POROUS THIN-FILM MEMBRANE, METHOD FOR PRODUCTION THEREOF AND ALSO POSSIBILITIES OF USE

Abstract

The subject of the invention is new membranes in which tailor-made membrane transport proteins (such as e.g. TCDB classified proteins) act as pore-forming proteins (e.g. FhuA) or peptides which act as pores in the membrane. The membranes can preferably be produced both by linking synthesised protein-polymer conjugates and by direct linking of the pore-forming proteins and peptides. Such membranes are distinguished by many outstanding features which existing membranes have not been able to offer to date.

Claims

1-18. (canceled)

19. A porous thin-film membrane made up of covalently crosslinked, pore-forming proteins or peptides forming continuous pores in the thin-film membrane.

20. The porous thin-film membrane according to claim 19, which has a pore density in the range of 1.Math.10.sup.8 channels/cm.sup.2 to 1.Math.10.sup.13 channels/cm.sup.2.

21. The porous thin-film membrane according to claim 19, whose pore size is in the range of 0.1 to 20 nm.

22. The porous thin-film membrane according to claim 19, in which the pore size of all the pores is essentially identical.

23. The porous thin-film membrane according to claim 19, wherein the thickness of the porous thin-film membrane is between 1 and 100 nm.

24. The porous thin-film membrane according to claim 19, wherein the pore-forming proteins or peptides are selected from the group consisting of transmembrane proteins and proteins or peptides of the TCDB classification categories TC #1-9.

25. The porous thin-film membrane according to claim 19, which is produced by crosslinking a protein/peptide polymer conjugate having crosslinkable functionalities.

26. The porous thin-film membrane according to claim 25, wherein the protein/peptide polymer conjugate is a conjugate of a polymer selected from the group consisting of polymers or statistical copolymers with groups which are crosslinkable by radiation, radical reactions, or click-chemical reactions.

27. The porous thin-film membrane according to claim 26, wherein the polymer is selected from the group consisting of poly(co)acrylamides and poly(co)acrylates with substituents which are crosslinkable by radiation, radical reactions, or click-chemical reactions.

28. The porous thin-film membrane according to claim 26, wherein the polymer is selected from the group consisting of poly(co)(N-isopropylacrylamide)(2-(dimethylmaleimido)-N-(ethylacrylamide)), poly(co)(N-isopropylacrylamide)(3,4-dimethylmaleinimidobutylacrylate), poly(co)(N,N-dimethylaminoethylmethacrylate)(3,4-dimethylmaleinimidobutylmethacrylate), and poly(co)(vinylcaprolactam)(3,4-dimethylmaleinimidobutylacrylate).

29. The porous thin-film membrane according to claim 27, wherein the polymers of the protein/peptide polymer conjugates are or become bonded covalently to the pore-forming protein or peptide by an initiator, a chain-transfer agent, or a catalyst, which is bonded covalently to the pore-forming protein or peptide.

30. The porous thin-film membrane according to claim 19, which is produced by crosslinking of amino acid radicals or glycosylating groups of the pore-forming proteins or peptides by at least one bi- or multifunctional crosslinker.

31. The porous thin-film membrane according to claim 30, wherein the crosslinker is selected from the group consisting of dialdehydes, dicarboxylic acids, N-hydroxysuccinimide-activated dicarboxylic acids, diacid halogenides, diamines, and diiso(thio)cyanates.

32. The porous thin-film membrane according to claim 19, wherein the pore-forming proteins or peptides are functionalised on the inner pore surface.

33. The porous thin-film membrane according to claim 19, wherein the thin-film membrane is on a porous carrier structure.

34. A method for producing a porous thin-film membrane according to claim 19, which involves crosslinking the pore-forming proteins or peptides to each other covalently.

35. The method according to claim 34, wherein a) at least one initiator, chain-transfer agent, or catalyst for ROMP is bonded covalently to each pore-forming protein or peptide via at least one amino acid radical, b) the initiator-, chain-transfer agent- or catalyst-functionalised, pore-forming protein or peptide is reacted with monomers, and protein/peptide polymer conjugates are formed, in which polymers or statistical copolymers with groups which are crosslinkable by radiation, radical reactions or click-chemical reactions are formed, and c) the protein/peptide polymer conjugates is crosslinked.

36. A method of separating molecules comprising contacting the porous thin-film membrane according to claim 19 with the molecules and isolatng the molecules from one another according to charge, size, chemical composition, intermolecular interactions or chirality.

Description

[0048] There are hereby shown:

[0049] FIG. 1 a schematic view on a planar membrane according to the present invention

[0050] FIG. 2 a synthesis possibility, by way of example, for producing a thin-film membrane according to the invention

[0051] FIG. 3 the principles for producing a thin-film membrane according to the invention on a porous carrier surface

[0052] FIG. 4 a view in a modified channel of a pore-forming protein or peptide of a membrane according to the invention for the application possibility for separation of enantiomer mixtures.

[0053] No method has existed to date for producing membranes with high density of pore-forming proteins or peptides, in particular transmembrane proteins which form continuous pores in the thin-film membrane with exactly uniform size in the range of a few nm, which method is based on a different way from using the uniformity of pore-forming proteins or peptides.

[0054] In addition, no membranes exist in which the pore-forming proteins and peptides are bonded covalently so that continuous pores are formed in the thin-film membrane.

[0055] This defect is eliminated by the present invention.

[0056] FIG. 1 shows a plan view on a planar thin-film membrane according to the present invention, with high density of linked pore-forming proteins or peptides. The pore-forming proteins and peptides thereby typically have a pore internal diameter of 1 to 3 nm and are bonded together covalently via a network of polymers. As an alternative hereto (not illustrated), the pore-forming proteins and peptides can also be linked together directly via bi- or multifunctional, monomolecular linkers.

[0057] Membranes, in which pore-forming proteins and peptides such as FhuA sit with high density with an open channel of approx. 1-3 nm diameter can be produced in two different ways: by crosslinking the polymer chains of protein/peptide copolymer conjugates (FIG. 2) and also by direct crosslinking of the pore-forming proteins and peptides with a bi- or multifunctional linker.

[0058] The preparation of protein/peptide polymer conjugates is described numerous times with globular and soluble proteins and also viruses (Polym. Chem. 2015, 6, 5143 and Chem. Commun. 2011, 47, 2212 give an overview). The conjugate synthesis with pore-forming proteins or peptides by the grafting from approach is however not known. The used grafting from has the advantage over different strategies for synthesis of protein/peptide polymer conjugates that a comparatively high number of polymer chains can be grown from the protein/peptide surface.

[0059] FIG. 2 shows, by way of example, the synthesis of protein/peptide polymer conjugates by binding-on an initiator unit to the amino acid radicals (left) and subsequent polymer synthesis of the protein/peptide surface. In the example, the copolymerisation of N-isopropylacrylamide (NIPAAm) with approx. 5% 2-(dimethylmaleimido)-N-ethylacrylamide (DMIAAm) is shown. The side chains of DMIAAm can be crosslinked in a [2+2] cycloaddition using UV light. The crosslinking effected by the UV light thereby produces the final membrane.

[0060] For the synthesis shown in FIG. 2, firstly an initiator for the polymerisation is bonded to an amino acid radical, in the example to the lysin radicals of FhuA. The subsequent polymerisation is shown by NIPAAm, but can also be effected with other monomers. In order to make possible the crosslinking of the polymer chains, a corresponding comonomer such as DMIAAm at approx. 5% is added. The maleimide units can link polymer chains by irradiation with UV light in a [2+2] cycloaddition.

[0061] Pore-forming proteins and peptides have an intrinsic interface activity due to their hydrophilic and hydrophobic regions. In contrast to unmodified proteins, the interface activity of protein/peptide polymer conjugates is generally again significantly higher. The conjugates self-assemble at the air/water interface from greatly diluted solution and can be bonded by linking the polymer chains to each other to form a stable, thin membrane. After evaporation of the water phase, this membrane is situated in a planar manner on the used support.

[0062] In this connection, FIG. 3 shows, by way of example, the principle of self-assembly of membrane protein-polymer conjugates at the water-air interface and linking of the polymer chains. After evaporation of the water, the membrane sits on a porous carrier.

[0063] In addition to the linking of covalently bonded polymer chains, pore-forming proteins and peptides can also be linked directly by crosslinkers. The crosslinkers must have at least two functionalities which are separated by a short spacer and react with amino acid radicals. One example is glutaraldehyde, which reacts with the amino groups of the amino acid lysin. In this way, the spacing of the pore-forming proteins or peptides in the membrane is again smaller and the greatest possible density of the protein pores can be achieved.

[0064] One possible application of such membranes is use for separation of enantiomer mixtures. The components are smaller than the channel diameter in order to ensure a throughflow of the enantiomers, preferably only one enantiomer being allowed to pass through the channel. This is achieved by chemical and/or genetic modifications in the channel interior, which modifications interact differently with the different enantiomers. Possible substance classes are enantiomeric amino acids but also amines, epoxides and terpenes. FIG. 4 shows, by way of example, tryptophan in the channel for chiral separation of amino acids.

[0065] FIG. 4 thereby shows various strategies for FhuA Engineering in order A) to separate sterically non-demanding (FhuA Wildtype) and B) sterically demanding (FhuA 1-159 with fluorescein marking) enantiomer mixtures (cross-sectional view of Fhua).

[0066] Commercial membranes with covalently bonded pore-forming proteins or peptides as pores are a new class of membranes. The main field of application is separations of all types. By means of the uniform size of the protein/peptide channels, firstly a separation with a precision not achieved to date is possible because of a size exclusion. Particles, the size of which is below the 2-3 nm diameter of the pores, can pass through the membrane, whilst larger particles are held back. In addition, the low throughflow resistance makes possible flows which have not been achieved to date via the membrane with low energy requirement. By introducing functionalities into the channel interior, separations can be implemented which go beyond purely a size exclusion. An essential field of application resides here in the separation of an enantiomer mixture. The membranes therefore allow a new approach for producing enantiomer-pure compounds, which approach offers a significant advantage with respect to costs and efficiency relative to existing methods.