N-dialkyl-acrylamides, ethoxylated acrylates and methacrylates, polyethyleneglycol-mono methacrylates and polyethyleneglycolmonomethylether methacrylates, hydroxyl-substituted (lower alkyl)acrylamides and methacrylamides, hydroxyl-substituted lower alkyl vinyl ethers, sodium vinylsulfonate, sodium styrenesulfonate, 2-acrylamido-2- methylpropanesulfonic acid, N-vinylpyrrole, N-vinyl-2-pyrrolidone, 2-vinyloxazoline,

[0078] 2-vinyl-4,4’-dialkyloxazolin-5-one, 2- and 4-vinylpyridine, vinylically unsaturated carboxylic acids having a total of 3 to 5 carbon atoms, am ino( lower alkyl)- (where the term amino also includes quaternary ammonium), monollowcr alkylamino)(lower alkyl) and di(lower alkylamino)(lower alkyl) acrylates and methacrylates, allyl alcohol.

[0079] 3 -trimethylammonium 2-hydroxypropylmethacrylate chloride (Blemer,QA, for example from Nippon Oil), dimethylaminoethyl methacrylate (DMAEMA), dimethylaminoethylmethacrylamide, glycerol methacrylate, and N-(l,l-dimethyl-3- oxobutyl)acrylamide.

[0080] Specific examples of hydrophilic monomers from which such polymers can be made are cyclic imino ethers (2-Ci-3alkyloxazoline, e.g. 2-methyloxazoline), vinyl ethers (for example methyl vinyl ether, ethyl vinyl ether and methoxy ethyl vinyl ether), cyclic ethers including epoxides, cyclic unsaturated ethers, N-substituted aziridines, P- lactones and P- lactams. Further suitable monomers include ketene acetals, vinyl acetals and phosphoranes. Suitable cyclic imino ethers include 2-oxazoline. If a 2- oxazoline having an alkenyl group in 2 position is used as hydrophilic monomer, a polymerizable unsaturated group is provided within segment A (in a side chain) of the amphiphilic segmented copolymer to serve as the polymerizable unsaturated group necessary for the final polymerization to obtain a polymeric product or as an additional polymerizable unsaturated group which offers the possibility of direct crosslinking in the preparation of the polymer.

[0081] The hydrophilic portion of the block copolymer may include a single type of polymer or more than one type of polymer, such as two or more of those mentioned above. Typically, in an embodiment, the hydrophilic portion of a block copolymer of the present invention includes only a single type of polymer.

[0082] The mean molecular weight (g / mol) of one hydrophilic segment A (or C if present) may be in the range from about 150 to about 50,000, e.g. in the range from about 200 to about 15,000, from about 250 to 5,000, from about 300 to about 1,000, or from about 300 to 500.

[0083] Synthesis of block copolymers by polymerisation is well known, and the length of the one or more segments which are to be copolymerized on the starting segment can be easily controlled by controlling the amount of monomer (hydrophilic or hydrophobic) which is added for the copolymerization, and / or by the addition of suitable chain-terminating capping agents. In this way the size of the segments and their ratio can easily be controlled.

[0084] In another embodiment of this aspect, the A block is formed from a polyoxazoline (for example a (poly)2-Ci-3alkyl-2-oxazoline (PAOXA)), polyethylene glycol, polyethylene oxide, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, poly(meth)acrylic acid, a polyethylene oxide-co-polypropyleneoxide block copolymer, poly (vinylether), poly(N,N-dimethylacrylamide), polyacrylic acid, polyacyl alkylene imine, a polyhydroxyalkylacrylate such as hydroxyethyl methacrylate, hydroxyethyl acrylate, hydroxypropyl acrylate, or a polyol. In a further embodiment of this aspect, the A block is formed from a polyoxazoline, for example a (poly)2-Ci-3alkyl-2- oxazoline (PAOXA)), for example poly 2-methyl-2-oxazoline (PMOXA).

[0085] In a further embodiment of this aspect, the A block is formed from (poly)2-Ci- 3alkyl-2-oxazoline (PAOXA) (for example PMOXA), and the B block is formed from a polymer selected from the group consisting of polybutadiene (PB) and poly(dimethylsiloxane) (PDMS). In another embodiment of this aspect, the A block is formed from PAOXA (for example PMOXA), and the B block is formed from PB.

[0086] The absolute and relative lengths of the hydrophilic and hydrophobic blocks are important in determining the suitability of the copolymers for forming nanodiscs (so called polymer hydrophobic ratio). Further, the length of the blocks should be such that the thickness of the nanodisc is broadly comparable with the length of the membrane protein so that the protein can be readily incorporated into the nanodisc without the channel becoming blocked. In an embodiment, the lengths of the blocks within the copolymers are chosen such that the thickness of the nanodisc is from about 4 to about 12 nm, for example about 5 to 10 nm, for example about 8 to 10 nm. The thickness may be measured by cryo-Transmission Electron Microscopy (cryoTEM). CryoTEM is a microscopy method wherein nanoparticles are deposited on 3 mm diameter copper or gold circular grids are flash frozen i.e., vitrified in liquid ethane and stored below - 180°C in liquid nitrogen. These grids under their frozen state are then transferred into a cryo holder, which is inserted into the cryo microscope. Under vacuum, the specimen is exposed to a highly collimated electron beam, which passes through the frozen vitreous ice, producing a transmission image on a digital camera below the column. The images are digitised and can be further processed using standard image processing algorithms.

[0087] The length of the hydrophobic block B is particularly important, and this should be no greater than 200 repeat units, for example 100 or fewer repeat units, for example 50 or fewer repeat units, for example 20 or fewer repeat units.

[0088] Therefore, in another embodiment of this aspect, the A block has 200 or fewer units and the B block has 200 or fewer units, for example the A block has 150 or fewer units and the B block has 150 or fewer units, for example the A block has 100 or fewer units and the B block has 100 or fewer units. For example, the A block has 2-75 units and the B block has 2-75 units, for example the A block has 2-50 units and the B block has 2-50 units, for example the A block has 3-40 units and the B block has 3-40 units. In an embodiment of this aspect, the A block has 2-25 units and the B block has 5-25 units. In an embodiment of this aspect, the A block has 2-20 units, or 2 to 10 units, for example 3 to 6 units. In an embodiment of this aspect, the B block has 5 to 50 units, for example 10 to 40 units. In an embodiment of this aspect, the A block has 2 to 8 units and the B block has 8 to 14 units.

[0089] The choice of end groups for block copolymers suitable for use in a membrane assembly as described herein may promote nanodisc formation or may provide functionality for onward reaction of the polymer. Suitable functional groups may be present following initial synthesis of the copolymer or may be introduced following the copolymer synthesis. If not present following initial synthesis, it is possible to introduce an appropriate end group by suitable reactions at the end of the relevant block (to produce functional end groups). For this purpose, the polymerization of the growing segment may be terminated after a suitable chain length is reached and the initiator group present at the chain end capped. For example, capping using water will result in an -OH end group, while capping with an appropriate amine will lead to an amine end group. Alternatively, hydroxyl end groups can be converted to primary amine via a Mitsunobu reaction (Kuo et al., Journal of Polymer Science: Part A: Polymer Chemistry, Vol. 46, 3108-3119 (2008); Park et al., Macromolecules 2004, 37, 6786- 6792). In a further alternative, amine end groups may be introduced using a method as described in US 3,758,636, the contents of which are incorporated by reference. Capping may also be carried out using any other desired terminator, and the required end group may be introduced using known chemistry. For example, termination may be carried out using KOH / MeOH or unsaturated groups at the end of the growing segment. The end group(s) may then be reacted using conventional chemistry to introduce the required groups.

[0090] It is not necessary that all the block copolymer molecules used in the present invention should have reactive end groups. The proportion of block copolymer molecules having reactive end groups is not critical, provided that there are sufficient groups to react with reactive groups either in a second population of nanodiscs and / or on the surface of the support. For example, at least 10%, for example at least 20%, for example at least 30 %, for example at least 40%, for example up to 60%, or up to 100%, of the block copolymer molecules used in the invention may have reactive end groups. Similarly, it is not required that only one type of reactive end group is present. For example, blends of block copolymers may be used, one containing one reactive end group, for example an end group including an -NH2 group, and the second containing a different reactive end group.

[0091] The end groups on any particular polymer molecule may be the same as each other, or they may be different, but preferably they are the same. For example, one end group may be a reactive end group, while the other end group may be a non-reactive group. The exact nature of the groups will depend on the desired reactivity of the block copolymers and vesicles formed from the block copolymers.

[0092] Suitable reactive groups include for example amine groups (reactive with for example carboxylic acid, activated carboxylic acid and / or azide groups), carboxylic acid, activated carboxylic acid and / or azide groups (reactive with for example amine groups), thiol groups (reactive with for example alkene groups), hydroxyl groups (reactive with for example amine and alkyl halide groups), methacrylate groups (reactive with for example thiol and alkene groups), and “click chemistry” groups (for example azide or alkyne groups, which are respectively reactive with alkyne and azide groups). A wide variety of amine-based end groups is available, and these may contain -NH2 (primary amine) and / or NH (secondary amine) groups. In an embodiment of this aspect of the invention, the A block of the block copolymer is a PAOXA comprising an end group selected from a carboxylic acid, an activated carboxylic acid, an alkyne, an amine, a hydroxyl, a thiol, a methacrylate, or an azide group. In an embodiment of this aspect of the invention, the A block of the block copolymer is a PAOXA comprising an amine end group, for example a primary and / or a secondary amine group. In an embodiment, the A block of the block copolymer is a PAOXA comprising a primary amine end group.

[0093] In an embodiment of the invention, the amphiphilic block copolymer comprises at least one hydrophilic block comprising poly(4-vinyl-N-methylpyridine iodide). In an embodiment of the invention, the amphiphilic block copolymer comprises at least one hydrophobic block comprising hydrogenated polybutadiene (HPBD). In an embodiment of the invention, the amphiphilic block copolymer comprises at least one hydrophilic block comprising poly(4-vinyl-N-methylpyridine iodide) and at least one hydrophobic block comprising hydrogenated polybutadiene (HPBD). In an embodiment of the invention, the amphiphilic block copolymer is the triblock copolymer HPBD-b-(poly(4-vinylpyridine)28)2.

[0094] In an embodiment of the invention, the amphiphilic block copolymer comprises at least one hydrophilic block comprising (poly)2-Ci-3alkyl-2-oxazoline. In an embodiment of the invention, the amphiphilic block copolymer comprises at least one hydrophobic block comprising polybutadiene. In an embodiment of the invention, the amphiphilic block copolymer comprises at least one hydrophilic block comprising (poly)2-Ci-3alkyl-2-oxazoline and at least one hydrophobic block comprising polybutadiene. In an embodiment of the invention, the amphiphilic block copolymer is a diblock copolymer AB in which (poly)2-Ci-3alkyl-2-oxazoline forms the A block and polybutadiene forms the B block. In an embodiment of the invention, the amphiphilic block copolymer is a diblock copolymer AB in which (poly)2-Ci-3alkyl-2-oxazoline forms the A block and polybutadiene forms the B block, and which has at least one end group at the end of a (poly)2-Ci-3alkyl-2-oxazoline block which is selected from carboxy, activated carboxy, amine, methacrylate, thiol, azide, and alkyne. belt molecules

[0095] As described above, the purpose of a stabilising belt molecule within a nanodisc is to hold the lipids / lipid-like molecules of the nanodisc together and to provide a hydrophobic surface on the inside of the belt facing the hydrophobic portions of the lipids / lipid-like molecules, and a hydrophilic surface on the outside of the belt facing the aqueous medium (i.e. the stabilising belt molecule is amphipathic). The stabilising belt molecule therefore also ensures that the nanodisc is soluble in aqueous solution. In an embodiment of the invention, the nanodiscs comprise at least one stabilising belt molecule. In an embodiment of the invention, a nanodisc as described herein comprises at least one stabilising belt molecule surrounding, e.g. covering, a hydrophobic surface of the lipid portion of the nanodisc. In an embodiment of the invention, the at least one stabilising belt molecule is selected from the group consisting of: (i) a membrane scaffold protein (MSP) or a derivative thereof; (ii) a synthetic belt polymer, i.e. an amphipathic non-peptide-based or non-protein-based polymer; and (iii) an amphipathic peptide.

[0096] In an embodiment of the invention, the at least one stabilising belt molecule is an amphipathic peptide, for example an amphipathic a-helical peptide, for example apolipoprotein Al-derived peptide 18A originally designed by Anantharamaiah et al.,

[0097] Journal of Biological Chemistry, v. 260(18), p. 10248 (1985) (DOI: dimer or polymer thereof, for example a dimer or polymer of peptide 18 A. The sequence of peptide 18A is

[0098] DWLKAFYDKVAEKLKEAF (SEQ ID NO: 1).

[0099] In an embodiment of the invention, the at least one stabilising belt molecule is an MSP, or a derivative thereof. MSPs can be truncated forms of human apolipoprotein Apo Al (usually some form of modification of the original Apo Al sequence is required to form homogeneous, stable and monodisperse nanodiscs with controlled size). Examples of various modified Apo Al sequences are provided in Denisov and Sligar, Chem. Rev., 2017, 117(6): 4669-4713. In an embodiment of the invention, the at least one stabilising belt molecule is an MSP selected from the group consisting of MSP1, MSP1D1, MSP1D2, MSP1E(1,2,3)D1, MSP2N(1,2,3)D1, MSPD1AH4,

[0100] MSP1D1AH5, MSP1D1AH4H5, MSP1D1AH4-H6, MSP1E1, MSP1E2, MSP1E3,

[0101] MSP1E3D1, MSPN1, MSP2N2 and MSP2N3 (for an explanation of the nomenclature, see Figure 4 and Table 1 of Denisov and Sligar, Chem. Rev., 2017, 117(6): 4669-4713). In an embodiment of the invention, the at least one stabilising belt molecule is an MSP selected from the group consisting of MSP ID 1 and MSP1D1 AH5. Examples of MSPs suitable for use in the present invention can be found in Denisov and Sligar, Chem. Rev., 2017, 117(6): 4669-4713; Hagn, F. et al., J. Am. Chem. Soc. (2013), 135:1919- 1925; Pandit A., et al., Biophys. J. (2011) 101:2507-2515 and Grinkova, Y.V., et al., Protein Engineering, Design & Selection (2010) 23(l l):843-848. The size of an MSP nanodisc can range between 7-17 nm. The size is determined by the length of the MSP which is used to form the nanodisc. Suitable MSPs can be obtained from Cube Biotech GmbH, Monheim, Germany.

[0102] In an embodiment of the invention, the at least one stabilising belt molecule is a synthetic polymer, i.e. the nanodisc is a synthetic nanodisc. As used herein, a synthetic polymer refers to a stabilising belt molecule which is amphipathic, but which is not a protein-based or a peptide-based polymer. In an embodiment of the invention, the at least one stabilising belt molecule is a synthetic amphipathic polymer. In an embodiment of the invention, the at least one stabilising belt molecule is a polyacrylic acid-based copolymer or a polymaleic acid-based copolymer. In an embodiment of the invention, the at least one stabilising belt molecule is a synthetic polymer selected from the group consisting of a poly(diisobutylene-aZt-maleic acid) (DIBMA) polymer, a poly(styrene-co-maleic acid) (SMA) copolymer, a poly(acrylic acid-co-styrene) copolymer (AASTY) and an amphipol, for example an amphipol which is capable of solubilising a membrane protein directly from a membrane, for example a polyacrylic acid-based copolymer having the structure of formulae V or VI as described elsewhere herein.

[0103] DIBMA is an alternating copolymer of maleic acid and diisobutylene (2,4,4- trimethylpent-l-ene) that is commercially available under the trade name Sokalan CP9 (BASF, Germany). In an embodiment, the at least one stabilising molecule is a poly(diisobutylene-aZt-maleic acid) (DIBMA) polymer. In an embodiment, the at least one stabilising belt molecule is a DIBMA polymer of Formula I:

[0104] Formula I wherein n is the number of moieties in the polymer. In an embodiment, n is about 40, for example n is from about 35 to about 45, for example about 36, about 37, about 38, about 39, about 40, about 41, about 42 or about 43. In an embodiment, the diisobutylene:maleic acid ratio is 1:1.

[0105] In an embodiment, the at least one stabilising belt molecule is a poly(styrene- co-maleic acid) (SMA) copolymer. In an embodiment, the at least one stabilising belt molecule is a SMA copolymer of Formula II:

[0106] Formula II wherein x:y is the ratio of maleic acid: styrene subunits and n is the repeating number of the two subunits in the copolymer.

[0107] In an embodiment, the maleic acid:styrene ratio is 1:2 or 1:3.

[0108] In an embodiment, x is 1 and y is 2.

[0109] In an embodiment, x is 1 and y is 3. A suitable SMA copolymer is available under the trade name SMA2000 (Cray Valley (TotalEnergies), Grand Junction, CO, USA).

[0110] In an embodiment, the at least one stabilising belt molecule is a poly(acrylic acid-co- styrene) copolymer (AASTY). In an embodiment, the at least one stabilising belt molecule is an AASTY copolymer of Formula III:

[0111] Formula III wherein, x and y are the molar percentages of each type of subunit; wherein x is from about 40 to about 60, y is from about 40 to about 60 and the sum of x and y equals 100.

[0112] Amphipol polymers

[0113] Amphipols were first described in Tribet et al., 1996, PNAS, vol. 93, p. 15047- 15050. They are a class of polymers which are designed to keep membrane proteins soluble in aqueous solution in the absence of detergent. Thus, in an embodiment of the invention, said membrane mimetic structures which comprise an amphiphilic molecule (e.g. an amphipol molecule) are essentially, or completely, free of detergent. A review of amphipols is provided in Popot et al. , Annu. Rev. Biophys. 2011. 40:379^408, which specifies that amphipols are short amphiphilic polymers (i.e. polymers which comprise both hydrophobic and hydrophilic moieties), which can keep membrane proteins water soluble in detergent-free aqueous solutions as small individual entities by adsorbing onto their hydrophobic surface. In this context, “short” is intended to mean amphipol molecules having e.g. 10-1000, 10-800, 10-600, 10-400, 10-300 or 10-250 monomer subunits / amphipol molecule. As will be apparent, the hydrophobic moieties of the amphipol adsorb to the hydrophobic surface of the membrane protein (e.g. the transmembrane domain of a channel protein) and the hydrophilic moieties of the amphipol interact with the aqueous environment, thus solubilising the membrane protein (e.g. the channel protein) in aqueous solution. As described elsewhere herein, the amphipol molecule or molecules form a ring structure around the (external) hydrophobic surface of the membrane protein (e.g. the transmembrane domain of a channel protein) thus solubilising the channel protein in aqueous solution. Amphipols present a distinctive feature compared to most other amphiphilic polymers: they can self-assemble into well-defined particles comprising a small number of amphipol molecules (e.g. 10 or fewer, 8 or fewer, 5 or fewer, e.g. 4 or 3), which resemble detergent micelles. This results from the favourable combination of: (i) a short backbone chain length and an appropriate number and size of hydrophobic segments (so that the equilibrium size of the hydrophobic core can be reached with the amount of hydrophobic groups carried by just a few chains, while avoiding the otherwise typical formation of large networks of interconnected polymers due to hydrophobic selfassociation); (ii) flexibility allowing a small radius of curvature of the interface with the solution, and (iii) a limited polydispersity in composition (to avoid large populations of chains with too few or too many hydrophobic groups compared to the optimum). Amphipols may be selected from homopolymers wherein each polymer subunit includes both hydrophobic and hydrophilic moieties, e.g. hydrophobic and hydrophilic pendant groups on the polymer backbone (with such amphipols being referred to as homopolymeric amphipols) or heteropolymeric copolymers wherein each polymer subunit includes a hydrophobic or a hydrophilic moiety, e.g. a hydrophilic or a hydrophobic pendant group on the polymer backbone (with such amphipols being referred to as heteropolymeric amphipols). The hydrophobic pendant groups can be selected from the group consisting of short alkyl side groups (e.g. C2-C15, C3-C15, C5-C15, C3-C12, C3-C8, C5-C12, C8-C12 or C6-C8), cycloalkyl (e.g. C5-C14, C5- C10, C6-C10, C5-C8 or C6-C8) and aryl side groups (e.g. C5-C14, C5-C10, C6-C10, C5-C8 or C6-C8). The hydrophilic pendant groups can be selected from the group consisting of carboxylate and sulfonate. In an embodiment, the hydrophilic and hydrophobic subunits in a heteropolymeric amphipol are arranged randomly along the polymer chain, for example there is a mixed arrangement of hydrophilic and hydrophobic subunits along the polymer chain (i.e. there are not blocks of hydrophobic and hydrophilic subunits). For example, the hydrophobic and hydrophilic subunits in a heteropolymeric amphipol may be arranged such that the hydrophobic subunits are separated by (e.g. one to four) hydrophilic subunits.

[0114] In an embodiment, the heteropolymeric amphipol polymer is a modified polyacrylic acid, a modified polyacrylamide or a modified copolymer of acrylic acid and acrylamide (i.e. a polymer comprising both acrylamide-derived and acrylic acid- derived subunits), wherein said modified polyacrylic acid comprises subunits which have been modified with a hydrophobic pendant group, wherein said modified polyacrylamide comprises acrylamide-derived subunits which have been modified with a hydrophobic pendant group and acrylamide-derived subunits which have been modified with a hydrophilic pendant group, and wherein said modified copolymer of acrylic acid and acrylamide comprises acrylamide-derived subunits which have been modified with a hydrophobic pendant group. Such amphipol polymers accordingly have amphiphilic character due to the presence of both hydrophobic and hydrophilic pendant groups on the polymer backbone (in the modified poly aery lie acid and the modified copolymer of acrylic acid and acrylamide, the carboxylate groups of the acrylic acid- derived subunits provide the hydrophilic pendant groups on the polymer backbone). In an embodiment, the hydrophobic pendant group is selected from the group consisting of an alkyl, a cycloalkyl and an aryl group. In an embodiment, the hydrophilic pendant group is selected from the group consisting of carboxylate and sulfonate. In an embodiment, the hydrophobic and the hydrophilic pendant groups are attached to the polymer backbone via an amide bond.

[0115] In an embodiment, the amphipol polymer comprises a copolymer according to formula IV:

[0116] Formula IV wherein,

[0117] Ri is carboxylate, or V-phosphorylcholinc-V’- ethylenedioxybis(ethyl)acrylamide;

[0118] R2 is a Ci-Ce alkylene, or is a bond;

[0119] R3 is a Ci-Ce alkyl, a C3-C9 cycloalkyl, a Ce-Cu aryl which is optionally substituted with a C1-C4 alkyl;

[0120] R4 is a Ci-Ce alkyl, or ethylsulfonate; wherein x, y and z are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85, y is from about 10 to about 85 and z is from 0 to about 60 and wherein the sum of x, y and z equals 100;

[0121] In an embodiment, the total number of subunits present in a copolymer of Formula IV is from about 10 to about 1000.

[0122] In another embodiment, Ri is carboxylate. In another embodiment, Ri is N- phosphorylcholine-V’-ethylenedioxybis(ethyl)acrylamide.

[0123] In another embodiment, R2 is a C1-C4 alkylene, or is a bond, for example R2 is a C1-C2 alkylene or is a bond, for example R2 is a Ci alkylene, a C2 alkylene, or is a bond.

[0124] In another embodiment, R3 is a C2-C6 alkyl, a Ce-Cs cycloalkyl or a Ce-Cs aryl, wherein any of said aryl groups may be optionally substituted with a C1-C2 alkyl, for example at the para position.

[0125] In another embodiment, R3 is a C2-C6 alkyl, for example R3 is a C4-C6 alkyl, for example R3 is a Ce alkyl, for example hexyl.

[0126] In another embodiment, R3 is a Ce-C8cycloalkyl, for example R3 is a Ce cycloalkyl, a C7 cycloalkyl or a C8cycloalkyl.

[0127] In another embodiment, R3 is a C6-C12 aryl, for example R3 is a Ce-Cio aryl or R3 is a C6-C8aryl, for example, R3 is a Ce aryl, a C7 aryl or a C8aryl, wherein any of said aryl groups may be optionally substituted with a C1-C2 alkyl, for example at the para position.

[0128] In another embodiment, R4 is a C1-C4 alkyl, for example, a C3 alkyl, for example isopropyl.

[0129] In another embodiment, x is from about 25 to about 75, for example x is from about 35 to about 60 or from about 50 to about 55, for example x is about 35, about 40, about 50 or about 75.

[0130] In another embodiment, y is from about 25 to about 75, for example y is from about 35 to about 60 or from about 50 to 55, for example y is about 25, about 30, about 50 or about 75. In another embodiment, z is from about 0 to about 50, for example z is from about 0 to about 40, or from about 0 to about 30, or from about 10 to about 40, or from about 15 to about 40, or from about 20 to about 50, or from about 30 to 45, for example z is 0, about 15, about 30 or about 40.

[0131] In another embodiment, x is from about 40 to about 60, y is from about 40 to about 60 and z is from about 10 to about 20.

[0132] In another embodiment, x is from about 40 to about 60, y is from about 40 to about 60 and z is 0.

[0133] In another embodiment, the total number of subunits present in a copolymer of Formula IV is from about 10 to 800, for example from about 10 to about 600, from about 10 to about 400, from about 10 to about 300, or from about 10 to about 250.

[0134] In another embodiment, Ri is carboxylate; R2 is a C1-C4 alkylene or is a bond; R3 is a Cs-Cs cycloalkyl, or R3 is a Cs-Cs aryl, wherein said aryl is optionally substituted with a C1-C2 alkyl, for example at the para position; R4 is a C2-C4 alkyl; x is from about 15 to about 75; y is from about 25 to about 85; z is from 0 to about 25; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0135] In another embodiment, Ri is carboxylate; R2 is a C1-C2 alkylene or is a bond; R3 is a Ce-Cs cycloalkyl, or R3 is a Ce-Cs aryl, wherein said aryl is optionally substituted with a C1-C2 alkyl, for example at the para position; R4 is a C3 alkyl; x is from about 30 to about 40; y is from about 45 to about 50; z is from about 10 to about 15; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0136] In another embodiment, Ri is carboxylate; R2 is a C1-C2 alkylene or is a bond; R3 is a Ce-Cs cycloalkyl or a Ce-Cs aryl, wherein said aryl is optionally substituted with a C1-C2 alkyl, for example at the para position; x is from about 15 to about 75, for example from about 40 to about 75; y is from about 25 to about 85, for example from about 25 to about 60; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0137] In another embodiment, Ri is carboxylate; R2 is a C1-C2 alkylene or is a bond; R3 is a Ce-Cs cycloalkyl; x is from about 40 to about 60; y is from about 40 to about 60; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 500. In another embodiment, Ri is carboxylate; R2 is a Ci-Ce alkylene or is a bond; R3 is a Ci-Ce alkyl; R4 is a C2-4 alkyl; x is from about 25 to about 85; y is from about 15 to about 35; z is from 0 to about 60; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0138] In another embodiment, Ri is A-phosphorylcholine-A7- ethylenedioxybis(ethyl)acrylamide; R2 is a Ci-Ce alkylene; R3 is a Ci-Ce alkyl; R4 is a C2-C4 alkyl; x is from about 35 to about 75; y is from about 20 to about 40; z is from 0 to about 50; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0139] In another embodiment, Ri is carboxylate; R2 is a Ci-Ce alkylene; R3 is a Ci-Ce alkyl; R4 is ethyl sulfonate; x is from about 25 to about 50; y is from about 20 to about 35; z is from about 25 to about 50; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0140] In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is a Ce cycloalkyl; x is about 50; y is about 50; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0141] In another embodiment, Ri is carboxylate; R2 is a bond; R3 is a Cs cycloalkyl; x is about 50; y is about 50; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0142] In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is hexyl; R4 is isopropyl; x is about 35; y is about 25; z is about 40; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0143] In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is hexyl; x is about 75; y is about 25; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0144] In another embodiment, Ri is A-phosphorylcholine-A7- ethylenedioxybis(ethyl)acrylamide; R2 is a C2 alkylene; R3 is hexyl; R4 is isopropyl; x is from about 35 to about 45; y is from about 25 to about 35; z is from about 25 to about 30; and the total number of subunits present in the copolymer is from about 10 to about 1000. In another embodiment, Ri is V-phosphorylcholine-V- ethylenedioxybis(ethyl)acrylamide; R2 is a C2 alkylene; R3 is hexyl; x is about 70; y is about 30; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0145] In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is hexyl; R4 is ethyl sulfonate; x is about 35; y is about 25; z is about 40; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0146] In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is hexyl; R4 is ethyl sulfonate; x is about 42; y is about 25; z is about 33; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0147] In an embodiment, the amphipol polymer comprises a copolymer according to formula IV: wherein,

[0148] RI is carboxylate, or V- pho sphorylcholine- N ’ - ethylenedioxybis(ethyl)acrylamide;

[0149] R2 is a Ci-Ce alkylene, or is a bond;

[0150] R3 is a Ci-Ce alkyl;

[0151] R4 is a Ci-Ce alkyl, or ethylsulfonate; wherein x, y and z are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85, y is from about 10 to about 85 and z is from 0 to about 60 and wherein the sum of x, y and z equals 100. In an embodiment, the total number of subunits present in a copolymer of Formula IV is from about 10 to about 1000.

[0152] In another embodiment, Ri is carboxylate. In another embodiment, Ri is N- phosphorylcholine-V’-ethylenedioxybis(ethyl)acrylamide.

[0153] In another embodiment, R2 is a C1-C4 alkylene, or is a bond, for example R2 is a C1-C2 alkylene or is a bond, for example R2 is a Ci alkylene, a C2 alkylene, or is a bond. In another embodiment, R2 is a C2-C6 alkylene.

[0154] In another embodiment, R3 is a C2-C6 alkyl, for example R3 is a C4-C6 alkyl, for example R3 is a Ce alkyl, for example hexyl.

[0155] In another embodiment, R2 and R3 together make a Ce-Cio alkyl, for example, a C7, a Cs or a C9 alkyl, for example octyl.

[0156] In another embodiment, R4 is a C1-C4 alkyl, for example, a C3 alkyl, for example isopropyl.

[0157] In another embodiment, x is from about 25 to about 75, for example x is from about 35 to about 60 or from about 50 to about 55, for example x is about 35, about 40, about 50 or about 75.

[0158] In another embodiment, y is from about 25 to about 75, for example y is from about 35 to about 60 or from about 50 to 55, for example y is about 25, about 30, about 50 or about 75.

[0159] In another embodiment, z is from about 0 to about 50, for example z is from about 0 to about 40, or from about 0 to about 30, or from about 10 to about 40, or from about 15 to about 40, or from about 20 to about 50, or from about 30 to 45, for example z is 0, about 15, about 30 or about 40.

[0160] In another embodiment, the total number of subunits present in the copolymer is from about 10 to 800, for example from about 10 to about 600, from about 10 to about 400, from about 10 to about 300, or from about 10 to about 250.

[0161] In another embodiment, Ri is carboxylate; R2 is a Ci-Ce alkylene or is a bond;

[0162] R3 is a Ci-Ce alkyl; R4 is a C2-4 alkyl; x is from about 25 to about 85; y is from about 15 to about 35; z is from 0 to about 60; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0163] In another embodiment, Ri is A-phosphorylcholine-A7- ethylenedioxybis(ethyl)acrylamide; R2 is a Ci-Ce alkylene; R3 is a Ci-Ce alkyl; R4 is a C2-C4 alkyl; x is from about 35 to about 75; y is from about 20 to about 40; z is from 0 to about 50; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0164] In another embodiment, Ri is carboxylate; R2 is a Ci-Ce alkylene; R3 is a Ci-Ce alkyl; R4 is ethyl sulfonate; x is from about 25 to about 50; y is from about 20 to about 35; z is from about 25 to about 50; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0165] In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is hexyl; R4 is isopropyl; x is about 35; y is about 25; z is about 40; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0166] In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is hexyl; x is about 75; y is about 25; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0167] In another embodiment, Ri is A-phosphorylcholine- 7- ethylenedioxybis(ethyl)acrylamide; R2 is a C2 alkylene; R3 is hexyl; R4 is isopropyl; x is from about 35 to about 45; y is from about 25 to about 35; z is from about 25 to about 30; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0168] In another embodiment, Ri is A-phosphorylcholine- 7- ethylenedioxybis(ethyl)acrylamide; R2 is a C2 alkylene; R3 is hexyl; x is about 70; y is about 30; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0169] In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is hexyl; R4 is ethyl sulfonate; x is about 35; y is about 25; z is about 40; and the total number of subunits present in the copolymer is from about 10 to about 1000. In another embodiment, Ri is carboxylate; R2 is a C2 alkylene; R3 is hexyl; R4 is ethyl sulfonate; x is about 42; y is about 25; z is about 33; and the total number of subunits present in the copolymer is from about 10 to about 1000.

[0170] In an embodiment, the amphipol polymer comprises a copolymer according to formula V:

[0171] Formula V wherein,

[0172] Ri is a Ci-Ce alkylene, or is a bond;

[0173] R2 is a C3-C9 cycloalkyl, or a C6-C14 aryl which is optionally substituted with a C1-C4 alkyl, for example at the para position;

[0174] R3 is a Ci-Ce alkyl; wherein x, y and z are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85, y is from about 10 to about 85 and z is from 0 to about 60 and wherein the sum of x, y and z equals 100;

[0175] In an embodiment, the total number of subunits present in a copolymer of Formula V is from about 10 to about 1000, for example about 10 to about 500.

[0176] In another embodiment, Ri is a C1-C4 alkylene, or is a bond, for example Ri is a C1-C2 alkylene or is a bond, for example Ri is a Ci alkylene, a C2 alkylene, or is a bond.

[0177] In another embodiment, R2 is a Ce-Cs cycloalkyl or a Ce-Cs aryl, wherein any of said aryl groups may be optionally substituted with a C1-C2 alkyl, for example at the para position. In another embodiment, R2 is a Ce-Cs cycloalkyl, for example R2 is a Ce cycloalkyl, a C7 cycloalkyl or a Cs cycloalkyl.

[0178] In another embodiment, R2 is a C6-C12 aryl, for example R2 is a Ce-Cio aryl or R2 is a Ce-Cs aryl, for example, R2 is a Ce aryl, a C7 aryl or a Cs aryl, wherein any of said aryl groups may be optionally substituted with a C1-C2 alkyl, for example at the para position.

[0179] In another embodiment, R3 is a C1-C4 alkyl, for example, a C3 alkyl, for example isopropyl.

[0180] In another embodiment, x is from about 25 to about 75, for example x is from about 35 to about 60 or from about 50 to about 55, for example x is about 35, about 40, about 50 or about 75.

[0181] In another embodiment, y is from about 25 to about 75, for example y is from about 35 to about 60 or from about 50 to 55, for example y is about 25, about 30, about 50 or about 75.

[0182] In another embodiment, z is from about 0 to about 50, for example z is from about 0 to about 40, or from about 0 to about 30, or from about 10 to about 40, or from about 15 to about 40, or from about 20 to about 50, or from about 30 to 45, for example z is 0, about 15, about 30 or about 40.

[0183] In another embodiment, x is from about 40 to about 60, y is from about 40 to about 60 and z is from about 10 to about 20.

[0184] In another embodiment, x is from about 40 to about 60, y is from about 40 to about 60 and z is 0.

[0185] In another embodiment, the total number of subunits present in the copolymer is from about 10 to about 400, from about 10 to about 300, or from about 10 to about 250.

[0186] In another embodiment, Ri is a C1-C4 alkylene or is a bond; R2 is a Cs-Cs cycloalkyl, or R2 is a Cs-Cs aryl, wherein said aryl is optionally substituted with a Ci- C2 alkyl, for example at the para position; R3 is a C2-4 alkyl; x is from about 15 to about 75; y is from about 25 to about 85; z is from 0 to about 25; and the total number of subunits present in the copolymer is from about 10 to about 500. In another embodiment, Ri is a C1-C2 alkylene or is a bond; R2 is a Ce-Cs cycloalkyl, or R2 is a Ce-Cs aryl, wherein said aryl is optionally substituted with a Ci- C2 alkyl, for example at the para position; R3 is a C3 alkyl; x is from about 30 to about 40; y is from about 45 to about 50; z is from about 10 to about 15; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0187] In another embodiment, Ri is a C1-C2 alkylene or is a bond; R2 is a Ce-Cs cycloalkyl or a Ce-Cs aryl, wherein said aryl is optionally substituted with a C1-C2 alkyl, for example at the para position; x is from about 15 to about 75, for example from about 40 to about 75; y is from about 25 to about 85, for example from about 25 to about 60; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0188] In another embodiment, Ri is a C1-C2 alkylene or is a bond; R2 is a Ce-Cs cycloalkyl; x is from about 40 to about 60; y is from about 40 to about 60; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0189] In another embodiment, Ri is a C2 alkylene; R2 is a Ce cycloalkyl; x is from about 40 to about 60; y is from about 40 to about 60; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0190] In another embodiment, Ri is a bond; R2 is a Cs cycloalkyl; x is about 50; y is about 50; z is 0; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0191] In an embodiment, the amphipol polymer comprises a copolymer according to formula VI:

[0192] Formula VI wherein,

[0193] Ri is a Ci-C6alkylene, or is a bond;

[0194] R2 is a C3-C9 cycloalkyl, or a C6-C14 aryl which is optionally substituted with a C1-C4 alkyl, for example at the para position; wherein x and y are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85 and y is from about 15 to about 85 wherein the sum of x and y equals 100.

[0195] In an embodiment, the total number of subunits present in a copolymer of Formula VI is from about 10 to about 500.

[0196] In another embodiment, Ri is a C1-C4 alkylene, or is a bond, for example Ri is a C1-C2 alkylene or is a bond, for example Ri is a Ci alkylene, a C2 alkylene, or is a bond.

[0197] In another embodiment, R2 is a Ce-Cs cycloalkyl or a Ce-Cs aryl, wherein any of said aryl groups may be optionally substituted with a C1-C2 alkyl, for example at the para position.

[0198] In another embodiment, R2 is a Ce-Cs cycloalkyl, for example R2 is a Ce cycloalkyl, a C7 cycloalkyl or a Cs cycloalkyl.

[0199] In another embodiment, R2 is a C6-C12 aryl, for example R2 is a Ce-Cio aryl or R2 is a Ce-Cs aryl, for example, R2 is a Ce aryl, a C7 aryl or a Cs aryl, wherein any of said aryl groups may be optionally substituted with a C1-C2 alkyl, for example at the para position.

[0200] In another embodiment, x is from about 25 to about 75, for example x is from about 35 to about 60 or from about 50 to about 55, for example x is about 35, about 40, about 50 or about 75.

[0201] In another embodiment, y is from about 25 to about 75, for example y is from about 35 to about 60 or from about 50 to about 55, for example y is about 25, about 30, about 50 or about 75. In another embodiment, x is from about 40 to about 60 and y is from about 40 to about 60.

[0202] In another embodiment, the total number of subunits present in the copolymer is from about 10 to about 400, from about 10 to about 300, or from about 10 to about 250.

[0203] In another embodiment, Ri is a C1-C2 alkylene or is a bond; R2 is a Ce-Cs cycloalkyl or a Ce-Cs aryl, wherein said aryl is optionally substituted with a C1-C2 alkyl, for example at the para position; x is from about 15 to about 75, for example from about 40 to about 75; y is from about 25 to about 85, for example from about 25 to about 60; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0204] In another embodiment, Ri is a C1-C2 alkylene or is a bond; R2 is a Ce-Cs cycloalkyl; x is from about 40 to about 60; y is from about 40 to about 60; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0205] In another embodiment, Ri is a C2 alkylene; R2 is a Ce cycloalkyl; x is from about 40 to about 60; y is from about 40 to about 60; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0206] In another embodiment, Ri is a bond; R2 is a Cs cycloalkyl; x is from about 40 to about 60; y is from about 40 to about 60; and the total number of subunits present in the copolymer is from about 10 to about 500.

[0207] An example of a homopolymeric amphipol is:

[0208] wherein n is from about 10 to about 120.

[0209] Membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein

[0210] In an embodiment of the invention, in said membrane mimetic structures which comprise an amphiphilic molecule, e.g. an amphipol molecule as described herein, said amphiphilic molecule contacts, e.g. directly contacts said membrane protein (e.g. a channel protein as described herein). In an embodiment of the invention, in said membrane mimetic structures which comprise an amphiphilic molecule, e.g. an amphipol molecule as described herein, said amphiphilic molecule adsorbs to the membrane (i.e. hydrophobic) surface of said membrane protein (e.g. the transmembrane domain of a channel protein as described herein). In an embodiment of the invention, in said membrane mimetic structures which comprise an amphipol polymer, wherein said amphipol polymer contacts said membrane protein (e.g. a channel protein), said amphipol polymer is a polymer according to formula IV, formula V, formula VI or formula VII, as described elsewhere herein. It is not necessary for the entirety of the amphiphilic molecule to contact the membrane (e.g. channel) protein and, in certain embodiments, only a portion of the amphiphilic molecule will contact the membrane (e.g. channel) protein.

[0211] As an alternative to nanodiscs, membrane proteins can be stabilised in an aqueous environment in membrane mimetic structures which comprise an amphiphilic molecule, e.g. an amphipol molecule as described herein, that contacts said membrane protein (e.g. channel protein). These structures can be formed by extracting the membrane protein (e.g. channel protein) from the membrane using a detergent, then replacing the detergent with the amphiphilic molecule, e.g. the amphipol. This replacement is achieved via a tertiary complex whereby both the detergent and amphipol can bind to the membrane protein (e.g. a channel protein), then the detergent is removed from the complex by any appropriate method, for example using polystyrene beads or by diluting the solution to below the critical micelle concentration of the detergent. As described elsewhere herein, the hydrophobic portions of the amphipol adsorb to the hydrophobic surfaces of the membrane protein (e.g. the transmembrane domain of a channel protein) and the hydrophilic portions interact with the aqueous environment, thus solubilising the membrane protein (e.g. the channel protein) in aqueous solution.

[0212] An alternative way of forming membrane mimetic structures which comprise an amphiphilic molecule, e.g. an amphipol molecule, that contacts said membrane protein (e.g. said channel protein) is by extracting the membrane protein using a denaturant such as urea or sodium dodecyl sulphate (SDS) before adding the amphiphilic molecule, e.g. the amphipol, which helps to refold protein, and removing the denaturant. As described elsewhere herein, the amphiphilic molecule, e.g. an amphipol molecule forms a ring structure around the hydrophobic surface of the membrane protein, e.g. the transmembrane domain of a channel protein.

[0213] The amount of amphipol that is required to stabilise membrane proteins (e.g. channel proteins) varies depending on factors such as the overall size of the protein and how accessible its hydrophobic regions are. The ratio of amphipol to protein can be in the region of 4-5 g of amphipol per 1 g of protein for small proteins that are found deeply embedded in the lipid membrane, but for larger proteins that have extended hydrophobic regions as little as about 0.1 g of amphipol per 1 g of protein can be sufficient.

[0214] As described elsewhere herein, individual amphipol molecules will selfassemble in solution into small, well-defined particles containing a small number of individual amphipol molecules. These particles contact the hydrophobic surfaces of the membrane protein (e.g. a channel protein) forming a layer of amphipol that does not form an extended corona as is typical of detergents. This layer can be about 1.5 to 2 nm thick. Unlike when stabilised in aqueous solution with a detergent, membrane proteins (e.g. channel proteins) that are stabilised in a membrane mimetic structure which comprise an amphipol which contacts said membrane / channel protein typically retain their native functionality. They also are typically more stable in such structures than when stabilised with a detergent, with a rate of denaturation which is decreased. It is hypothesised that a reason for this is that the membrane proteins (e.g. channel proteins) in the membrane mimetic structures which comprise an amphipol which contacts said membrane / channel protein are able to retain more of their native cofactors and other lipid molecules which help to regulate the structure and function of the membrane / channel protein.

[0215] In an embodiment of the invention, the plurality of membrane mimetic structures is a mixture of nanodiscs as defined herein and membrane mimetic structures which comprise an amphiphilic molecule, e.g. an amphipol polymer as described herein, which contacts said membrane / channel protein as defined herein.

[0216] As will be evident from the present disclosure, amphipol molecules as described herein may form membrane mimetic structures in two different ways. Certain amphipol molecules can be used to form nanodiscs. For example, some amphipol molecules (e.g. amphipol molecules according to Formula V or Formula VI) are capable of solubilising membrane proteins (e.g. channel proteins) direct from a membrane (e.g. a cell membrane). Such membrane mimetic structures are nanodiscs because when the protein is solubilised from the membrane, it is surrounded by a disc of lipid (i.e. native lipids from the cell membrane) and fall within the definition of nanodiscs as referred to herein. Alternatively, amphipol molecules can solubilise membrane proteins (e.g. channel proteins) by contacting (e.g. directly contacting) the external hydrophobic surface of the membrane / channel protein to form the membrane mimetic structure. As described above, such membrane mimetic structures are formed by first extracting the membrane protein (e.g. channel protein) from the membrane with a detergent or a denaturant and then replacing the detergent / denaturant with an amphipol. Such membrane mimetic structures do not have a disc of lipid surrounding the membrane (e.g. channel) protein which is characteristic of nanodiscs (although they may still contain small amounts of lipid which are bound to the membrane / channel protein). Such membrane mimetic structures are referred to herein as amphipol- stabilised membrane mimetic structures. Formation of a of membrane mimetic structures on the surface of the

[0217] In an embodiment of the invention, the plurality of membrane mimetic structures forms a layer, for example a planar layer or a monolayer, on the surface of the porous support. In an embodiment, the thickness of the layer is from about 3 to about 20 nm, for example from about 4 nm to about 10 nm, for example from about 5 nm to about 6 nm. In an embodiment of the invention, the plurality of membrane mimetic structures forms a continuous layer on the surface of the porous support. In an embodiment of the invention, the membrane mimetic structures which form a layer on the porous support are covalently linked to each other. In an embodiment of the invention, the membrane mimetic structures which form a layer on the porous support are covalently linked to the porous support. In an embodiment of the invention, the membrane mimetic structures which form a layer on the porous support are covalently linked to each other and to the porous support. Such covalent linking may be achieved by any suitable method and appropriate chemistries are well-known to a person of skill in the art. For example, suitable reactive groups such as click chemistry groups may be added to the membrane mimetic structures and / or the surface of the porous support. Alternatively, where the membrane mimetic structures comprise a protein (e.g. an MSP) or a peptide, a cysteine residue which is naturally present or which has been introduced by mutation can be used to form disulphide bonds between the membrane mimetic structures.

[0218] Synthetic peptide-stabilised membrane mimetic structures

[0219] In an embodiment of the invention, said membrane mimetic structures comprising a membrane protein (e.g. a channel protein) are stabilised using an amphiphilic molecule which is a synthetic peptide, i.e. the one or more amphiphilic molecules is / are a synthetic peptide. A synthetic peptide may also be referred to as A / e novo designed” and in the context of synthetic peptide-stabilised nanodiscs, they are engineered to have optimal hydrophilic and hydrophobic moieties. Such peptides stabilise membrane proteins (e.g. channel proteins) by mimicking their natural membrane environment, without the need for detergent, thus stabilising the membrane proteins (e.g. channel proteins) and retaining their activity in detergent-free aqueous solutions. Specific examples include the amphiphilic double-helical peptides NSP (nanodisc scaffold peptide) and NSPr (reversed nanodisc scaffold peptide). When using NSPr, such membrane mimetic structures may be referred to as “Peptidiscs” (see Carlson et al. eLife 2018; 7:e34085. DOI: https: / / doi.org / 10.7554 / eLife.3408). The method generally uses multiple copies of the peptide. The hydrophobic parts of the peptides wrap around the hydrophobic parts of the membrane (e.g. channel) protein thus shielding them from the aqueous solution, with the hydrophilic parts of the peptides interacting with the aqueous solution. NSP contains two repeats of the ApoAl -derived 18A peptide joined by a flexible linker proline in addition to two leucine residues which are substituted by phenylalanines to increase lipid affinity. NSPr is a reversed sequence form of NSP, has greater solubility in aqueous solutions and is capable of stabilising both a-helical and P-barrel membrane proteins into soluble, detergent-free Peptidiscs. The sequences of NSPr and NSP are as follows (linker proline highlighted in bold and substituted Phe residues highlighted with underlining):

[0220] NSPr - FAEKFKEAVKDYFAKFWDPAAEKLKEAVKDYFAKLWD (SEQ ID NO: 3)

[0221] NSP - DWLKAFYDKVAEKLKEAAPDWFKAFYDKVAEKFKEAF (SEQ ID NO: 2)

[0222] A detailed method for preparing synthetic peptide-stabilised membrane mimetic structures is provided in Carlson et al. However, briefly, in one method (termed the “on-column” method), the membrane protein (e.g. channel protein) is mixed with NSP or NSPr in a solution comprising a suitable detergent (e.g. dodecyl maltoside) and then the mixture is applied onto a size exclusion column equilibrated in a detergent-free buffer. The synthetic peptide-stabilised membrane mimetic structures can then be collected from the column. Alternatively, an “in-gel” method can be used. In this method, the peptide is mixed with the membrane protein (e.g. channel protein) in nonionic detergent solution and the resulting mixture loaded onto a native gel. Removal of the detergent occurs during electrophoresis when the protein-peptide mixture enters the detergent-free part of the gel. The target protein / peptide membrane mimetic structure migrates in a soluble form to its expected molecular weight position. A further method which saves on peptide consumption and minimises exposure of the target protein to detergent is called the “on-beads” reconstitution method, wherein affinity purification of the target protein and complexing with the peptide occur simultaneously. When the target protein is still bound to the beads, the peptide is added in excess, followed by detergent dilution and eventually elution in a detergent-free buffer. In an embodiment, the membrane mimetic structures of a membrane assembly as described herein are membrane mimetic structures (comprising a membrane protein such as a channel protein) stabilised using NSP or NSPr.

[0223] Saposin-stabilised membrane mimetic structures

[0224] Alternatively, in an embodiment of the invention, said membrane mimetic structures comprising a membrane protein (e.g. a channel protein) are stabilised using a saposin protein, for example Saposin A, B, C or D. In an embodiment, said membrane mimetic structures comprising a membrane protein (e.g. a channel protein) are stabilised using Saposin A, e.g. human Saposin A. Such saposin-stabilised membrane mimetic structures may also be referred to as “Salipro” structures (see Frauenfeld et al. 2016 Nature Methods 13:345-351).

[0225] Saposins (also referred to as sphingolipid activator proteins or SAPS) are small membrane- active proteins that normally facilitate the degradation of lipids in the lysosome. Since saposin proteins can form lipid complexes in the native environment, they can act as an amphiphilic scaffold protein which can reconstitute and purify membrane proteins (such as channel proteins) into soluble and functional lipid nanomembrane particles directly from cells. The technology can preserve the native structure and function of the membrane (e.g. channel) protein in stable, lipid-containing nanoparticles. The lipids in these nanoparticles are generally annular lipids, i.e. lipids that preferentially interact with the surface of membrane proteins (e.g. channel proteins) in biological cell membranes. Annular lipids thus form a ‘shell’ around the membrane protein, which stabilises the membrane protein while simultaneously being able to interact with saposin proteins surrounding the membrane protein (e.g. channel protein).

[0226] One of the key advantages of a saposin scaffold is that it is flexible and adapts to a variety of membrane proteins (e.g. channel proteins), of differing size and shape. The principle is that the saposin forms a ring structure around the hydrophobic surface of the lipid nanoparticle containing the membrane (e.g. channel) protein thus stabilising the lipid / protein complex in aqueous solution.

[0227] Detergent-stabilised membrane mimetic structures

[0228] In another embodiment of the invention, said membrane said membrane mimetic structures comprising a membrane protein (e.g. a channel protein) are stabilised using detergents as described elsewhere herein. Non-denaturing detergents are particularly suitable for preparing membrane assemblies comprising a membrane protein (e.g. a channel protein), since they are formulated to maintain the native structure and function of proteins during solubilization. This facilitates downstream applications of the solubilised channel proteins (e.g. filtration applications) which rely on the preserved state of the proteins. Suitable detergents that may be used in membrane mimetic structures in a membrane assembly of the invention may also include non-ionic and zwitterionic detergents, which are known to be less denaturing than ionic detergents, and are often used to solubilize membrane proteins and channel proteins where it is important to retain native protein function. In an embodiment, the detergent in a detergent-stabilised membrane mimetic structure is CHAPS. In another embodiment, the detergent in a detergent membrane mimetic structure is a Triton-X non-ionic detergent. In another embodiment, the detergent in a detergent-stabilised membrane mimetic structure is selected from the group consisting of: DDM (n- Dodecyl-P-D-Maltopyranoside), OG (n-Octyl-P-D-Glucopyranoside), LDAO (n- Dodecyl-N,N-Dimethylamine-N-Oxide), n-Octylpolyoxyethylene, Lauryl Maltose Neopentyl Glycol, and Decyl Maltose Neopentyl Glycol. For example, the detergent in a detergent-stabilised membrane mimetic structure is DDM.

[0229] Membrane / channel proteins

[0230] A membrane protein as referred to herein is a protein which is associated with the membrane of a cell or organelle inside the cell. They are divided into peripheral and integral proteins. Peripheral membrane proteins are temporally associated with the lipid bilayer but do not usually fully span the membrane. Integral membrane proteins are permanently embedded in the membrane, usually fully span the entire lipid bilayer, and contain hydrophobic alpha-helical or beta-barrel structures residing inside the membrane. They can be further subdivided into groups such as receptors or channel proteins depending on their cellular function. The majority of membrane proteins comprise both hydrophilic regions (which interact with the environment outside the membrane) and hydrophobic regions (which interact with the hydrophobic interior of the membrane lipid bilayer). Thus, most membrane proteins show an amphipathic character. This amphipathic character additionally produces a signature by which integral membrane proteins can often be identified. This is due to their primary structures containing 19-23 hydrophobic amino acids in their linear sequences, required to span the hydrophobic interior of a membrane. P-barrels with hydrophobic residues pointing to the outside of the barrel can also act as a good indicator of a membrane protein. The hydrophobic regions of a membrane protein as referred to herein (e.g. the transmembrane domain of a channel protein as referred to herein) also facilitate its stabilisation within the lipid / lipid-like environment of the membrane mimetic structures of the membrane assembly of the invention, as described hereinabove.

[0231] Membrane proteins perform many different biological functions, for example transport of substances across the membrane, enzymatic reactions, signal transduction, cell-cell recognition, intercellular junctions, and anchorage / attachment (for example to the cytoskeleton, extracellular matrix, or other proteins). Therefore, a membrane protein (e.g. a channel protein) comprised in at least a portion of the membrane mimetic structures of the membrane assembly of the invention may for example act as a transporter or channel (e.g. ATP-binding cassette transporters, membrane-bound ATPases, solute carrier transporters, ion channels or water channels) to allow water or other substances to pass through the membrane mimetic structures. Alternatively, a membrane protein comprised in at least a portion of the membrane mimetic structures of the membrane assembly of the invention may for example act as an enzyme, signalling molecule, receptor, or adhesive protein.

[0232] As mentioned above, there are two general classes of membrane proteins, peripheral and integral membrane proteins, either of which may be used in a membrane mimetic structure of the membrane assembly of the invention. Integral membrane proteins may be classified according to their position relative to the lipid bilayer as: (i) integral monotopic proteins; or (ii) transmembrane proteins. An integral monotopic protein as referred to herein is permanently embedded within the membrane, but only from one side. A transmembrane protein (e.g. a channel protein) as referred to herein is an integral membrane protein which spans the entire width of a membrane, in which some residues are exposed on each side of the membrane and a transmembrane domain spans the membrane.

[0233] Non-limiting examples of membrane proteins (including channel proteins) which may be used in the present invention are given in Table 1 below. Variants of such membrane proteins, including naturally or non-naturally occurring variants and orthologs or paralogs of such proteins may also be used.

[0234] In an embodiment, the membrane protein comprised in at least a portion of the membrane mimetic structures of the membrane assembly of the invention is an integral membrane protein, for example a transmembrane protein, such as a channel protein. In an embodiment, the transmembrane protein (e.g. a channel protein) is selected from the group consisting of: aquaporins and glyceroporins; beta-barrel transmembrane proteins; and alpha-helical transmembrane proteins. In an embodiment, the transmembrane protein (e.g. a channel protein) is an aquaporin, for example Aquaporin Z.

[0235] Aquaporins are biological cell transmembrane proteins whose function is to selectively transport water through a cell membrane (i.e. they are channel proteins as referred to herein); the transport channel of the protein is a two-way channel through which water can flow in either direction. They are expressed by many human and other animal cell types, and also by bacterial and plant cells. Any of the different members of the aquaporin family of proteins can be used in a membrane assembly of the present invention. Suitable aquaporins for use in the present invention include Aquaporin 4 (Aqp4) (e.g. Homo sapiens Aqp4, GenBank ID KAI4045862.1), Aquaporin 3 (Aqp3) (e.g. Homo sapiens aquaporin-3, GenBank ID: CAD38526.1), Aquaporin 1 (Aqpl) (e.g. Homo sapiens Aqpl, GenBank ID UQX14437.1 and, especially, Aquaporin Z (Aqp Z) (e.g. Rhodobacter sphaeroides (also known as Cereibacter sphaeroides) AqpZ (referred to herein as RsAqpZ), GenBank ID ABA78939.1; Escherichia coli AqpZ (referred to herein as EcAqpZ), GenBank ID BAA08441.1).

[0236] Aquaporins may exist in monomeric, dimeric, tetrameric and higher oligomeric forms, as well as mutated, conjugated and truncated versions of the primary sequence. Provided that the biological function of the aquaporin, i.e. the selective transport of water, is maintained, any of these may be used in a membrane assembly of the present invention. Therefore, in an embodiment of the invention, the membrane protein (e.g. a channel protein) comprised in at least a portion of the membrane mimetic structures of the membrane assembly of the invention as described herein is an aquaporin, for example Aquaporin Z, for example RsAqpZ or EcAqpZ.

[0237] Aquaglyceroporins as referred to herein form a subset of aquaporin proteins. The aquaglyceroporins encompass AQP3, AQP7, AQP9 and AQP10 and are integral membrane proteins which conduct water, glycerol and other small, uncharged solutes through the cell membrane. Therefore, in an embodiment of the invention, the membrane protein (e.g. a channel protein) comprised in at least a portion of the membrane mimetic structures of the membrane assembly of the invention as described herein is an aquaglyceroporin, for example AQP7 or AQP10.

[0238] In addition, any other membrane protein or channel protein having desirable transport properties may be used in the present invention. Variants of such membrane proteins, including naturally or non-naturally occurring variants and orthologs or paralogs of such proteins may be used. Such proteins include for example the proteins in Table 1 below. Table 1. Membrane and channel proteins (not all the membrane proteins referred to in the following table are channel proteins).

[0239] In an embodiment of the invention, a portion or substantially all the membrane proteins (e.g. channel proteins) present in the membrane assembly are oriented orthogonally to the surface of the porous support. For example, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% of the membrane proteins (e.g. channel proteins) present in the membrane assembly are oriented orthogonally to the surface of the porous support. In an embodiment of the invention, all (100%) of the of the membrane proteins (e.g. channel proteins) present in the membrane assembly are oriented orthogonally to the surface of the porous support. The orientation of the membrane proteins (e.g. channel proteins) in the membrane assembly relative to the surface of the porous support can be determined by the use of known microscopy techniques, for example atomic force microscopy (AFM).

[0240] A channel protein as referred to herein is a protein which allows the transport of a substance, e.g. a hydrophilic substance such as water, ions, or small molecules, across the membrane of a cell or an organelle inside the cell. Channel proteins form pores / channels across the membrane, e.g. hydrophilic pores / channels that, when open, allow the relevant substance(s) to travel across the membrane. Channel proteins may also be ‘gated’, meaning that part of the channel protein can move in response to a stimulus to open or close the pore / channel. Gated channels include mechanically-gated channels (where the stimulus is a mechanical force), voltage-gated channels (where the stimulus is a change in membrane potential), and ligand-gated channels (where the stimulus is the binding or removal of a ligand to a binding site on the channel protein).

[0241] In an embodiment, the channel protein is a transmembrane channel protein. As described herein, a transmembrane channel protein is a type of integral membrane protein spanning the entire width of the membrane in which it is inserted and forming a pore / channel (e.g. a hydrophilic pore) across the membrane.

[0242] In an embodiment, the membrane or the channel protein as referred to herein is selected from the group consisting of:

[0243] • Aquaporin AqpZ (Escherichia coli)

[0244] • Aquaporin AQPO (Ovis aries)

[0245] • Aquaporin AQP1 (red blood cell; Homo sapiens)

[0246] • Aquaporin AQP2 (kidney; Homo sapiens (expressed in Pichia pasloris))

[0247] • Aquaporin AQP4 (glial cell; Rattus norvegicus)

[0248] • Aquaporin AQP4 (Homo sapiens (expressed in Pichia pasloris))

[0249] • Aquaporin AQP5 (HsAQP5) (Homo sapiens)

[0250] • Aquaglyceroporin AQP7 (Homo sapiens (expressed in Komagataella pastoris) • Aquaglyceroporin AQP10 (Homo sapiens (expressed in 5. cerevisiae)

[0251] • Aquaporin AqpM (Methanothermobacter marburgensis)

[0252] • GlpF glycerol facilitator channel (Escherichia coli)

[0253] • Aquaglyceroporin AQP (Plasmodium falciparum)

[0254] • OmpX (Escherichia coli)

[0255] • PagP (Escherichia coli)

[0256] • OmpW (Escherichia coli)

[0257] • OmpT (Escherichia coli)

[0258] • EspP autotransporter beta domain (Escherichia coli)

[0259] • OmpLA (Escherichia coli)

[0260] • OmpG (Escherichia coli)

[0261] • OmpF (Escherichia coli)

[0262] • FhuA (Escherichia coli)

[0263] • T7 DNA ejectosome periplasmic tunnel (Escherichia phage T7)

[0264] • Aerolysin (Aeromonas hydrophila)

[0265] • Main porin MspA (Mycolicibacterium smegmatis)

[0266] • Alpha-hemolysin (Staphylococcus aureus)

[0267] In an embodiment, the channel protein is selected from the group consisting of: Aquaporin AqpZ (Escherichia coli), Aquaporin AQPO (Ovis aries), Aquaporin AQP1 (red blood cell; Homo sapiens), Aquaporin AQP2 (kidney; Homo sapiens (expressed in Pichia pastoris)), Aquaporin AQP4 (glial cell; Rattus norvegicus), Aquaporin AQP4 (Homo sapiens (expressed in Pichia pastoris)), Aquaporin AQP5 (HsAQP5) (Homo sapiens), Aquaglyceroporin AQP7 (Homo sapiens (expressed in Komagataella pastoris), Aquaglyceroporin AQP10 (Homo sapiens (expressed in 5. cerevisiae), Aquaporin AqpM (Methanothermobacter marburgensis), GlpF glycerol facilitator channel (Escherichia coli), Aquaglyceroporin AQP (Plasmodium falciparum), OmpX (Escherichia coli), OmpW (Escherichia coli), OmpT (Escherichia coli), OmpLA (Escherichia coli), OmpG (Escherichia coli), OmpF (Escherichia coli). For example, the channel protein is selected from Aquaporin AqpZ (GenBank ID U38664) or OmpG (GenBank ID P76045).

[0268] For the avoidance of doubt, the channel protein as referred to herein is not OmpA of E. coli or a homolog thereof, or the transmembrane domain thereof (tOmpA) or a homolog thereof. OmpA is a well-known transmembrane protein found in the outer membrane of the Gram-negative bacterium E. coli. It is an 8-stranded P-barrel, which is attached via a flexible linker to a periplasmic C-terminal domain that noncovalently binds the E. coli cell wall. However, OmpA’s exact role in the outer membrane has until very recently remained poorly-understood, despite several decades of research, and despite its known association with processes such as virulence, adhesion, and maintaining the integrity of the outer membrane. More recent research indicates that OmpA plays a structural role in the Gram-negative outer membrane, by ordering the immobile lattice of other outer membrane proteins and directly coupling this lattice with the underlying cell wall; see Benn et al. 2024, PNAS, 121 (50), e2416426121.

[0269] In an embodiment, the channel protein is selected from the group consisting of: aquaporins and glyceroporins; beta-barrel channel proteins; and alpha-helical channel proteins, for example where the channel protein is an aquaporin, for example Aquaporin Z.

[0270] Porous support

[0271] The porous support may be made of any suitable microporous material. It may for example be based on a conventional membrane support, as used in reverse osmosis or ultrafiltration membranes.

[0272] In an embodiment of the invention, the porous support comprises a polymer selected from the group consisting of polysulfone (PS), polyethersulfone (PES), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), polyimide, poly(ether imide) (PEI), poly aery lie acid (PAA), polyamide (for example aromatic polyamide), polycarbonate (PC), polyethylene (PE), polypropylene (PP), poly(phthalazinone ether sulfone ketone) (PPESK), poly etheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), poly(vinyl butyral), polyvinyl alcohol (PVA), poly(2,6-dimethyl-l,4-phenylene oxide), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), polypiperazine, polybenzimidazoline, polyols (for example polyphenol), polyolefins, cellulose acetates (for example cellulose triacetate), cellulose nitrates, cellulose esters, regenerated cellulose, and cellulose.

[0273] In an embodiment of the invention, the porous support comprises a polymer selected from the group consisting of polysulfone (PS), polyethersulfone (PES), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), polyimide, poly(ether imide) (PEI), polyacrylic acid (PAA), polyamide, polycarbonate (PC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polypiperazine, cellulose acetates, cellulose nitrates, and cellulose esters.

[0274] In an embodiment of the invention, the porous support comprises a polymer selected from the group consisting of polysulfone (PS), polyethersulfone (PES), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), polyethylene (PE), polypropylene (PP) and polypiperazine. In an embodiment of the invention, the porous support comprises polysulfone (PS), polyethersulfone (PES) or polypiperazine, for example polysulfone (PS).

[0275] Additional features of the membrane

[0276] In an embodiment of the first and third aspects of the invention, the membrane mimetic structures are adsorbed to, covalently attached to (e.g. covalently cross-linked to) or attached via charge -based interactions to the surface of the porous support. In an embodiment, the membrane mimetic structures are covalently attached to (e.g. covalently cross-linked) the surface of the porous support. Such covalent attachment may be achieved by conjugating suitable reactive groups on the membrane mimetic structures to suitable reactive groups on the surface of the porous support. Such conjugation reactions are well-known to a person of skill in the art.

[0277] In an embodiment of the first and third aspects of the invention, the membrane assembly further comprises a coating such as a protective coating. Without being bound by theory, the present inventors hypothesise that such a coating has the advantage of further stabilising the membrane mimetic structures and / or the membrane proteins within the membrane assembly. For example, the coating may be on (e.g. overlayered on) the membrane mimetic structures, such that the membrane mimetic structures (e.g. nanodiscs) are enclosed between the coating and the porous support, for example between the surface of the coating facing the environment external to the membrane assembly and the surface of the porous support facing the coating. For example, the coating is on (e.g. overlayered on) the membrane mimetic structures and is on (e.g. overlayered on) any areas of the porous support surface that are not covered by membrane mimetic structures, such that the coating both surrounds the membrane mimetic structures and completely covers the surface of the membrane mimetic structures exposed to an environment which is external to the membrane assembly. Alternatively, the coating may be on (e.g. overlayered on) the surface of the porous support facing the membrane mimetic structures (e.g. nanodiscs), such that the coating surrounds the membrane mimetic structures but does not fully enclose them (i.e. the coating is on the areas of the porous support surface that are not covered by membrane mimetic structures, and at least partially encapsulates the membrane mimetic structures, but at least a part of the membrane mimetic structures’ surface is exposed to the environment external to the membrane assembly). Without being bound by theory, the present inventors hypothesise that such a coating, for example a polyamide coating, has the advantage of contributing to the salt rejection of membrane assemblies according to the invention. The coating may also help to protect the membrane mimetic structures.

[0278] In an embodiment, the coating comprises a polyamide. Such a polyamide coating may be prepared via any suitable technique known in the art, such as interfacial polymerisation, which is used to produce thin film composite (TFC) membranes. Interfacial polymerisation involves first treating a surface, e.g. the surface of the porous support of a membrane assembly as described herein and / or the surface of the plurality of membrane mimetic structures which are on said porous support) with an aqueous, alkaline solution of amine monomers, and subsequently exposing the treated support surface with a solution of acyl halide (e.g. acyl chloride) monomers in an organic solvent. When the monomers reach the interface between the two solutions, they polymerise, forming a thin polyamide film. Interfacial polymerisation is described for example in Seah et al. Polymers 2020, 12(12), 2817; In an embodiment, the coating comprises a polyamide produced by piperazine / trimesoyl chloride polymerisation. In an embodiment, the coating comprises a thin film polyamide composite, for example a thin film polyamide composite produced by m-phenylenediamine / trimesoyl chloride (MPD / TMC) polymerisation.

[0279] In an embodiment, amine groups may also be present on the membrane mimetic structures as described herein and / or on the membrane protein comprised within the membrane mimetic structures. When interfacial polymerisation is used to produce a coating as described above, these further amine groups may also participate in the interfacial polymerisation reaction, leading to the formation of chemical cross-linking involving the membrane mimetic structures. For example, in membrane mimetic structures comprising a membrane protein which is an aquaporin, there are typically around 27 lysine residues per aquaporin monomer, some of which will be exposed to solvent; in membrane mimetic structures which are nanodiscs comprising a membrane scaffold protein (MSP), there are typically around 19 lysine residues per MSP molecule. Therefore, in an embodiment of this aspect, the membrane mimetic structures as described herein are chemically cross-linked within the coating, e.g. a polyamide matrix as described above. The membrane mimetic structures may be cross-linked laterally, i.e. to each other, and / or they may be cross-linked to the polyamide matrix.

[0280] The support in a membrane assembly disclosed herein may be cast onto a backing, for example a fabric layer, typically nonwoven polyester or polypropylene, although any form of backing may be used. In an embodiment of this invention, the membrane assembly disclosed herein further comprises a backing, for example a nonwoven layer, e.g. a non-woven polyester backing fabric, wherein the backing is on the opposite surface of the porous support from the plurality of membrane mimetic structures, i.e. the porous support is located between the plurality of membrane mimetic structures and the backing.

[0281] Methods of making a membrane assembly

[0282] In a further aspect, the present invention also provides a method of making a membrane assembly as described herein. In an embodiment, the invention provides method of making a membrane assembly, as described herein, which comprises the steps of: a. providing a porous support; and b. applying an aqueous solution to the surface of the porous support, the solution comprising a plurality of membrane mimetic structures as described herein; thereby forming the membrane assembly.

[0283] For example, in an embodiment of this aspect, the present invention provides a method of making a membrane assembly comprising: a) a porous support; and b) a plurality of membrane mimetic structures on a surface of the support, wherein at least a portion of said membrane mimetic structures each comprise a membrane protein; wherein the membrane mimetic structures are selected from the group consisting of: (i) nanodiscs; (ii) membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein, wherein said amphiphilic polymer is not a lipid or an amphiphilic block copolymer and (iii) a mixture of (i) and (ii); and wherein said method comprises the steps of: a) providing the porous support; b) applying an aqueous solution of the membrane mimetic structures to a surface of the porous support; thereby forming the membrane assembly.

[0284] In a further example, the present invention provides a method of making a membrane assembly comprising: a) a porous support; and b) a plurality of membrane mimetic structures on a surface of the support, wherein the membrane mimetic structures comprise: (i) a channel protein which comprises a transmembrane domain having a hydrophobic surface, and (ii) one or more amphiphilic molecules; and wherein the one or more amphiphilic molecules form a ring structure around the hydrophobic surface of said transmembrane domain of the channel protein; wherein said method comprises the steps of: a) providing the porous support; b) applying an aqueous solution of the membrane mimetic structures to a surface of the porous support; thereby forming the membrane assembly.

[0285] For the avoidance of doubt, the features of a membrane assembly according to the first and third aspects of the invention (including the porous support and the membrane mimetic structures as well as the materials from which they are made and methods of their production) are equally applicable to the method of making a membrane assembly according to the second and fourth aspects of the invention, and vice versa.

[0286] Membrane casting

[0287] The porous support used in a method of making a membrane assembly according to the invention may be made of any suitable microporous material. It may for example be based upon a conventional membrane support, as used in reverse osmosis or ultrafiltration membranes. As described above, the features of the porous support of the membrane assembly according to the first aspect of the invention are equally applicable to the method according to the second aspect of the invention.

[0288] The porous support used in a method of making a membrane assembly according to the invention may be fabricated using any known casting technique. Casting is the precipitation of a membrane-forming polymer from a casting solution (also known as the casting dope), which is a mixture of the polymer, together with any additives, in a solvent. Non-limiting examples of casting techniques include:

[0289] • Immersion precipitation / non- solvent induced phase separation process - where polymer and additives are dissolved in the solvent, and subsequently immersed into a non-solvent medium, thus forming a membrane. The most common non-solvent medium used is water, but other liquids for example alcohols, may also be used.

[0290] • Thermal precipitation - where polymer is dissolved in the solvent at a given temperature, cast, and allowed to cool to form a precipitated membrane.

[0291] • Evaporation induced precipitation - where polymer and additives are dissolved in the solvent and volatiles are allowed to evaporate after the solution is cast onto a support or running belt.

[0292] • Electrospinning.

[0293] • Stretching.

[0294] • Track etching.

[0295] • Interfacial polymerization.

[0296] A membrane-forming polymer in a membrane assembly disclosed herein may be cast onto a backing, for example a fabric layer, typically nonwoven polyester or polypropylene, although any form of backing may be used. Therefore, in an embodiment of this aspect, the method for making a membrane assembly according to the invention further comprises a step of casting a membrane-forming polymer onto a backing, for example a non-woven substrate, for example a non-woven polyester backing fabric, prior to method step (a) as described hereinabove. In another embodiment of this aspect, the casting step prior to method step (a) as described hereinabove comprises casting a membrane-forming polymer on a non-woven substrate via a non- solvent induced phase separation process.

[0297] A casting process used to prepare the porous support used in method step (a) as described hereinabove can achieve membranes in various configurations, for example flat-sheet membranes (for spiral wound elements), made using a doctor blade or a diecoater, involving extrusion of polymer solution through extrusion knives; hollowfibre membranes (for hollow-fibre elements) where viscous dope is pushed through an extrusion nozzle into quenching solution / solutions on the outside and inside of the fibre; or tubular membranes.

[0298] The porous support in step (a) of a method of making a membrane assembly as disclosed herein may comprise any suitable membrane-forming polymer, as described in detail above in the context of the first aspect of the invention. In an embodiment of the second aspect of the invention, the porous support in method step (a) comprises a polymer selected from the group consisting of polysulfone (PS), polyethersulfone (PES), polyacrylonitrile (PAN), polyvinylidene fluoride (PVDF), polyimide, poly(ether imide) (PEI), polyacrylic acid (PAA)), polyamide (for example aromatic polyamide), polycarbonate (PC), polyethylene (PE), polypropylene (PP), poly(phthalazinone ether sulfone ketone) (PPESK), poly etheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), poly(vinyl butyral), polyvinyl alcohol (PVA), poly(2,6-dimethyl-l,4-phenylene oxide), poly(methyl methacrylate) (PMMA), polydimethylsiloxane (PDMS), polypiperazine, polybenzimidazoline, polyols (for example polyphenol), polyolefins, cellulose acetates (for example cellulose triacetate), cellulose nitrates, cellulose esters, regenerated cellulose, and cellulose.

[0299] Amine and acyl chloride solutions

[0300] As described above in the context of the first and third aspects of the invention, treatment of a surface (e.g. the surface of the porous support in method step a) with an aqueous solution of amine monomers, followed by exposing the treated surface with a solution of acyl chloride monomers in an organic solvent, will form a polyamide film through interfacial polymerisation. Such a polyamide film can function as a coating (e.g. a protective coating), which may for example be arranged such that the membrane mimetic structures are located between the coating and the porous support. Therefore, in an embodiment, the method of making a membrane assembly according to the second aspect or fourth aspect of the invention further comprises between steps a) and b) the step of:

[0301] A. applying an aqueous solution comprising an amine to a surface of the porous support; and further comprises after step b) the step of:

[0302] B. applying a solution comprising an acyl halide and a non-polar solvent to the surface of the support comprising the plurality of membrane mimetic structures, thus forming a polyamide via interfacial polymerisation. Any suitable combination of amine (in step A), acyl halide and non-polar solvent (both in step B), together with any other suitable additives, may be used. In an embodiment of this aspect, the aqueous solution in step A comprises a diamine, for example a non-aromatic cyclic diamine such as piperazine, or for example an aromatic diamine such as m-phenylenediamine (MPD). For example, the aqueous solution in step A comprises piperazine or m-phenylenediamine (MPD), for example piperazine. In an embodiment of this aspect, the acyl halide in step B is an acyl chloride, for example trimesoyl chloride (TMC). Any suitable non-polar solvent may be used in step B, for example cyclohexane, hexane, heptane, or a mixture thereof, for example a mixture of cyclohexane, hexane and heptane. In an embodiment, the non-polar solvent in step B is hexane.

[0303] In an embodiment of this aspect, the amine in step A is piperazine; and the solution in B comprises trimesoyl chloride (TMC). In an embodiment of this aspect, in order to increase the efficiency of the interfacial polymerisation reaction, the aqueous solution in step A further comprises the additives triethylamine (TEA) and camphor sulfonic acid (CSA) in addition to piperazine. Without wishing to be bound by theory, the present inventors hypothesise that use of piperazine-based interfacial polymerisation (e.g. to produce a coating comprising a polyamide made from piperazine and TMC) produces a membrane assembly comprising a coating which is well-suited for applications such as nanofiltration.

[0304] In another embodiment of this aspect, the amine in step A is m- phenylenediamine and the solution in step B comprises trimesoyl chloride (TMC). Without wishing to be bound by theory, the present inventors hypothesise that use of MPD / TMC-based interfacial polymerisation (e.g. to produce a thin film polyamide composite coating according to the first aspect of the invention) produces a membrane assembly with a higher degree of cross-linking in the polyamide coating, which improves salt rejection of the finished membrane assembly.

[0305] Cross-linking of membrane mimetic structures

[0306] In addition to the amine groups present in the aqueous solution comprising an amine in step A, further amine groups may also be present on the membrane mimetic structures as described herein. These further amine groups may also participate in the formation of the polyamide in step B, leading to the formation of chemical cross-linking (e.g. covalent cross-linking) involving the membrane mimetic structures. For example, in membrane mimetic structures comprising a membrane protein which is an aquaporin, there are typically around 27 lysine residues per aquaporin monomer; in membrane mimetic structures which are nanodiscs comprising a membrane scaffold protein (MSP), there are typically around 19 lysine residues per MSP molecule. Therefore, in an embodiment of this aspect, the plurality of membrane mimetic structures themselves comprise amine groups and can participate in the formation of the polyamide in step B. For example, step B involves cross-linking (e.g. covalent cross -linking) of the membrane mimetic structures as described herein within the polyamide matrix formed by the reaction between the amine in step A and the acyl chloride in step B. The membrane mimetic structures may be cross-linked (e.g. covalently cross-linked) laterally, i.e. to each other, and / or they may be cross-linked to the polyamide matrix.

[0307] As described above in the context of the first and third aspects of the invention, the plurality of membrane mimetic structures in step b) may be adsorbed to, covalently attached to (e.g. covalently cross-linked to) or attached via charge -based interactions to the surface of the porous support, for example covalently attached to (e.g. covalently cross-linked) the surface of the porous support. Therefore, in an embodiment of the second aspect, step b) further comprises forming attachments between the membrane mimetic structures to the surface of the porous support, for example via adsorption, covalent attachment (e.g. covalent cross -linking) or charge-based interactions.

[0308] Uses of the membrane assembly and methods of filtration

[0309] The membrane assemblies disclosed herein find use in filtration applications (for example high-resolution selective separation) requiring the use of a semipermeable membrane. A solution or liquid may be passed through a membrane assembly disclosed herein, thereby separating solutes, colloids, and / or fine particulates from the solution. Suitable filtration applications therefore include but are not limited to microfiltration, ultrafiltration, nanofiltration, reverse osmosis and engineered osmosis applications. Such filtration applications are useful in (for example) water treatment (including drinking water purification, waste water treatment and desalination) and other industrial processes such as manufacturing.

[0310] The present inventors have found that the membrane assemblies according to the invention, when used for filtration of liquids, perform comparably with commercially available filtration membranes and show surprising enhancements of both flux (permeability) and selectivity (rejection), effectively overcoming the tradeoff between permeability and selectivity. Membrane assemblies made according to the invention also have surprisingly good robustness (e.g. when provided with a protective polyamide layer) when exposed to denaturing conditions (e.g. heat), which is beneficial in real- world filtration applications where environmental conditions may be difficult to control (e.g. desalination of water in hot climates).

[0311] Therefore, in a further aspect, the present invention also provides a use of a membrane assembly as described herein. In an embodiment, the invention provides a use of a membrane assembly as described herein in a filtration application. In an embodiment, the filtration application is selected from the group consisting of microfiltration, ultrafiltration, nanofiltration, reverse osmosis, and engineered osmosis).

[0312] In a further aspect, the present invention also provides a filtration method which uses a membrane assembly as described herein. In an embodiment, the invention provides a filtration method comprising the following steps: a. providing a membrane assembly as described herein; b. filtering a liquid or solution using the membrane assembly, for example filtering a liquid through the membrane assembly.

[0313] In the context of the present invention, a ‘solution’ refers to a liquid containing more than one substance, in which one or more substances (solutes) are uniformly distributed within another substance (solvent), which is a liquid. A ‘suspension’ is a heterogeneous mixture of a liquid and solid particles that are large enough for sedimentation (i.e. the particles will settle out of the suspension over time). A ‘colloid’ is a homogeneous mixture containing insoluble particles (‘colloidal particles’) that remain uniformly dispersed throughout a second substance, e.g. a liquid. Unlike the particles in a suspension, colloidal particles do not settle.

[0314] Therefore, in another embodiment, the invention provides a filtration method comprising the following steps: a. providing a membrane assembly as described herein; b. passing a solution, suspension or colloid through the membrane assembly; thereby separating solutes, colloidal particles, and / or other fine particulates from the solution. Whilst the present invention has been described and illustrated with reference to particular embodiments, it will be appreciated by those of ordinary skill in the art that the invention lends itself to many different variations not specifically illustrated herein. By way of example only, certain possible variations will now be described.

[0315] EXAMPLES

[0316] Example 1. Expression, purification and quality control of Rs-AqpZ and membrane scaffold proteins

[0317] This example describes the lab-scale process for producing and isolating Rhodobacter sphaeroides Aquaporin Z protein (RsAqpZ) and membrane scaffold proteins.

[0318] 1,1 Expression and Purification of Rs-AqpZ for incorporation in liposomes and polymeric vesicles

[0319] Both unlabelled and fluorescently tagged tetrameric aquaporin from Rhodobacter sphaeroides species (Rs-AqpZ) (including a GFP-tagged version (SEQ ID NO. 1) as well as a transport ‘dead’ mutant, R188A-RsAqpZ (SEQ ID NO.6)) were recombinantly expressed in E. coli using the pET28b(+) system under ampicillin resistance and T7 promoter control for expression using ImM IPTG at mid-log. Schematic diagrams of the protein expression constructs used to express and purify the RsAqpZ proteins and MSPs are shown in Figure 1A. The growth medium was Luria broth (LB) and the culture size used was 24L. Plasmids containing the desired aquaporin insert were transformed into a house keeping cell line (typically DH5a) for plasmid storage and amplification. 5 ml cultures of the housekeeping cell line containing the plasmid were then used for plasmid extraction using the Qiagen miniprep kit. The 1.5 mL centrifuge-based recovery method yielded anywhere between 250-500 ng / pl of plasmid DNA.

[0320] Once the plasmids were amplified and recovered, they were incorporated into the host strain BL21ZDE3 for overexpression under the control of a T7 promoter which commences expression upon the addition of the inducer IPTG. A single colony from an agar plate containing the transformed host strain was used to start a daytime culture in LB media with 100 pg / mL of Ampicillin present. This daytime culture was seeded into a 120 mL overnight culture to grow overnight. The next day, the overnight culture was used to inoculate 12 L growth media containing the 2XYT media (twice the amount of Yeast extract and tryptone compared to typical LB media). This growth media is supplemented with 100 pg / mL Ampicillin. The growth was performed in a 15 L Applikon Bioreactor at 37 °C for about 3-4 hours while the optical density (OD600) was measured every 30 minutes. When the OD600 reached 0.4, the circulating water bath which heats the bioreactor heating jacket was reduced to 20°C, with continuous monitoring of the OD.

[0321] At an OD600 of 0.75, the culture was induced with 1 mM IPTG to commence expression. After 16 hours of expression, the bacterial cells were spun down at 6000 rpm and the cell pellets were frozen at -80°C until further use. When required, the frozen pellets were removed and slowly allowed to thaw on ice. After thawing on ice, the cells were allowed to resuspend in lysis buffer in the presence of protease inhibitors (cOmplete™ Protease Inhibitor Cocktail 1 tablet + 1 mM PMSF) and enzymes Dnase and Rnase. The fully resuspended cells were then placed in a glass beaker surrounded by ice and sonicated for 30 minutes. The resulting lysate was centrifuged at 9300 x g, 4°C for 30 minutes to remove broken cell debris. The supernatant was further centrifuged using ultracentrifuge at 38000 rpm at 4°C for 1 hour. The resulting membranes were further broken apart by gentle stirring in solubilization buffer containing n-Octyl-P-D-Glucopyranoside (OG) for solubilization and removal of host lipids attached to the proteins overnight. The resulting solubilized protein was purified using 6x-Histidine-dependent Cobalt affinity chromatography or a Streptavidin resin (streptactin) affinity column. The bound protein was eluted using imidazole or Biotin. Imidazole was used when a cobalt resin (TALON) was used to bind 6xHistidine tagged protein. Biotin was used when a Streptavidin resin (Streptactin XT) was used to bind proteins carrying the Twin strep tag. The imidazole or biotin-rich protein solution was a buffer exchange to remove excess imidazole. The affinity-purified proteins thus obtained were stable at 4°C for several weeks. An example of purification of wild-type RsAqpZ and the R188A dead mutant is shown in Figure 1B-C.

[0322] For fluorescent labelling studies to visualize intact aquaporins in the membrane assembly of the invention, a labelling strategy was used where the primary amines of purified RsAqpZ (SEQ ID NO.2) and GFP-AqpZ (SEQ ID NO. 1) were crosslinked with Dylight 633 nm dye using the EDC / NHS crosslinking chemistry. The non crosslinked dye was removed by way of dialysis or Size exclusion chromatography (SEC), typically SEC (however the outcome and duration of each method is the same). The double-labelling strategy for GFP-RsAqpZ (i.e. a combination of GFP fusion and dye labelling) was used as a way of ascertaining the presence of intact proteins, as GFP loses its ability to fluoresce when unfolded (i.e. the presence of a GFP signal indicates correctly folded protein) with the red signal from Dylight 633 indicating the presence of the dye labelled aquaporin even if the aquaporin is unfolded.

[0323] 1.2 Expression and purification of Membrane scaffold proteins for nanodisc

[0324] Plasmids carrying the insert for the membrane scaffold proteins (MSPs) were designed and amplified (see Figure 1A for schematics of the MSP protein expression constructs used). As for the expression and purification of the RsAqpZ construct (described above), the MSPs were also transformed into the host strain BL21XDE3 for overexpression under the control of a T7 promoter which commences expression upon the addition of the inducer IPTG. Cultures were grown in 15 L bioreactors after which the biomass containing the overexpressed MSPs were isolated. The biomass was resuspended into a lysis buffer containing 20 mM phosphate buffer pH 7.4 in the presence of protease inhibitors (cOmplete™ Protease Inhibitor Cocktail 1 tablet + 1 mM PMSF) and enzymes Dnase and Rnase. After the cells were completely resuspended, the lysis process was performed by sonication. The lysate was clarified by centrifugation at 30,000 x g at 4°C for 30 minutes.

[0325] The protein was then purified from the resulting clarified lysate by affinity chromatography. The metal chelating affinity resin (a cobalt-NTA (TALON) resin) was equilibrated with 40 mM phosphate buffer, pH 7.4. The clarified lysate was loaded onto the column using a peristaltic pump of a Fast protein liquid chromatography pump, typically an AKTA Pure. The flow rate was around 5 mL / min. After loading the lysate and flowing it through, the column was washed with 250 mL of the following buffers: 40 mM Tris / HCl, 0.3 M NaCl, 1% Triton X-100, pH 8.0, 40 mM Tris / HCl, 0.3 M NaCl, 50 mM Na-cholate, 20 mM imidazole, pH 8.0 and 40 mM Tris / HCl, 0.3 M NaCl, 50 mM imidazole, pH 8.0. The protein was eluted with 40 mM Tris / HCl, 0.3 M NaCl, 0.4M imidazole, pH 8.0. The elution fractions were analyzed for the presence of the MSP protein using SDS-PAGE gels and colorimetric assays to determine the protein concentration. The peak fractions were then pooled together for dialysis against an aqueous buffer containing 20 mM Tris / HCl, 0.1 M NaCl, 0.5 mM EDTA, pH 7.4 at 4°C. Table 2 below shows amino acid sequences of the RsAqpZ and MSPs which may be used in the context of the present invention.

[0326] Table 2: MSP and RsAqpZ sequences.

[0327] 1.3 Liposome and polymcrsomc quality control of MSP and RsAqpZ protein

[0328] For quality control of MSPs, the elution fractions of MSP proteins were analysed using SDS / PAGE (as described above) and dynamic light scattering. For quality control of the purified RsAqpZ protein prior to incorporation into nanodiscs, aquaporin proteins were first incorporated back into liposomes which mimic their native bilayer. Liposomes were synthesised by making lipid stocks from E. coli polar lipids. The vial containing the lipid stock solution was first sonicated in a water bath with argon being flushed into it gently for 10 minutes. The sonicated lipid stocks were then added to a test tube placed in ice which contained either the protein solution (for proteoliposomes) or the protein processing buffer alone (for control liposomes) for 1 hour. After the hour-long incubation on ice, the sample was injected into a dialysis cassette and dialyzed against lOmM NaMOPS (pH 7.5) overnight. This process enabled liposomes to self-assemble with reconstituted proteins in them. The control liposomes and proteoliposomes were then filtered using a 1.2 pm filter followed by extrusion using a 0.1 pm filter. The liposomes were then used in stopped-flow light scattering to ensure the incorporation of RsAqpZ. The liposomes were also examined using dynamic light scattering (DLS) and turbidity measurements using absorbance at A660nm. At a Lipid to Protein ratio of 100 (LPR100), the proteoliposomes had a permeability of >-1100 pm / s. (This was to ensure that the detergent solubilization process used in the extraction of the tetramer had not permanently damaged the protein complex.) Example 2. Production of protein-containing vesicles and nanodiscs

[0329] This example describes the generation of RsAqpZ polymer vesicles (proteopolymersomes) and nanodiscs used in the experiments of the application.

[0330] 2, 1 Production of proteopolymersome vesicles

[0331] Once the purified aquaporin protein had passed the liposome quality control, as described above, the protein was used for incorporation into a polybutadiene (PB): poly 2-methyl-2-oxazoline (PMOXA) di-block polymer (molecular weight 800 Dalton, PDI 1.14; Polymer Source) proteopolymersomes using the polymer film sonication (PFS) method at 1.25mg / ml of polymer concentration. This sonication process involved making a thin film of the polymer by dissolving the polymer in chloroform, followed by subjecting the solution to vacuum suction (which evaporates the chloroform and leaves a thin film). To this thin film, a buffer containing lOmM NaMOPS (pH 7.5) was added along with a protein processing buffer which contained either the detergent or the protein solution itself before sonicating at an amplitude of 20% for 5 minutes (1 sec ON: 1 sec OFF). As the sonication progressed, the thin polymeric film dissociated from the bottom of the vial and slowly self-assembled thereby reconstituting the protein in its monolayer. This process is called sonication mediated protein reconstitution.

[0332] Following sonication, the vesicles were allowed to cool at room temperature for 30 minutes. The vesicle turbidities were measured as the absorbance at 660nm, then examined under dynamic light scattering (DLS) to ascertain their monodispersity. The vesicles were filtered using a 1.2pm filter followed by extrusion with 0.1pm filter. The extruded vesicles were then transferred into a dialysis cassette and allowed to dialyze against lOmM NaMOPS (pH 7.5 overnight). This dialysis step not only removes small micelles, but it also equalizes the osmotic strength inside and outside the vesicles.

[0333] Following dialysis, the samples were subjected to stopped flow light scattering experiments. The proteopolymersomes had a substantial increase in permeability at a polymer to protein ratio of 100 (PoPRIOO) Stopped-flow light scattering analysis of proteopolymersomes was also used to confirm that the R188A dead RsAqpZ mutant had decreased permeability compared to wild-type RsAqpZ under osmotic stress (Figure 3). 2.2 Strategy for RsAqpZ reconstitution into nanodiscs

[0334] Nanodiscs for biomimetic coating of membrane assemblies according to the invention were produced using biologically derived scaffolds (Membrane Scaffold proteins (MSPs) as described above). The biologically derived membrane scaffold proteins (MSPs), which are analogous to the human apolipoprotein molecules, possess an amphipathic characteristic, such that when combined with lipids, detergent- solubilized RsAqpZ protein and bio-beads, they will assemble into stable nanodiscs under slow dialysis. This dialysis process is thought to enable the bio-beads to bind the free or bound detergent molecules which are either in solution or being released from the protein as the concentration of the detergent slowly drops below the critical micelle concentration (CMC).

[0335] Prior to assembling the components to make the MSP nanodiscs, two sets of aqueous buffers were prepared, namely, Nanodisc Protein Buffer which contained 20 mm Tris base at pH 7.4, and Nanodisc Lipid Buffer which contained 20 mm Tris base at pH 7.4 and 100 mM sodium cholate. 200 pL of the purified RsAqpZ solution (obtained using the protocol of Example 1.1) at a concentration of 2-5 mg / mL was mixed with the membrane scaffold protein where the MSP concentration was ~20 times the molar quantity of RsAqpZ. Lipids resuspended in aqueous buffer were added to the mixture at a [RsAqpZ:MSP:lipid] ratio of [1:20:1600]. This mixture was incubated at 4 °C for 2 hours. For all nanodiscs used in the experiments described herein, MSP1D1 was used as the MSP and DMPC was used as the lipid.

[0336] As the equilibration of the nanodisc mixture was ongoing, 2.5 g of adsorbent (SM2 Biobeads (Bio-Rad)) was incubated in the nanodisc protein buffer with degassing to remove any solubilized oxygen. Once this step was completed, the protein / lipid nanodisc mixture was added to the degassed Biobeads solution and incubated at 4°C for 8-12 hours. Once the incubation was completed, the solution was centrifuged at 10- 12,000 x g for 2 min and the supernatant transferred to a fresh tube. This process was repeated twice more, after which the supernatant was filtered using a 0.45-micron filter to remove precipitates that might have formed during incubation. (Optionally, the nanodisc mixture could be injected into a size exclusion chromatography column to fractionate the empty nanodiscs from RsAqpZ incorporated nanodiscs. This step was performed prior to generating negative stain TEM images of the nanodiscs as set out below.) Schematics of aquaporin protein incorporated into polymer vesicles and nanodiscs are shown in Figure 4A and 4B respectively. In addition, the RsAqpZ incorporated vesicles and nanodiscs were imaged under high resolution using transmission electron cryo microscopy (cryo-TEM) and negative stain transmission electron microscopy (TEM), respectively.

[0337] For cryo-TEM, 4-5 pL of vesicles (1.25 mg / mL) was deposited on a freshly glow discharged 1.2 / 1.3 C-Flat Holey carbon copper grid fastened between a pair of tweezers mounted on a FEI Vitrobot. The Vitrobot conditions were kept at room temperature (for vesicles) and 4°C (for nanodiscs) and a humidity of 100%. The samples were incubated on the grid for 2 minutes (vesicles) and 30 seconds (nanodiscs) before blotting using the vitrobot with a blot force of 1 and immediately plunged into liquid ethane at cryo temperatures. Once the grids were flash frozen in liquid ethane, they were carefully transferred into liquid nitrogen and placed in the grid storage box all under cryo conditions.

[0338] For negative stain TEM, before preparing the samples, 37.5 mg of uranyl formate was weighed out in a 20 mL scintillation vial and wrapped in aluminium foil to prevent light from entering. To this vial containing uranyl formate, 5 mL of deionized water was added and stirred with a small stir bar on a stir plate. 5 M NaOH as added dropwise to raise the pH slowly up to 4.5 (after which the uranyl formate precipitates). Once this pH is reached, the solution was filtered using a 0.2 pm syringe filter and set aside. A 6” by 3” strip of parafilm was cut out and placed on the laboratory bench and 0.2 mL droplets of freshly made uranyl formate and optionally buffer solutions were placed carefully separating each droplet by 0.5”. A set of 5 to 6 carbon coated 400 mesh copper grids purchased from Electron microscopy services, Inc. were glow discharged using a Ted Pella glow discharger for 30 sec to make the carbon surface hydrophilic. After the grids were glow discharged, a single grid was grappled by a pair of negative tension tweezers and placed with the carbon side facing upwards. To this carbon side a droplet of 4-5 microliter of nanodisc solution was deposited and allowed to incubate for 1 min. after the incubation step, the grid was allowed to float on the droplet of uranyl formate (carbon side touching the droplet) for 10 seconds for the staining to take place. After this step, the grid was removed from the stain and any excess stain was wicked away using a filter paper and the grid was allowed to air dry for 10 minutes. The concentration of the nanodisc samples stained using uranyl formate was between 0.1- 0.5 mg / mL.

[0339] Once ready for imaging, the grids were inserted into an FEI TALOS 200 kV TEM using the side entry sample insertion port. The grids were mounted on a single tilt holder (for negatively stained samples) and a Gatan 626 cryo holder for cryo samples. The microscope was prepared for imaging by performing direct alignments to provide near parallel illumination and images were collected at a defocus of -2 to -5 microns. The images were collected using a 4k x 4k Ceta or Falcon camera and were processed using the single particle imaging software EMAN. As shown in Figure 4C-D, the vesicles were around 120 nm in diameter and 3-dimensional in shape (Figure 4C) and the nanodiscs were around 10 nm in diameter and planar in shape (Figure 4D), with most nanodiscs in a flat orientation (i.e. one where the aquaporin protein would be oriented orthogonally to the grid).

[0340] Example 3. Fabrication of membrane assemblies

[0341] This Example describes the production of membrane assemblies according to the invention.

[0342] 30 kDa polysulfone (PS) supports were fabricated in-house using the phase inversion process using a roll-to-roll caster. Briefly, polysulfone was dissolved in dimethylformamide (DMF) and cast onto the surface of non-woven polyester or polypropylene fabric. After fabrication, the poly sulfone supports were then soaked in an overflow bath of deionised water for 1 hr at room temperature, then used to fabricate biomimetic membrane assemblies using piperazine chemistry. According to this approach, control membranes (without vesicles or nanodiscs) were fabricated by first subjecting the PS support layers to a soaking process step, wherein the supports were first soaked in an aqueous amine solution containing 2% piperazine, 2% triethylamine (TEA) and 4% camphor sulfonic acid (CSA, 0-0.3% SDS) for 5 minutes. This was followed by dip-coating the PS support in 0.1% trimesoyl chloride (TMC)Zhexane solution for 1 minute. After this step the support was placed in deionised water in the cold room (4 °C) for an overnight incubation step until further performance or fluorescence testing. A polyamide coating was formed on the PS support via interfacial polymerisation reaction between the piperazine and the TMC.

[0343] The same approach as above was used for embedding the polymer vesicles or MSP1D1 / DMPC nanodiscs in the polyamide layer via the addition of a hand spray step, which was introduced between the two dip coating steps, wherein the desired amount (approximately 2 mL (0.5 mL x 4 sprays)) of the appropriate biomimetic solution was sprayed onto the support. (Biomimetic solutions were obtained using the protocols in Example 2; vesicle concentration (at a polymer to protein ratio of 100) was 1.25 mg / mL, and the concentration of nanodiscs was 0.03 mg / mL).

[0344] Example 4: Fluorescence analysis of RsAiipZ protein on the membrane surface This Example compares the protein coverage on the surface of membrane assemblies fabricated using either vesicles (proteopolymersomes) or nanodiscs.

[0345] Membrane assemblies coated with either RsAqpZ-proteopolymersomes (vesicles) or RsAqpZ-incorporated MSP1D1 / DMPC nanodiscs were prepared according to the protocol in Example 3, using GFP-RsAqpZ double-labelled with dye and imaged in both the GFP channel (Figure 5A) or the channel corresponding to the cross-linked dye (Figure 5B). For the vesicle and nanodisc solutions applied to the membrane assemblies, the vesicle concentration (at a polymer to protein ratio of 100) was 1.25 mg / mL and the concentration of nanodiscs was 0.03 mg / mL. Fluorescence image scans of the membrane assemblies were collected using the LI-COR Odyssey M Imaging system. Briefly, hydrated membrane assemblies were placed onto the glass surface with the coated side face down. 10 mM MOPS buffer at pH 7.4 was subsequently added onto the assemblies to maintain hydration, and a polymer gel weight mat placed on top of the assemblies. A roller was used to gently flatten the assemblies to the surface evenly and remove excess buffer trapped beneath them. Images were then collected at the appropriate excitation and emission channels with 20 pm pixel resolution. Each image scan contains all membrane assemblies within the same image file and a negative control (i.e. empty nanodiscs with no aquaporin) was always included.

[0346] Fluorescence images were processed using the Fluorescence Imaging for Nanoparticle Detection (FIND) on Biomimetic Surfaces analysis pipeline. Briefly, each of the fluorescence scan TIFs along with an image collection meta- text fie from the LI- COR Empiria software was paired with a layout CSV file and information meta-JSON file. The locations of the membrane assemblies within the image were determined by finding the background image intensity, applying the Sobel edge detection filter, and subsequently applying the circle Hough transform algorithm. The fluorescence intensity of the background was determined by the negative control membrane assembly included in the image. Visualizations and percent coverage calculations were determined based on this background intensity value (Figure 5C). As shown in Figure 5C, although the coverage of total RsAqpZ protein (assessed using the signal intensity of the dye, see Figure 5B) appeared similar for both aquaporin-containing vesicles and aquaporin-containing nanodiscs, membranes using nanodiscs had much higher coverage of correctly folded, intact RsAqpZ (assessed using the signal intensity of GFP, see Figures 5A and 5C).

[0347] 5: Denaturation studies of nanodisc-based membranes

[0348] This Example describes heat- and ethanol-based denaturation studies of nanodisc-based membranes.

[0349] Membrane assemblies containing GFP-RsAqpZ double-labelled with dye were prepared as described in Example 3 above and imaged as described in Example 4 above. The amount of nanodiscs applied to the membranes was varied by spraying different volumes (0.5 mF, 1.0 mF or 2.0 mF) of an 0.03 mg / mL nanodisc solution during the spray step. For heat denaturation, membranes were dried overnight in an 85°C oven and then rehydrated for 5 days in 10 mM NaMOPS solution at pH 7.4. After rehydration, the membranes were then soaked in 99% ethanol overnight at room temperature, followed by a second rehydration step. Imaging was performed before heat treatment (Figure 6A), after rehydration following heat treatment (Figure 6B), and after rehydration following ethanol treatment (Figure 6C). As can be seen in the figure, heat and ethanol treatment did not affect total protein coverage of the membranes. The persistence of the GFP signal following both heat and ethanol treatment, at all amounts of nanodisc application, indicated that correctly folded RsAqpZ within the nanodiscbased membranes could withstand overnight drying and ethanol-based denaturation.

[0350] Example 6: AFM analysis of the surface of nanodisc- and vesicle-based membranes

[0351] Example 6 describes the assessment of the surface roughness and topology of membrane assemblies according to the invention containing either nanodiscs or proteopolymersomes (vesicles), respectively.

[0352] Membrane assemblies coated with either RsAqpZ-proteopolymersomes (vesicles) or RsAqpZ nanodiscs were prepared according to the protocol in Example 3. The surface roughness and topology of the membranes were assessed using Atomic Force Microscopy (AFM). Initially, samples were affixed onto magnetic discs (faceup) and inserted into the Cypher ES AFM machine for analysis under the AR SPM software. Surface imaging was performed using “dry tapping mode,” where surface roughness was quantified utilizing force and height parameters as indicated by the Au- coated or bio-environment cantilever probe (respectively). For wet AFM, the AFM was set to the following parameters: Cantilever probe tip type: BioLever Mini (BL- AC40TS) - silicone nitride material Cr / Au(5 / 30) - 75-145kHz, 0.02-0.14N / m, Tapping Mode in “AC Water Topography” Environment; 5V, 20kHz in liquid, BlueDrive laser at 0.03x, Sample holder type: electromagnetic testing cell with “cup” holder (removed electrical components to use the cup-holder) and Objective lens set to 2. For the AFM analysis, membranes were cut to a 1 cm x 1 cm area and placed into a 20 mL vial of ultrapure water or 10 mM NaMOPS buffer solution before use. Once ready, the piece of membrane sample was placed into the sample holder with the sides fastened. Once the membrane is fastened, the Biolever probe tip was placed in the centre under the cantilever holder using a pair of tweezers. The sample holder was then placed onto the Cypher ES at the lowest setting and the Cantilever holder at the highest height setting to begin dry environment calibration. During the calibration step, the probe tip and sample focus points were calibrated using the SpotOn and BlueDrive lasers at the base of the probe tip. To recalibrate focus under a water environment, 200 pL of ultrapure water was added to the holder and a drop to the tip. Once this step was completed, the tip was moved to pre-engage. A thermal tune of the cantilever in liquid was captured and the phase and frequency were transferred to the normal tuning to calibrate around the current temperature. Once this step was completed, the setpoint amplitude is adjusted to 80% of drive amplitude before starting the tip approach to lower the top closer to the sample for data collection.

[0353] Image acquisition encompassed areas ranging from 10 pm2to 2 pm2to ensure comprehensive coverage of representative regions and defects (representative 2 x 2 pm images are shown in Figure 7, top row). Subsequently, Gwyddion software was utilized for image analysis, facilitating the determination of average area surface roughness and assessment of coating uniformity (see Figure 7, bottom row). Furthermore, statistical analyses were conducted to identify significant disparities between samples, thereby enabling a comprehensive examination of membrane surface texture and polyamide coating layer characteristics. As shown in Figure 7, there was a distinct difference between the surface topologies of nanodisc- and vesicle -based membranes, with the nanodisc-based membranes having a much smoother surface than either the vesicle-based membranes or the negative control (membranes having only the piperazine-based coating without nanodiscs or vesicles).

[0354] Without being bound by theory, the inventors hypothesise that the difference in surface topology may be due to the significantly different shapes of vesicles and nanodiscs, which when embedded on the surface of the porous support in the polyamide coating may give rise to differences in roughness and hence differences in total surface area. It is known that surface area is an important parameter that influences the pure water flux of a membrane. It is also known that aquaporin Z-containing vesicles produce membranes that are rougher (have a higher surface area) than membranes using empty vesicles without protein, i.e. the increase in flux observed when introducing aquaporin Z-containing vesicles may in part be due to the increased surface area. In contrast, the present inventors have found that aquaporin Z-containing nanodiscs do not increase the roughness (surface area) compared to empty nanodiscs (data not shown), meaning that an observed increase in flux will be due to the aquaporins.

[0355] Example 7. Morphological analysis of nanodisc- and vesicle-based membranes

[0356] Example 7 investigates the morphology of membrane assemblies according to the invention containing either nanodiscs or proteopolymersomes (vesicles), respectively.

[0357] Membrane assemblies coated with either RsAqpZ-proteopolymersomes (vesicles) or RsAqpZ nanodiscs were again prepared according to the protocol in Example 3. A control sample was also prepared, which was fabricated according to the protocol in Example 3 but without any membrane mimetic structures. The micro structure of the membranes was evaluated through scanning electron microscopy (SEM). Initially, the samples were sputter-coated with a 1 nm layer of Au / Pd using a sputter coater (Quorum Q150T ES) and securely mounted on appropriate stubs with double-sided carbon tape. Prior to imaging, the scanning electron microscope (SEM) (XEIA Tescan FEG) underwent meticulous calibration of zoom and focus to ensure optimal imaging conditions. Surface imaging (Figure 8A-C, bottom row) was conducted under a 5kV electron beam, capturing images at magnifications of lOkx, lOOkx, and 200kx to provide a comprehensive representation of the sample's surface features. For cross-sectional analysis (Figure 8A-C, top row), the samples underwent cryo-fracturing in liquid nitrogen to expose internal structures. Subsequently, a 2kV electron beam was employed, with images acquired at magnifications of 2kx, 30kx, and 60kx to scrutinize the coating layer within porous membrane structures. Image analysis was carried out using ImageJ software, encompassing the quantification of morphological features and assessment of coating uniformity. Similarly to the results of the AFM study in Example 6 above, the surface of the nanodisc-containing membrane (Figure 8C) was noticeably smoother than that of the vesicle-containing membrane (Figure 8B).

[0358] Example 8. Performance of membrane assemblies according to the invention

[0359] Biomimetic reverse osmosis membranes according to the invention were fabricated for performance testing using double-layer interfacial polymerisation.

[0360] First, a polysulfone (PSF) support (MWCO: 70-80 kDa) cast on a non-woven backing fabric was dried with an air knife at 20 psi for 1 minute. The initial aqueous phase with 0.15% piperazine (PIP), 2% triethylamine (TEA), 4% cellulose sulfate (CSA), and 0.10% sodium dodecyl sulfate (SDS) was then poured onto the PSF for 2 minutes, followed by drying with a squeegee. A solution of 0.015% trimesoyl chloride (TMC) in IsoparG was then applied for 1 minute and dried with an air knife for 2.5 minutes.

[0361] In the second aqueous phase, 0.04% m-phenylenediamine, 100 mM sodium chloride (NaCl), and 1.67 mM sodium MOPS (NaMOPS) was poured over the dried polyamide layer for 2 minutes, followed by air knife drying for 1 minute. Another layer of 0.015% TMC in IsoparG was then applied for 1 minute and dried again for 2 minutes. For membranes comprising membrane mimetic structures, solutions of the appropriate mimetic structure were also added to the second aqueous phase.

[0362] Specifically, for Aquaporin Z (AqpZ) nanodisc-based membranes, either ‘empty’ nanodiscs solution (control; i.e. nanodiscs without AqpZ protein) or AqpZ- incorporating nanodiscs was combined with the m-phenylenediamine solution, to achieve final loading concentrations of 0.08 mg / L (empty nanodiscs) or 0.08 mg / L and 0.21 mg / L (AqpZ- incorporating nanodiscs). The support used to fabricate both empty nanodisc-based membranes and AqpZ-nanodisc-based membranes in this experiment (see Figure 10) was an in-house PSF support (i.e. MWCO: 70-80 kDa, on a non-woven backing fabric) made using double-layered polyamide chemistry, as described above.

[0363] Similarly, for OmpG nanodisc-based membranes, 4 mL of OmpG nanodiscs was mixed with 236 mL of m-phenylenediamine solution, resulting in a concentration of 3.83 mM. The control used in this experiment (marked as ‘CTRL’, Figure 11) was an in-house PSF support (i.e. MWCO: 70-80 kDa, on a non-woven backing fabric) made using polyamide chemistry, as described above, without any membrane mimetic structures.

[0364] For membranes comprising detergent- stabilised mimetic structures, 1.6 mL of detergent-solubilised OmpG (0.23 pM SST-OmpGm solubilized in a solution containing 100 mM K2HPO4, 200 mM NaCl, 10% glycerol, 0.1% DDM, 5 mM sodium azide, pH 7.4) was combined with 238.4 mL of m-phenylenediamine solution to achieve a concentration of 1.53 mM. In this experiment, the control membrane (marked as ‘BW30’; Figure 12) was a commercially available flat sheet BW30 reverse osmosis membrane (DuPont Water Solutions), without any membrane mimetic structures. BW30 membranes are thin-film composite (TFC) structures with a polyamide selective layer supported by a polysulfone and polyester backing. They are commonly used for brackish water desalination and therefore serve as an appropriate comparator for membrane assemblies of the invention. The PSF support for the membrane assembly comprising detergent-stabilised membrane mimetic structures was again an in-house PSF support (i.e. MWCO: 70-80 kDa, on a non-woven backing fabric) made using polyamide chemistry, as described above. Both control and detergent- stabilised membranes were additionally bath sonicated for 10 minutes.

[0365] All the membranes described above were tested for permeability (GFD / psi) and salt rejection (%) at 25 °C in a cross-flow setup at 225 psi, with a salt concentration of 2000 ppm and a feed flow rate of 1 L / min. It can be seen from Figures 10-12 that membrane assemblies according to the invention comprising membrane mimetic structures (AqpZ nanodiscs, Figure 10; OmpG nanodiscs, Figure 11; detergent- stabilised OmpG, Figure 12) had overall greater permeability compared to controls, without a significant decrease in salt rejection.

[0366] Where in the foregoing description, integers or elements are mentioned which have known, obvious or foreseeable equivalents, then such equivalents are herein incorporated as if individually set forth. Reference should be made to the claims for determining the true scope of the present invention, which should be construed so as to encompass any such equivalents. It will also be appreciated by the reader that integers or features of the invention that are described as preferable, advantageous, convenient or the like are optional and do not limit the scope of the independent claims. Moreover, it is to be understood that such optional integers or features, whilst of possible benefit in some embodiments of the invention, may not be desirable, and may therefore be absent, in other embodiments.

Claims

Claims1. A membrane assembly comprising: a. a porous support; and b. a plurality of membrane mimetic structures on a surface of the support; wherein the membrane mimetic structures comprise: (i) a channel protein which comprises a transmembrane domain having a hydrophobic surface, and (ii) one or more amphiphilic molecules; and wherein the one or more amphiphilic molecules form a ring structure around the hydrophobic surface of said transmembrane domain of the channel protein.

2. The membrane assembly according to claim 1, wherein at least a portion of the hydrophobic moieties of the one or more amphiphilic molecules are oriented towards the channel protein and at least a portion of the hydrophilic moieties of the amphiphilic molecule are oriented away from the channel protein.

3. The membrane assembly according to claim 1 or claim 2, wherein the ring structure formed by the one or more amphiphilic molecules is capable of shielding the hydrophobic surface of the transmembrane domain of the channel protein from an aqueous environment such that the membrane mimetic structures are soluble when exposed to an aqueous environment.

4. The membrane assembly according to any preceding claim, wherein the diameter of the membrane mimetic structures is from about 0.2 nm to about 90 nm, for example from about 5 to about 25 nm.

5. The membrane assembly according to any preceding claim, wherein the density of the channel proteins on the surface of the support is from about 500 proteins / pm2to about 20,000 proteins / pm2, for example from about 500 proteins / pm2to about 2500 proteins / pm2or from about 8000 proteins / pm2to about 15,000 proteins / pm2.

6. The membrane assembly according to any preceding claim, wherein the membrane mimetic structures each comprise a single channel protein.

7. The membrane assembly according to any preceding claim, wherein the one or more amphiphilic molecules form an essentially planar ring structure around the hydrophobic surface of the transmembrane domain of the channel protein.

8. The membrane assembly according to claim 7, wherein the plane of the ring structure is essentially orthogonal to the axis of the channel protein.

9. The membrane assembly according to any preceding claim, wherein the membrane mimetic structures further comprise lipids or lipid- mimic polymers.

10. The membrane assembly according to claim 9 wherein the lipids or lipid-mimic polymers are located between the hydrophobic surface of the transmembrane domain of the channel protein and the one or more amphiphilic molecules.

11. The membrane assembly according to any preceding claim wherein at least a portion of the one or more amphiphilic molecules contacts the hydrophobic surface of the transmembrane domain of the channel protein.

12. The membrane assembly according to any preceding claim, wherein the membrane mimetic structures are selected from the group consisting of: nanodiscs, membrane mimetic structures stabilised using nanodisc scaffold peptide (NSP) or reversed nanodisc scaffold peptide (NSPr), detergent- stabilised membrane mimetic structures, saposin-stabilised membrane mimetic structures and amphipol-stabilised membrane mimetic structures.

13. The membrane assembly according to any preceding claim, wherein the membrane mimetic structure is an amphipol-stabilised membrane mimetic structure and the one or more amphiphilic molecules is an amphipol polymer.

14. The membrane assembly according to claim 13, wherein the amphipol polymer directly contacts the channel protein.

15. The membrane assembly according to claim 13 or claim 14, wherein the amphipol polymer is a polymer according to Formula IV:Formula IV wherein,RI is carboxylate,V- pho s pho ry 1 cho 1 i nc- V ethylenedioxybis(ethyl)acrylamide;R2 is a Ci-Ce alkylene, or is a bond;Ra is a Ci-C6alkyl, a C3-C9 cycloalkyl, a Ce-Cu aryl which is optionally substituted with a C1-C4 alkyl;R4 is a Ci-Ce alkyl, or ethylsulfonate; wherein x, y and z are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85, y is from about 10 to about 85 and z is from 0 to about 60 and wherein the sum of x, y and z equals 100.

16. The membrane assembly according to any preceding claim, wherein the channel protein is selected from the group consisting of: an aquaporin, an aquaglyceroporin, OmpX (Escherichia coli), PagP (Escherichia coli), OmpW (Escherichia coli), OmpT (Escherichia coli), EspP autotransporter beta domain (Escherichia coli), OmpLA (Escherichia coli), OmpG (Escherichia coli), OmpF (Escherichia coli), FhuA (Escherichia coli), T7 DNA ejectosome periplasmic tunnel (Escherichia phage T7), Aerolysin (Aeromonas hydrophila), Main porin MspA (Mycolicibacterium smegmatis), and Alpha-hemolysin (Staphylococcus aureus).

17. The membrane assembly according to any of claims 1 to 15, wherein the channel protein is selected from the group consisting of: aquaporins and glyceroporins; beta-barrel channel proteins; and alpha-helical channel proteins.

18. The membrane assembly according to claim 17, wherein the channel protein is an aquaporin, for example Aquaporin Z.

19. The membrane assembly according to any preceding claim, wherein substantially all the channel proteins present in the membrane assembly are oriented orthogonally to the surface of the porous support.

20. A membrane assembly comprising: a. a porous support; and b. a plurality of membrane mimetic structures on a surface of the support, wherein at least a portion of said membrane mimetic structures each comprise a membrane protein; wherein the membrane mimetic structures are selected from the group consisting of:(i) nanodiscs;(ii) membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein, wherein said amphiphilic polymer is not a lipid or an amphiphilic block copolymer; and(iii) a mixture of (i) and (ii).

21. The membrane assembly according to claim 20, where the portion of said membrane mimetic structures which each comprise a membrane protein is at least 80%, for example at least 90%.

22. The membrane assembly according to claim 20 or claim 21, wherein the density of membrane proteins on the surface of the support is from about 500 proteins / pm2to about 10,000 proteins / pm2, from about 500 proteins / pm2to about 5000 proteins / pm2, from about 500 proteins / pm2to about 2500 proteins / pm2, from about 1000 proteins / pm2to about 2000 proteins / pm2, for example about 1500 proteins / pm2; or from about 5,000 proteins / pm2to about 20,000 proteins / pm2, from about 8000 proteins / pm2to about 15,000 proteins / pm2, from about 10,000 proteins / pm2to about 15,000 proteins / pm2, for example from about 10,000 proteins / pm2to about 12,000 proteins / pm2.

23. The membrane assembly according to any preceding claim, wherein the membrane mimetic structures have a diameter of from about 0.2 nm to about 10 nm, from about 0.2 nm to about 5 nm, from about 0.2 nm to about 1 nm, for example about 0.5 nm; or from about 7 nm to about 90 nm, for example from about 7 nm to about 50 nm, from about 7 nm to about 20 nm, from about 8 nm to about 15 nm, from about 10 nm to about 15 nm, from about 10 nm to about 12 nm or from 8 nm to about 11 nm, for example about 10 nm.

24. The membrane assembly according to any of claims 1 to 12 or 16 to 23, wherein the membrane mimetic structures are nanodiscs.

25. The membrane assembly according to claim 24, wherein the nanodiscs are selected from the group consisting of: lipid-based nanodiscs, polymer-based nanodiscs, hybrid lipid / polymer-based nanodiscs and a combination thereof.

26. The membrane assembly according to claim 25, wherein the lipid-based nanodiscs or hybrid lipid / polymer-based nanodiscs comprise one or more lipids selected from the group consisting of: l,2-dimyristoyl-5n-glycero-3- phosphocholine (DMPC); l,2-dimyristoyl-sn-glycero-3-phospho-(l'-rac- glycerol) (DMPG); l,2-dioleoyl-5n-glycero-3-phosphocholine (DOPC); 1- myristoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(l -glycerol)] (LMPG); 1-pahmtoyl-2-hydroxy-sn-glycero-3-[phospho-rac-(l -glycerol)] (LPPG); 1- palmitoyl-2-oleoyl-5«-glycero-3-phosphocholine (POPC); 1 -palmitoyl-2- oleoyl-sn-glycero-3-phosphoethanolamine (POPE); palmitoyl-oleoyl- phosphatidylglycerol (POPG); 1 -palmitoyl-2-oleoyl-5n-glycero-3-phospho-L- serine (POPS); phosphatidylethanolamine (PE), phosphatidylglycerol (PG), cardiolipins, cholesterols and native lipids (e.g. native lipids from E. coli).

27. The membrane assembly according to claim 25, wherein the polymer-based nanodiscs comprise one or more amphiphilic block copolymers.

28. The membrane assembly according to claim 27, wherein the amphiphilic block copolymer comprises at least one hydrophilic block and at least one hydrophobic block.

29. The membrane assembly according to claim 28, wherein the amphiphilic block copolymer comprises at least one hydrophilic block comprising poly(4-vinyl-N- methylpyridine iodide) and at least one hydrophobic block comprising hydrogenated polybutadiene (HPBD), for example the triblock copolymer HPBD-Z?-(poly(4-vinylpyridine)28)2.

30. The membrane assembly according to claim 28, wherein the amphiphilic block copolymer comprises at least one hydrophilic block comprising (poly)2- Ci-3alkyl-2-oxazoline and at least one hydrophobic block comprising polybutadiene, for example, a diblock copolymer AB in which (poly)2-Ci-3alkyl- 2-oxazoline forms the A block and polybutadiene forms the B block, and which has at least one end group at the end of a (poly)2-Ci-3alkyl-2-oxazoline block which is selected from carboxy, activated carboxy, amine, methacrylate, thiol, azide, and alkyne.

31. The membrane assembly according to any of claims 24 to 30, wherein the nanodiscs comprise at least 1 stabilising belt molecule surrounding a hydrophobic surface of the lipid portion of each nanodisc.

32. The membrane assembly according to claim 31, wherein the at least 1 stabilising belt molecule is selected from the group consisting of: a membrane scaffold protein (MSP) or a derivative thereof, a synthetic polymer and an amphipathic peptide.

33. The membrane assembly according to claim 32, wherein the at least 1 stabilising belt molecule is a membrane scaffold protein (MSP) or a derivative thereof, for example MSP1, MSP1D1, MSP1D2, MSP1E(1,2,3)D1, MSP2N(1,2,3)D1,MSPD1AH4, MSP1D1AH5, MSP1D1AH4H5, MSP1D1AH4-H6, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSPN1, MSP2N2 or MSP2N3.

34. The membrane assembly according to claim 32, wherein the at least 1 stabilising belt molecule is a synthetic polymer, for example a poly(diisobutylcnc-o / / - maleic acid) polymer (DIBMA), a poly(styrene-co-maleic acid) copolymer (SMA), a poly(acrylic acid- co -styrene) copolymer (AASTY) or an amphipol polymer, for example a polymer according to formula V or formula VI:Formula V wherein,Ri is a Ci-C6alkylene, or is a bond;R2 is a C3-C9 cycloalkyl, or a Ce-Cu aryl which is optionally substituted with a C1-C4 alkyl, for example at the para position;R3 is a Ci-Ce alkyl; wherein x, y and z are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85, y is from about 10 to about 85 and z is from 0 to about 60 and wherein the sum of x, y and z equals 100; orFormula VI wherein,Ri is a Ci-C6alkylene, or is a bond;R2 is a C3-C9 cycloalkyl, or a Ce-Cu aryl which is optionally substituted with a C1-C4 alkyl, for example at the para position; wherein x and y are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85 and y is from about 15 to about 85 wherein the sum of x and y equals 100.

35. The membrane assembly according to any of claims 20 to 23, wherein the membrane mimetic structures are (ii) membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein, wherein said amphiphilic polymer is not a lipid or an amphiphilic block copolymer, e.g. wherein said amphiphilic polymer is an amphipol polymer, e.g. wherein said amphiphilic polymer is capable of stabilising said membrane protein in aqueous solution.

36. The membrane assembly according to claim 35, wherein said amphiphilic polymer, e.g. an amphipol polymer, directly contacts said membrane protein.

37. The membrane assembly according to claim 35 or claim 36, wherein the amphiphilic polymer, e.g. an amphipol polymer, is a polymer according to Formula IV:Formula IV wherein,Ri is carboxylate, or V-phosphorylcholine-V- ethylenedioxybis(ethyl)acrylamide;R2 is a Ci-Ce alkylene, or is a bond;R3 is a Ci-Ce alkyl, a C3-C9 cycloalkyl, a Ce-Cu aryl which is optionally substituted with a C1-C4 alkyl;R4 is a Ci-Ce alkyl, or ethylsulfonate; wherein x, y and z are the molar percentages (mol %) of each type of subunit, randomly distributed along the copolymer; wherein x is from about 15 to about 85, y is from about 10 to about 85 and z is from 0 to about 60 and wherein the sum of x, y and z equals 100.

38. The membrane assembly according to any of claims 20 to 23, wherein the plurality of membrane mimetic structures is a mixture of nanodiscs as defined in any of claims 24 to 34 and membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein as defined in any of claims 35 to 37.

39. The membrane assembly according to any preceding claim, wherein the plurality of membrane mimetic structures forms a layer, for example a planar layer or a monolayer, on the surface of the porous support.

40. The membrane assembly according to claim 39, wherein the thickness of the layer is from about 3 to about 20 nm, for example from about 4 nm to about 10 nm, for example from about 5 nm to about 6 nm.

41. The membrane assembly according to any of claims 20 to 40, wherein the membrane protein is an integral membrane protein, for example a transmembrane protein.

42. The membrane assembly according to claim 41, wherein the transmembrane protein is selected from the group consisting of: aquaporins and glyceroporins; beta-barrel transmembrane proteins; and alpha-helical transmembrane proteins.

43. The membrane assembly according to claim 42, wherein the transmembrane protein is an aquaporin, for example Aquaporin Z.

44. The membrane assembly according to any of claims 20 to 43, wherein substantially all the membrane proteins present in the membrane assembly are oriented orthogonally to the surface of the porous support.

45. The membrane assembly according to any preceding claim, wherein the porous support comprises a polymer selected from the group consisting of polysulfone (PS), polyethersulfone (PES), polyacrylonitrile (PAN), poly vinylidene fluoride (PVDF), polyimide, poly(ether imide) (PEI), polyacrylic acid (PAA), polyamide, polycarbonate (PC), polyethylene (PE), polypropylene (PP), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polypiperazine, cellulose acetates, cellulose nitrates, and cellulose esters.

46. The membrane assembly according to any preceding claim, wherein the membrane mimetic structures are adsorbed to, covalently attached to (e.g. covalently cross-linked to) or attached via charge-based interactions to the surface of the porous support.

47. The membrane assembly according to any preceding claim, further comprising a coating on the surface of the support comprising the membrane mimetic structures.

48. The membrane assembly according to claim 47, wherein the membrane mimetic structures are at least partially encapsulated by the coating.

49. The membrane assembly according to claim 47 or 48, wherein the coating comprises a polyamide, for example a thin film polyamide composite, for example a polyamide made from polymerising piperazine and trimesoyl chloride.

50. A method of making a membrane assembly comprising: a) a porous support; and b) a plurality of membrane mimetic structures on a surface of the support,wherein at least a portion of said membrane mimetic structures each comprise a membrane protein; wherein the membrane mimetic structures are selected from the group consisting of: (i) nanodiscs; (ii) membrane mimetic structures which comprise an amphiphilic polymer which contacts said membrane protein, wherein said amphiphilic polymer is not a lipid or an amphiphilic block copolymer and (iii) a mixture of (i) and (ii); wherein said method comprises the steps of: a) providing the porous support; b) applying an aqueous solution of the membrane mimetic structures to a surface of the porous support; thereby forming the membrane assembly.

51. A method of making a membrane assembly comprising: a) a porous support; and b) a plurality of membrane mimetic structures on a surface of the support, wherein the membrane mimetic structures comprise: (i) a channel protein which comprises a transmembrane domain having a hydrophobic surface, and (ii) one or more amphiphilic molecules; and wherein the one or more amphiphilic molecules form a ring structure around the hydrophobic surface of said transmembrane domain of the channel protein; wherein said method comprises the steps of: a) providing the porous support; b) applying an aqueous solution of the membrane mimetic structures to a surface of the porous support; thereby forming the membrane assembly.

52. A method as claimed in claim 50, wherein the features of the membrane assembly are as defined in any of claims 20 to 49.

53. A method as claimed in claim 51, wherein the features of the membrane assembly are as defined in any of claims 1 to 19, 23, 24-34, 39-40 or 45-49.

54. A method of making a membrane assembly according to any of claims 50 to 53, which further comprises between steps a) and b) the step of:A. applying an aqueous solution comprising an amine to a surface of the porous support; and which further comprises after step b) the step of:B. applying a solution comprising an acyl halide and a non-polar solvent to the surface of the support comprising the plurality of membrane mimetic structures, thus forming a polyamide via interfacial polymerisation.

55. The method according to claim 54, wherein the aqueous solution in step A) comprises a diamine, e.g. a non-aromatic cyclic diamine, e.g. piperazine, or e.g. an aromatic diamine, e.g. m-phenylenediamine.

56. The method according to claim 54 or 55, wherein the acyl halide is an acyl chloride, for example trimesoyl chloride (TMC).

57. The method according to any of claims 54 to 56, wherein the plurality of membrane mimetic structures themselves comprise amine groups and can participate in the formation of the polyamide in step B.

58. The method according to any of claims 54 to 57, wherein the aqueous solution in step A) further comprises triethyl amine (TEA) and camphor sulfonic acid (CSA).

59. The method according to any of claims 50 to 58, wherein step b) further comprises forming attachments between the membrane mimetic structures to the surface of the porous support, for example via adsorption, covalent attachment (e.g. covalent cross -linking) or charge-based interactions.

60. A membrane assembly preparable by a method as claimed in any of claims 50 to 59.

61. Use of a membrane assembly according to any of claims 1 to 49 in a filtration application (for example microfiltration, ultrafiltration, nanofiltration, reverse osmosis, or engineered osmosis).

62. A method of filtration comprising the steps of: a. providing a membrane assembly according to any of claims 1 to 49; b. filtering a liquid using the membrane assembly.

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