Engineered proteins, compositions, devices comprising them and use thereof

Abstract

The present invention relates to Tetratricopeptide Repeat (TPR) and compositions comprising them, in particular film or membrane compositions. The invention also relates to a process for the preparation of said compositions, to the use thereof as a separator in a battery or a supercapacitor and to devices comprising said compositions.

Description

[0001] ENGINEERED PROTEINS, COMPOSITIONS, DEVICES COMPRISING THEM AND USE THEREOF

[0002] FIELD OF THE INVENTION

[0003] [1] The present invention relates to Tetratricopeptide Repeat (TPR) polypeptides and compositions comprising them, in particular film or membrane compositions. The invention also relates to a process for the preparation of said compositions, to the use thereof as a separator in a battery or a supercapacitor and to devices comprising said compositions.

[0004] BACKGROUND

[0005] [2] Protein engineering is gaining a growing attraction in the development of biological tools for medicine and pharmacology applications. A particular case of such use is the incorporation of proteins in batteries, in particular within the biomedical field, as proteins are generally compatible with living organisms, unlike certain materials known in the art. This technique allows introducing tailored functionalities and properties to a protein by site-selective modification of the peptide sequence of the protein, which may alter one or more of the following: (i) primary structure of the protein, which may play a role on the solvation of ions in a medium, such as an electrolyte, (ii) the secondary structure of the protein, which can have a role on improving the ionic conductivity of an electrolyte while having an effect on the solubility of the protein; (iii) tertiary structure of a protein (e.g. self-assembly of proteins).

[0006] [3] Engineered proteins have already been employed in batteries as major components of electrolytes to replace toxic materials. Usually, the main limitation of these systems relates to their synthetic availability at a relevant industrial scale. Small protein amounts can be added as additives to participate in the formation of protective layers on both cathodes and anodes and improve the electrochemical performance of the battery. Engineered proteins can also be employed as a catalyst and in the form of artificial protective films (SEI layer). Wang etal. review such uses of engineered proteins in battery electrolyte in sections 3 and 4 of Adv. Energy Mater. 2022, 12, 2202568.

[0007] [4] Another opportunity is to employ engineered proteins in compositions used to isolate cathodes and anodes from each other and provide a pathway for ions to move between electrodes, thus forming the so-called separator. For acting as a separator, the material must have excellent chemical / mechanical properties, interconnected microporous networks, and good electrochemical performance and stability. Currently, there is still no single ideal separator for all batteries that provides optimal performance under all operational circumstances, as conventional separators based on polyolefin materials, such as polyethylene, suffer from a poor wettability, in particular for aqueous electrolytes. This limitation is important, and it can impact ion conductivity through the separator as well as the overall performance of the battery. Also, the poor wettability of the separator generally leads to long manufacturing time of the battery. The biocompatibility of polyolefins also represents an area of improvement, as these may lead to the formation of a fibrous capsule around the material when incorporated in implantable medical devices. Thus, given the limitations of conventional separators, the preparation of separators using proteins has emerged during the past years, bearing in mind that a good separator shall possess a good wettability towards electrolyte (e.g. aqueous solution of potassium chloride) combined with a good redox stability to ensure that the protein is not denatured at the interface with either the cathode or the anode.

[0008] [5] In this regard, Fu et al. disclosed in 2018 in J. Phys. Chem. Lett. 2018, 9, 245 a Li-S battery comprising a separator which incorporates a self-assembled protein nanofilter based on gelatin that is employed to trap polysulfides. Such approach allowed improving the retention capacity of the battery from 65% to 75% if compared with the analogous battery deprived of the self-assembled protein. This approach is however limited to Li-S batteries.

[0009] [6] In a different approach, protein nanofibers have also been used by Nie and coworkers in ACS Appl. Energy Mater. 2019, 11, 32373 to coat a separator and prevent the growth of lithium dendrite in Li-based batteries while improving the wettability of the electrolyte.

[0010] [7] Chen et al. disclose in J. Mater. Chem. 2020,8, 7377 a lithium-sulfide battery incorporating protein-based nanofabric based on the “Janus” protein allowing for preventing the growth of lithium dendrites and trapping polysulfides. The fabric is engineered as a bi-layer material based on gelatin whereby one layer is conductive, while the other layer is insulator. This approach is however limited to Li-S batteries.

[0011] [8] In a further approach, Chen and co-workers report in Adv. Energ. Matter. 2020, 10, 1903642 a lithium-sulfide battery incorporating a separator that is a composite material of carbon nanofiber with denatured gelatin or zein. After 250 charging cycles, the cells incorporating gelatin / carbon nanofiber separators retained about 57% of the charge capacity.

[0012] [9] Serra and co-workers report in Electrochi mica Acta 2023, 462, 142746 a separator for lithium-ion batteries based on cellulose and soy protein, both of which are readily available waste materials. In this study, the authors show that cellulose and soy protein-based membranes show excellent compatibility with the electrolyte in terms of wettability and swelling. The ionic conductivity value, lithium transference number and battery performance in cathodic half-cells are ~ 5.8 mS-cm-1, 0.77, and 112 mAh g-1at 1C-rate, respectively. The same team of researchers disclosed later in Journal of Energy Storage 2024, 75, 109748 a lithium-ion battery comprising a separator based on a combination of wool and soy protein isolate.

[0013]

[0010] Gao and co-workers disclose in Journal of Power Sources 2022, 541, 231658 a high energy density supercapacitor a polymer electrolyte matrix consisting of soybean protein isolate modified with polyacrylic acid. The material combines the film-forming properties of soybean protein isolate and the hydrophilic character of the carboxyl groups of the polyacrylic acid, allowing for the formation of a polymer electrolyte membrane comprising lithium cations which can be used in a supercapacitor operated in a voltage range of 0 - 2 V. Said supercapacitor exhibits a single electrode specific capacitance of up to 293.08 F / g and an energy density of 38.76 Wh / kg at a power density of 734.38 W / kg.

[0014]

[0011] In a different approach, Mosa and co-workers disclose in Adv. Energy Mater.

[0015] 2017, 1700358 a biocompatible and non-toxic film having a high energy density and comprising chemically modified mammalian proteins (bovine serum albumin grafted with polyamines and heme protein myoglobin) sandwiched between sheets of reduced graphene oxide. The film was used as separator in a supercapacitor.

[0016]

[0012] On the other hand, Tetratricopeptide Repeats (TPR) are motifs that form part of structural domains in proteins and can act as an interaction point with other biomolecules such as proteins involved in transcription, protein translocation, and protein degradation. The TPR unit is composed of 34 amino acids that structurally encode a helix-turn-helix motif. The stack of the helices by self-assembly lead to tertiary structures. Amongst the 34 amino acids, only 8 appear to have structural relevance, which leaves plenty of room to protein engineering and the introduction of functional mutations. For this reason, Consensus Tetratricopeptide Repeats (CTPR) have gained attraction as a platform for protein engineering, as summarized in Acc. Chem. Res. 2021 54, 4166 by Uribe and coworkers.

[0017]

[0013] For instance, Sanchez-de Alcazar et al. disclose in Nanoscale Adv. 2019, 1, 3980 a strategy for the preparation of biocompatible, thin and flexible nanopatterned protein films by self-assembly of T etratricopeptide Repeats (TPR) over a nanopatterned surface. In particular, the film is formed by drying of an aqueous solution of TPR and poly(ethylene)glycol PEG-400 deposited on a surface, followed by cross-linking of glutaraldehyde by vapour diffusion. A film was then prepared as an optical device by incorporation of TPR modified with a dye (rhodamine 6G). The resulting nanopattern behaved as second order surface emitting distributed feedback, showing the success of the approach in bringing functionality to TPR assembly. However, the authors are silent about the use of CTPR and engineered TPRs in battery applications, e.g. as a separator.

[0014] From what is disclosed in the art, it appears that there is still a need for separator compositions for batteries that possess inter alia, high wettability, redox stability and biocompatibility.

[0018] SUMMARY OF THE INVENTION

[0019]

[0015] The inventors have found that membranes of Tetratricopeptide Repeats (TPR) polypeptides are particularly useful as separator in electrochemical devices, such as supercapacitors. Surprisingly, these membranes combine the desired properties of separators in terms of wettability, robustness and mechanical properties. They are also highly biocompatible, which makes them particularly suitable for being used in biomedical devices incorporating batteries or supercapacitors. The high wettability of the separator membranes of the invention allows assembling electrochemical devices in a fast manner and with no need for the use of surfactants.

[0020]

[0016] The inventors have also developed engineered TPR polypeptides bearing a larger number of hydrophilic residues by incorporating glutamic acid in the sequence of TPR polypeptides and membranes comprising these proteins. These polypeptides constitute a first aspect of the invention, which relates to a Tetratricopeptide Repeat (TPR) polypeptide having the sequence according to [Xi X2 X3 WX5 Xe X7G X9 X10Y X12 X13 X14 X15X16 Y Xis X19 A X21 X22 X23Y X25X26A X28X29X30X31 P X33 X34]n (SEQ ID NO: 1), wherein a glutamic acid (E) or aspartic acid (D) is present in between 9 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, and wherein n equals to or is more than 1. These polypeptides are particularly useful in the formation of films or membrane compositions useful as separators in electrochemical devices.

[0021]

[0017] Thus, a second aspect of the invention relates to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide and a plasticizer that is a polymer having a molecular weight of at least 1 ,000 g / mol; preferably comprised between 1 ,000 g / mol and 10,000 g / mol wherein the Tetratricopeptide Repeat (TPR) polypeptide has the sequence [Xi X2X3W X5X6X7G X9X10 Y X12 X13 X14 X15 X16 Y Xis X19 A X21 X22 X23Y X25X26A X28X29 X30 X31 P X33 X34]n (SEQ ID NO: 1), wherein a glutamic acid (E) or aspartic acid (D) is present in between 7 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, and wherein n equals to or is more than 1.

[0022]

[0018] A third aspect of the invention relates to a method for the preparation of a composition according to the second aspect of the invention comprising the steps of: (i) providing an aqueous solution of the Tetratricopeptide Repeat (TPR) polypeptide and the plasticizer of the second aspect of the invention;

[0023] (ii) depositing the solution provided in (i) on a surface; and

[0024] (iii) drying the product of step (ii).

[0019] A fourth aspect of the invention relates to the product obtainable by the method of the third aspect of the invention.

[0025]

[0020] A fifth aspect of the invention relates to the use of the composition according to the second or the fourth aspect of the invention as a separator for a battery or for a supercapacitor. The inventors have particularly found that the capacity retention of supercapacitors bearing the membrane according to the invention, in particular the membrane comprising a TPR polypeptide having a sequence according to SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8, in particular SEQ ID NO: 6, SEQ ID NO: 8, preferably SEQ ID NO: 6, is higher than conventional separators such as glass fiber. The inventors have also found that supercapacitors comprising the membrane according to the second or the fourth aspect of the invention are faster to charge than devices comprising conventional separators such as glass fiber.

[0026]

[0021] A sixth aspect of the invention relates to a device, such as a battery or a supercapacitor, comprising the composition according to the second or fourth aspect of the invention.

[0027] BRIEF DESCRIPTION OF THE DRAWINGS

[0028]

[0022] Fig. 1 shows ribbon representation of the structure of engineered TPR polypeptide E-variants with the incorporated glutamic acid residues (E) shown as sticks. Structures are based on the crystal structure of the wild type CTPR4 (SEQ ID NO: 9) protein (PDB ID: 2HYZ). The structure of one repeat is shown, while the number of identical repeats that comprise each CTPR4 protein variant (4) is indicated. The corresponding sequences for each TPR polypeptide unit are shown with the incorporated mutations (E) underlined (SEQ ID NO: 3, 6 and 7, for left, middle and right panel respectively).

[0029]

[0023] Fig. 2 shows (a) a scheme of the process for the preparation of the self-standing Tetratricopeptide Repeats (TPR) membrane for supercapacitors; and (b) photography of a membrane prepared according to the procedure described herein.

[0030]

[0024] Fig. 3 shows photographs of the cross-linked product of a composition prepared according to the procedure disclosed in the Examples by replacing Tetratricopeptide Repeats (TPR) polypeptide by Bovine Serum Albumin (BSA) (top) or lysozyme (LYS) (bottom).

[0031]

[0025] Fig. 4 shows (a) Electrochemical Impedance Spectroscopy plots of supercapacitors comprising as a separator glass fiber or a membrane comprising a Tetratricopeptide Repeats (TPR) polypeptide having as sequence SEQ ID NO: 3, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID N:8 and (b) the high frequency amplification region of the plots in which the Z'axis intercept of the plots in the high frequency region refers to the equivalent series resistance (Rs), the size of the semicircular plots between the high frequency and intermediate frequency region reflects the interfacial charge transfer resistance (Ret), the length of the straight line in the intermediate frequency region determines the ion transfer between the electrolyte and porous electrode surface, and the slope of the plots in the low frequency region determines the ion diffusion through electrode microporosity.

[0032]

[0026] Fig. 5 shows cyclic voltammetry curves of supercapacitors comprising as a separator glass fiber or a membrane comprising a TPR polypeptides having as sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID N:8 at different scan rates of 10 mV / s, 20 mV / s, 50 mV / s, 100 mV / s or 200 mV / s.

[0033]

[0027] Fig. 6 shows the charge / discharge profile of supercapacitors comprising as a separator glass fiber or a membrane comprising a TPR polypeptides having as sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID N:8, and when carried out at current densities of 0.1 A / g, 1 A / g, 10 A / g and 20 A / g.

[0034]

[0028] Fig. 7 shows (a) Specific capacitance and voltage drop of supercapacitors comprising as a separator glass fiber or a membrane comprising a TPR polypeptides having as sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID N:8 when carried out at current densities of 0.1 A / g, 1 A / g, 10 A / g and 20 A / g, and (b) Ragone plot (per mass) of supercapacitors comprising as a separator glass fiber or a membrane comprising a TPR polypeptides having as sequence SEQ ID NO:3, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID N:8.

[0035]

[0029] Fig. 8 shows the evolution of capacity retention (circle symbol), expressed as a percentage, and coulombic efficiency (square symbol) of supercapacitors comprising as a separator glass fiber (filled symbols) or a membrane comprising a TPR polypeptides having as sequence SEQ ID NO: 6 (empty symbols).

[0036]

[0030] Fig. 9 shows (top) Electrochemical Impedance Spectroscopy plots of supercapacitors comprising as a separator glass fiber or a membrane comprising a Tetratricopeptide Repeats (TPR) polypeptide having as sequence SEQ ID NO:8 (wild type) and 5% PEG-400 (left, comparative example) or 5% PEG-4000 (right) as plasticizer; and (bottom) cyclic voltammetry curves of supercapacitors comprising a membrane comprising a TPR polypeptides having as sequence SEQ ID NO:8 and 5% PEG-400 (left, comparative example) or 5% PEG-4000 (right) as plasticizer at different scan rates of 10 mV / s, 20 mV / s, 50 mV / s, 100 mV / s.

[0037]

[0031] Fig. 10 shows a) Schematic representation of the NaCI doped CTPR4-4E film preparation and final EDLC device (SS: Stainless-Steel; TB: Teflon Body; AC: activated carbon electrodes), b) Electrochemical impedance spectroscopy (EIS) of the salt-doped protein devices before cycling, c) Cyclic Voltammetry (CV) of the devices with different salt content sweeping at 10 mV / s. d) Galvanostatic charge-discharge cycling of the devices at 0.1 A / g. e) Specific capacitance vs. current density (normalized to the mass of activated carbon in the electrode) for the same EDLCs, extracted from galvanostatic charge-discharge cycling, f) Ragone plot of the EDLCs fabricated with protein films doped with salt as membrane.

[0038] DETAILED DESCRIPTION

[0039]

[0032] All terms as used herein in this application, unless otherwise stated, shall be understood in their ordinary meaning as known in the art. Other more specific definitions for certain terms as used in the present application are as set forth below and are intended to apply uniformly throughout the specification and claims unless an otherwise expressly set out definition provides a broader definition.

[0040]

[0033] For the purposes of the invention, any ranges given include both the lower and the upper end-points of the range. Ranges or values given, such as temperatures, times, molar ratio, volume ratio and the like, should be considered approximate when they are defined by the term “about” (i.e. with a 5% margin of variation around indicated point).

[0041]

[0034] The term “tetratricopeptide repeat”, or its acronym “TPR”, as used herein refers to a structural motif consisting of at least 2 tandem repeats of 34 amino acid residues, preferably at least 3 tandem repeats of 34 amino acids, which adopts a helix-turn-helix arrangement in its tri-dimensional structure. The term “TPR” is also meant to encompass the term “consensus tetratricopeptide repeat”, or its acronym “CTPR”. The term “tetratricopeptide” refers to the 34 amino acid sequence which forms the basic unit of the TPR. The term “CTPR” refers to a specific TPR sequence based on the consensus, i.e. the most prevalent amino acid at each position derived from multiple aligned TPR sequences. The TPR is characterized by a basic unit of the TPR according to the wildtype CTPR sequence AEAWYNLGNAYYKQGDYDEAIEYYQKALELDPRS (SEQ ID NO: 9) or modifications thereof as disclosed herein . The term TPR4 refers to 4 repeats of said TPR.

[0042]

[0035] The terms “peptide”, as used herein, refers to a sequence of amino acids, analogues or mimetics having substantially similar or identical functionality. The term “peptide” also includes analogues having synthetic and natural amino acids joined together by peptide bonds. A peptide, as used herein and in the claims, is also intended to include analogues, derivatives, salts, retro-inverso isomers, mimics, mimetics, or peptidomimetics thereof. For example, a peptidic structure of a modulator of the invention may be further modified to increase its stability, bioavailability, solubility, etc. "Analog", "derivative" and "mimetic" include molecules which mimic the chemical structure of a peptidic structure and retain the functional properties of the peptidic structure. Approaches to designing peptide analogues, derivatives and mimetics are known in the art. For example, see Farmer, P. S. in Dmg Design (E. J. Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball, J. B. and Alewood, P. F. (1990) J. Mol. Recognition 3:55. Morgan, B. A. and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R. M. (1989) Trends Pharmacol. Sci. 10:270. See also Sawyer, T. K. (1995) Peptidomimetic Design and Chemical Approaches to Peptide Metabolism in Taylor, M. D. and Amidon, G. L. (eds.) Peptide-Based Drug Design: Controlling Transport and Metabolism, Chapter 17; Smith, A. B. 3rd, et al. (1995)J. Am. Chem. Soc.

[0043] 117:11113-11123; Smith, A. B. 3rd, etal. (1994) J. Am. Chem. Soc. 116:9947-9962; and Hirschman, R., et al. (1993) J. Am. Chem. Soc. 115:12550-12568. A "derivative" (e.g., a peptide or amino acid) includes forms in which one or more reaction groups on the compound have been derivatised with a substituent group. Examples of peptide derivatives include peptides in which an amino acid side chain, the peptide backbone, or the amino- or carboxy-terminus has been derivatised (e.g., peptidic compounds with methylated amide linkages). An "analogue" of a compound X includes compounds which retain chemical structures necessary for functional activity, yet which also contains certain chemical structures which differ. An example of an analogue of a naturally-occurring peptide is a peptide which includes one or more non-naturally-occurring amino acids. A "mimetic" of a compound includes compounds in which chemical structures of the compound necessary for functional activity have been replaced with other chemical structures which mimic the conformation of the compound. Examples of peptidomimetics include peptidic compounds in which the peptide backbone is substituted with one or more benzodiazepine molecules (see e.g., James, G. L. et al. (1993) Science 260:1937-1942).

[0044]

[0036] Peptides sequences and protein sequences in general, can be defined by the amino position, wherein the first position (1) is characteristically the N-terminus amino acid of the peptide and the last position (n) is characteristically the last position on the C-terminus of the peptide. Therefore, amino X4, wherein X refers to any amino acid, corresponds to the fourth amino acid of the amino acid sequence, counting from the N-terminal amino acid.

[0045]

[0037] The term “peptide” as used herein refers to amino acid sequences with 50 or less amino acids, while the term “polypeptide” as used herein refers to an amino acid sequence that has more than 50 amino acids. Therefore, the definition of “peptide” is equally valid for the term “polypeptide”.

[0046]

[0038] The term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. The term amino acid includes naturally occurring amino acids (Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Vai), uncommon natural amino acids, nonnatural (synthetic) amino acids. The amino acids are preferably in the L configuration, but also D configuration, or mixtures of amino acids in the D and L configurations. The term “natural amino acids” comprises aliphatic amino acids (glycine, alanine, valine, leucine and isoleucine), hydroxylated amino acids (serine and threonine), sulfured amino acids (cysteine and methionine), dicarboxylic amino acids and their amides (aspartic acid, asparagine, glutamic acid and glutamine), amino acids having two basic groups (lysine, arginine and histidine), aromatic amino acids (phenylalanine, tyrosine and tryptophan) and cyclic amino acids (proline). As used herein the term “non-natural amino acid” refers to a carboxylic acid, or a derivative thereof, substituted at position a with an amine group and being structurally related to a natural amino acid. Illustrative nonlimiting examples of modified or uncommon amino acids include 2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2’-diaminopimelicacid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylasparagine, hydroxylysine, allohydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylglycine, N-methylisoleucine, 6-N-methyl-lysine, N-methylvaline, norvaline, norleucine, ornithine, etc.

[0047]

[0039] In the context of the invention, the term "cross-linked" refers to the presence of a covalent bond between a first polypeptide chain and a second polypeptide chain or to the presence of a group for covalently linking said chains. The term "cross-linking reagent" refers to an entity or agent which is an intermediate molecule to catalyze the covalent linkage of two polymers or polypeptides. The term “cross-linked” when referring to a TPR refers to the fact that a residue comprised in a side chain of said peptide is covalently bonded to a residue comprised in a side chain of the same and / or another peptide molecule. In the context of the invention, the cross-linked polypeptides have preferably substantially the same sequence. Several cross-linking sites are known in the art, and include for instance, amino or thiol groups on the side chain of the peptide backbone. Suitable agents for promoting the cross-linking of peptides are known in the art. For instance, aldehydes, such as glutaraldehyde, are known cross-linking agent for covalently binding amino groups comprised in a peptide side chain.

[0048]

[0040] In the context of the invention, the term “plasticizer” refers to a chemical substance suitable for improving the flexibility, durability and handling properties of the composition comprising it. In the context of the invention, the plasticizer is selected from the plasticizers known in the art as being suitable components for separator membranes or films in electrochemical devices. Such plasticizers include for instance organic molecules such as (tere)phthalates (e.g. DBP, DEHP), benzoate esters, trimellitate esters, polyesters, citrates, azelates, sebacates, dimethylcarbonate and adipates (e.g. DOA), or polymers, such as poly(ethylene)glycol, poly(propylene) glycol, poly(vinyl acetate), or polyesters. In the preferred embodiments of the invention, the plasticizer is a polymer. Said polymer is preferably selected from the group consisting of poly(ethylene)glycol, poly(propylene) glycol and poly(vinyl acetate) and preferably has an average molecular weight of between 1,000 g / mol and 10,000 g / mol.

[0049]

[0041] In the context of the invention, the term “separator” refers to an electrically insulating component of an electrochemical device that is in electrochemical contact with the anode and the cathode of said device and is suitable for allowing the flow of ions between said electrodes. Said separator may have the form of a film or a membrane. In the context of the invention, both “film” and “membrane” are sufficiently porous to allow for the exchange of ions between the electrodes.

[0050]

[0042] As mentioned above, a first aspect of the invention relates to a Tetratricopeptide Repeat (TPR) polypeptide having the sequence according to [Xi X2 X3 W X5 Xe X7G X9 X10 Y X12 X13 X14 X15X16 Y Xis X19 A X21 X22 X23Y X25X26A X28X29X30X31 P X33 X34]n (SEQ ID NO: 1), wherein a glutamic acid (E) or aspartic acid (D) is present in between 9 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, and wherein n equals to or is more than 1. In a preferred embodiment of the first aspect of the invention, the TPR polypeptide is one wherein a glutamic acid or an aspartic acid amino acid is present in at least positions 2, 5, 6, 16, 18, 19, 22, 29 and 31).

[0051]

[0043] In a preferred embodiment of the first aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide according is one wherein a glutamic acid or an aspartic acid amino acid is present in 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence.

[0052]

[0044] In a preferred embodiment of the first aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide is one having the sequence according to [Xi E X3WEE X7G X9X10Y X12 X13 X14 X15 DYDEA X21E X23Y X25X26A X28E X30DP X33 X34]n (SEQ ID NO: 2), wherein n > 1.

[0053]

[0045] In a further embodiment of the first aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide is one having the sequence according to SEQ ID NO: 3.

[0054]

[0046] SEQ ID NO: 3

[0055] AEAWE E LGNAYYKQGDYDEAI EYYQKAL E LDPRSAEAWE E LGNAYYKQGDYDEAI EYYQKAL E LDPRSAE AWE E LGNAYYKQGDYDEAI EYYQKAL E LDPRSAE AWE E LGNAYYKQGDYDEAI EYYQKAL E LDPRS

[0056]

[0047] In yet another embodiment of the first aspect of the invention, the Tetratricopeptide Repeat (TPR) is one having the sequence according to [Xi E X3 WEE X7G X9X10 Y X12 X13 X14 X15 DYDEA X21E X23Y X25X26A X28E X30DP X33 X34]n (SEQ ID NO: 2), wherein n > 1 or having the sequence according to SEQ ID NO: 3, and further comprising a glutamic acid or an aspartic acid amino acid in at least one, at least two, at least three or at least four of positions 9, 12, 25 and 26.

[0057]

[0048] In one more embodiment of the first aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide is one having the sequence according to [Xi E X3WEE X7G E X10 Y E X13 X14 X15 DYDEA X21E X23Y X25X26A X28E X30DP X33 X34]n (SEQ ID NO: 4), wherein n > 1.

[0058]

[0049] In another embodiment of the first aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide is one having the sequence according to [Xi E X3 WEE X7 G E X10 Y E X13 X14 X15 DYDEA X2IE X23Y E E A X28E X30DP X33 X34]n (SEQ ID NO: 5), wherein n > 1.

[0059]

[0050] In an embodiment of the first aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide is one having a sequence according to SEQ ID NO: 6 or SEQ ID NO: 7.

[0060]

[0051] SEQ ID NO: 6

[0061] AEAWE E LG EAYEKQGDYDEAI EYYQKAL E LDPRSAEAWE E LGEAYEKQGDYDEAI EYYQKAL E LDPRSAE AWE E LGEAYEKQGDYDEAI EYYQKAL E LDPRSAE AWE E LG EAYEKQGDYDEAI EYYQKAL E LDPRS

[0062]

[0052] SEQ ID NO: 7 AEAWEELGEAYEKQGDYDEAIEYYEEALELDPRSAEAWEELGEAYEKQGDYDEAIEYYEEALELDPRSAE AWE E LGEAYEKQGDYDEAI EYYE E AL E LDPRSAE AWE E LG EAYEKQGDYDEAI EYYE EAL E LDPRS

[0063]

[0053] In another embodiment of the first aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide is one wherein n= 1. In another embodiment of the first aspect of the invention, the TPR polypeptide is one wherein n is 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. In another embodiment of the first aspect of the invention, the TPR polypeptide is one wherein n=4. In another embodiment of the first aspect of the invention, the TPR polypeptide is one wherein n> 4.

[0064]

[0054] As mentioned above, the Tetratricopeptide Repeat (TPR) polypeptide of the invention is suitable for forming films or membranes which may act as separator in electrochemical devices.

[0065] The second aspect of the invention thus relates to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide and a plasticizer that is a polymer having a molecular weight of at least 1 ,000 g / mol; preferably comprised between 1 ,000 g / mol and 10,000 g / mol wherein the Tetratricopeptide Repeat (TPR) polypeptide has the sequence [Xi X2X3W X5X6X7G X9X10 Y Xi2X13 X14 X15 X16 Y Xi8X19 A X2iX22X23Y X25X26A X28X29 X30 X31 P X33 X84]n(SEQ ID NO: 1), wherein a glutamic acid (E) or aspartic acid (D) is present in between 7 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, and wherein n equals to or is more than 1.

[0055] In a preferred embodiment of the second aspect of the invention, the composition is provided in the form of a film or a membrane.

[0066]

[0056] In a preferred embodiment of the second aspect of the invention, the polypeptide of the composition is one wherein a glutamic acid or an aspartic acid amino acid is present in 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the Xi amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence. Preferably, the Tetratricopeptide Repeat (TPR) polypeptide is one wherein a glutamic acid or an aspartic acid amino acid is present in at least positions 2, 5, 6, 16, 18, 19, 22, 29 and 31.

[0067]

[0057] In another preferred embodiment of the second aspect of the invention, the composition is one wherein the Tetratricopeptide Repeat (TPR) polypeptides as defined in any of the preferred embodiments of the first aspect of the invention described above.

[0068]

[0058] In a further preferred embodiment of the second aspect of the invention, the composition is one wherein the Tetratricopeptide Repeat (TPR) polypeptides are crosslinked.

[0069]

[0059] Thus, preferred embodiments of the second aspect of the invention relate to a composition provided as a film or a membrane and comprising cross-linked Tetratricopeptide Repeat (TPR) polypeptides having the sequence as defined in any of the embodiments of the first aspect of the invention.

[0070]

[0060] In a preferred embodiment of the second aspect, the composition provided as a film or a membrane and comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptides having the sequence having a sequence selected from SEQ ID NO:3, SEQ ID NO:6, or SEQ ID NO:7.

[0071]

[0061] In a particular embodiment of the second aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide is cross-linked and is further selected from one having the sequence [Xi X2 X3 W X5 Xe X7G X9 X10Y X12 X13 X14 X15X16Y Xis X19A X21 X22 X23 Y X25 X26 A X28 X29 X30 X31 P X33 X34]n (SEQ ID NO: 1), wherein a glutamic acid (E) or aspartic acid (D) is present in between 7 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, and wherein n equals to or is more than 1 and wherein a glutamic acid or an aspartic acid amino acid is present in at least positions 2, 5, 6, 16, 18, 19, 22, 29 and 31.

[0072]

[0062] In a more particular embodiment, the Tetratricopeptide Repeat (TPR) polypeptide may further be selected from the Consensus Tetratricopeptide Repeats (TPR) having the sequence SEQ ID NO: 8, said TPR polypeptide being preferably cross-linked.

[0073]

[0063] SEQ ID NO: 8 AEAWYNLGNAYYKQGDYDEAIEYYQKALELDPRSAEAWYNLGNAYYKQGDYDEAIEYYQKALELDPRSAE AWYNLGNAYYKQGDYDEAIEYYQKALELDPRSAEAWYNLGNAYYKQGDYDEAIEYYQKALELDPRS

[0064] Further preferred embodiments of the second aspect of the invention relate to a composition provided as a film or a membrane and comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having the sequence SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7 or SEQ ID NO: 8.

[0074]

[0065] Methods of cross-linking proteins are well known in the art and will become apparent to the skilled person upon reduction to practice of the invention. Such methods are summarized for instance by Jayachandran et al. in Molecules 2022, 27, 8124. It is preferred that the chosen cross-linking method does not involve carboxylic acid groups on the side chain of the peptide sequence, as it is believed that these groups are useful in enhancing the wettability of the film and / or enhancing ion transport through said composition. For instance, cross-linking of amino groups of the peptide side chain is preferred. Suitable reagents for the cross-linking of amino groups are well known in the art and will become apparent to the skilled person upon reduction to practice of the invention; those include for instance, glutaraldehyde, epoxy compounds, formaldehyde, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and disuccinimidyl suberate (DSS). It is preferred that the cross-linking is the result of the treatment of the composition with glutaraldehyde. Preferably, said treatment is carried out by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0075]

[0066] In preferred embodiments, the second aspect of the invention relate to a composition provided as a film or a membrane and comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptides having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7, preferably SEQ ID NO: 3, wherein the crosslinking is the result of the treatment of the composition with glutaraldehyde.

[0076]

[0067] Suitable plasticizers for forming part of the composition of the second aspect of the invention are well known in the art and will become apparent to the skilled person upon reduction to practice of the invention. Those include for instance phthalates such as dibutyl phthalate, diethyl phthalate, triethyl citrate, poly(ethylene)glycol, poly(propylene) glycol, poly(vinyl acetate) and any other chemical substance imparting a plastic effect to the composition of the second aspect of the invention.

[0077]

[0068] In preferred embodiments of the second aspect of the invention, the composition is one wherein the plasticizer is a polymer. Said polymer is preferably selected from the group consisting of poly(ethylene)glycol, poly(propylene) glycol, polyesters and poly(vinyl acetate) and / or has an average molecular weight of at least 1,000 g / mol.

[0078]

[0069] In preferred embodiments of the second aspect of the invention, the composition is one wherein the plasticizer is a polymer having a molecular weight of no more than 10,000 g / mol.

[0070] In preferred embodiments of the second aspect of the invention, the composition is one wherein the plasticizer is selected from the group consisting of poly(ethylene)glycol, poly(propylene) glycol and poly(vinyl acetate).

[0079]

[0071] In further preferred embodiments of the second aspect of the invention, the composition is one wherein the plasticizer is poly(ethylene)glycol.

[0080]

[0072] When the plasticizer of the second aspect of the invention is a polymer, this one preferably has a molecular weight comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol.

[0081]

[0073] Thus, preferred embodiments of the second aspect of the invention relate to a composition wherein the plasticizer is poly(ethylene) glycol and the molecular weight of poly(ethylene)glycol is preferably of about 4,000 g / mol.

[0082]

[0074] Thus, preferred embodiments of the second aspect of the invention relate to a composition provided as a film or a membrane and comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, or SEQ ID NO: 7,

[0083] wherein the cross-linking is the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0084]

[0075] Other preferred embodiments of the second aspect of the invention relate to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID N°: 8; and a plasticizer that is poly(ethylene glycol).

[0085]

[0076] In other preferred embodiments, the second aspect of the invention relates to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and a plasticizer that is poly(ethylene glycol) wherein the molecular weight of poly(ethylene)glycol is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol.

[0086]

[0077] In other preferred embodiments, the second aspect of the invention relates to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from the group consisting of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8, and a plasticizer that is poly(ethylene glycol) wherein the molecular weight of poly(ethylene)glycol is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol.

[0087]

[0078] In other preferred embodiments, the second aspect of the invention relates to a composition provided as a film or a membrane comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7or SEQ ID NO: 8, and a plasticizer that is poly(ethylene glycol).

[0088]

[0079] In other preferred embodiments, the second aspect of the invention relates to a composition provided as a film or a membrane comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:8, and a plasticizer that is poly(ethylene glycol) wherein the molecular weight of poly(ethylene)glycol is comprised between 1 ,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol.

[0089] In other preferred embodiments, the second aspect of the invention relates to a composition provided as a film or a membrane comprising a cross-linked TPR polypeptide having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8, and a plasticizer that is poly(ethylene glycol), and wherein the cross-linking is the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0090]

[0080] In other preferred embodiments, the second aspect of the invention relates to a composition provided as a film or a membrane comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and a plasticizer that is poly(ethylene glycol) wherein the molecular weight of poly(ethylene)glycol is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol, and wherein the cross-linking is the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0091]

[0081] In other preferred embodiments, the second aspect of the invention relates to a composition comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and a plasticizer that is poly(ethylene glycol) wherein the molecular weight of poly(ethylene)glycol is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol.

[0092]

[0082] In other preferred embodiments, the second aspect of the invention relates to a composition comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having the sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8 and a plasticizer that is poly(ethylene glycol) wherein the molecular weight of poly(ethylene)glycol is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol; and wherein the cross-linking is the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0083] In other more preferred embodiments, the second aspect of the invention relates to a composition provided as a film or a membrane comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having the sequence SEQ ID NO: 3 or SEQ ID NO: 6 and a plasticizer that is poly(ethylene glycol) wherein the molecular weight of poly(ethylene)glycol is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol, and wherein the cross-linking is the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0093]

[0084] In other more preferred embodiments, the second aspect of the invention relates to a composition provided as a film or a membrane comprising a cross-linked Tetratricopeptide Repeat (TPR) polypeptide having a sequence selected from SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8 and a plasticizer that is poly(ethylene glycol) wherein the molecular weight of poly(ethylene)glycol is preferably of about 4,000 g / mol, and wherein the cross-linking is the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0094]

[0085] In other embodiments of the second aspect of the invention, the composition is one wherein the amount of the Tetratricopeptide Repeat (TPR) polypeptide is of between 5 and 50 .mol of TPR polypeptide per each gram of plasticizer in the composition. Preferably, the composition is one wherein the amount of the TPR polypeptide is of between 5 and 20 .mol of TPR polypeptide per each gram of plasticizer in the composition; more preferably of about 12 .mol of TPR polypeptide per each gram of plasticizer in the composition.

[0095]

[0086] In a preferred embodiment, the second aspect of the invention relates to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide and a plasticizer wherein:

[0096] (i) the TPR polypeptide has a sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; preferably the sequence is selected from SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; more preferably, the sequence is SEQ ID NO: 6; (ii) the plasticizer is selected from poly(ethylene)glycol, poly(propylene) glycol and poly(vinyl acetate); preferably it is poly(ethylene) glycol; and

[0097] (iii) the molecular weight of the plasticizer is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol.

[0098]

[0087] In a more preferred embodiment, the second aspect of the invention relates to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide and a plasticizer wherein: (i) the TPR polypeptide has a sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; preferably the sequence is selected from SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; more preferably, the sequence is SEQ ID NO: 6; (ii) the plasticizer is selected from poly(ethylene)glycol, poly(propylene) glycol and poly(vinyl acetate); preferably it is poly(ethylene) glycol;

[0099] (iii) the molecular weight of the plasticizer is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol; and

[0100] (iv) the composition is provided as a membrane or a film and / or the TPR polypeptide is cross-linked, wherein the cross-linking is preferably the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0101]

[0088] In further embodiments of the second aspect of the invention, the composition is provided as a membrane or a film having a thickness comprised between 10 and 100 .m; preferably between 30 and 50 .m. A suitable method to measure said thickness is scanning electron microscopy (SEM).

[0102]

[0089] Thus, in a more preferred embodiment, the second aspect of the invention relates to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide and a plasticizer wherein:

[0103] (i) the TPR polypeptide has a sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8; preferably the sequence is selected from SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 8; more preferably, the sequence is SEQ ID NO: 6; (ii) the plasticizer is selected from poly(ethylene)glycol, poly(propylene) glycol and poly(vinyl acetate); preferably it is poly(ethylene) glycol;

[0104] (iii) the molecular weight of the plasticizer is comprised between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol; and

[0105] (iv) the composition is provided as a membrane or a film having a thickness comprised between 10 and 100 .m; preferably between 30 and 50 .m;

[0106] (v) the TPR polypeptide is cross-linked, wherein the cross-linking is preferably the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0107]

[0090] In a further more preferred embodiment, the second aspect of the invention relates to a composition comprising a Tetratricopeptide Repeat (TPR) polypeptide and a plasticizer wherein:

[0108] (i) the TPR polypeptide has a sequence selected from SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 8; preferably the sequence is SEQ ID NO: 3, SEQ ID NO: 6; more preferably, the sequence is SEQ ID NO: 6;

[0109] (ii) the plasticizer is selected from poly(ethylene)glycol, poly(propylene) glycol and poly(vinyl acetate); preferably it is poly(ethylene) glycol; (iii) the molecular weight of the plasticizer is between 1,000 g / mol and 10,000 g / mol; more preferably of about 4,000 g / mol; and

[0110] (iv) the composition is provided as a membrane or a film having a thickness comprised between 10 and 100 .m; preferably between 30 and 50 .m;

[0111] (v) the TPR polypeptide is cross-linked, wherein the cross-linking is preferably the result of the treatment of the composition with glutaraldehyde, preferably by exposure of the composition of the second aspect of the invention to vapour comprising glutaraldehyde.

[0112]

[0091] As mentioned above, the composition of the second aspect of the invention is particularly suitable for use as separator in electrochemical devices. The role of separators in such devices is to isolate electrically the cathode from the anode while allowing for the transport of ions from an electrode compartment to the other during charging and discharging cycles. Said ions are generally comprised in the electrolyte composition with which the separator is soaked or impregnated. However, a viable alternative is that wherein the separator composition itself comprises ions.

[0113]

[0092] Thus, in a further embodiment, the composition of the second aspect of the invention further comprises ions. Said ions are particularly provided as salts. Suitable salts are well known in the art and will become apparent to the skilled person upon reduction to practice of the invention on the basis of common general knowledge.

[0114]

[0093] In a further embodiment, the composition of the second aspect of the invention further comprises a salt selected from the group consisting of alkaline salts of an anion selected from the group consisting of chloride, sulfate and hydroxide.

[0115]

[0094] In a further embodiment, the composition of the second aspect of the invention further comprises a salt selected from the group consisting of sodium chloride and potassium chloride.

[0116]

[0095] The amount of such salt in said composition is preferably adjusted such that the composition of the second aspect maintains the aspect of a film while ensuring the maximal amount of salt within the composition.

[0117]

[0096] In a further embodiment, the composition of the second aspect of the invention further comprises a salt in an amount such that the molar ratio of the salt to the TPR polypeptide is comprised from 3:1 to 1000:1; preferably from 100:1 to 500:1: more preferably of about 333:1.

[0118]

[0097] A method for the preparation of the composition of the second aspect of the invention is also part of the invention and forms the third aspect of the invention.

[0119]

[0098] As defined above, the third aspect of the invention relates to a method for the preparation of a composition according to the second aspect of the invention comprising the steps of: (i) providing an aqueous solution of the Tetratricopeptide Repeat (TPR) polypeptide and the plasticizer wherein the plasticizer and the amount of TPR polypeptide are preferably as defined in the first and second aspects of the invention;

[0120] (ii) depositing the solution provided in (i) on a surface; and

[0121] (iii) drying the product of step (ii).

[0122]

[0099] In preferred embodiments of the method of the third aspect of the invention, the plasticizer of the solution provided in step (i) is as defined in any of the embodiments of the second aspect of the invention defining the plasticizer described above.

[0123]

[0100] In preferred embodiments of the method of the third aspect of the invention, the Tetratricopeptide Repeat (TPR) polypeptide of the solution provided in step (i) is as defined in any of the embodiments of the first or second aspect of the invention defining the TPR polypeptide described above.

[0124]

[0101] In preferred embodiments of the method of the third aspect of the invention, the relative amounts of Tetratricopeptide Repeat (TPR) polypeptide and plasticizer in the solution provided in step (i) are as defined in any of the embodiments of the second aspect of the invention defining said amounts.

[0125]

[0102] In further embodiments of the method of the third aspect of the invention, the concentration of Tetratricopeptide Repeat (TPR) polypeptide in the solution of step (i) is of between 100 iM and 1,000 .M; preferably of between 400 iM and 800 .M; more preferably of 600 iM.

[0126]

[0103] In preferred embodiments of the method of the third aspect of the invention, the relative amounts of Tetratricopeptide Repeat (TPR) polypeptide and plasticizer in the solution provided in step (i) are as defined in any of the embodiments of the second aspect of the invention defining said amounts and the concentration of TPR polypeptide in the solution of step (i) is of between 100 iM and 1,000 .M; preferably of between 400 .M and 800 .M; more preferably of 600 .M.

[0127]

[0104] In particular embodiments of the method of the third aspect of the invention, step (ii) is carried out by drop-casting. Nevertheless, as will be obvious to the skilled person, any method known in the art as being suitable for depositing a precursor solution of a film formed by evaporation of said precursor solution may be used to carry out step (ii) of the method of the third aspect of the invention.

[0128]

[0105] In particular embodiments of the method of the third aspect of the invention, step (iii) is carried out by drying at room temperature. Nevertheless, as will be obvious to the skilled person, any method known in the art as being suitable for drying a precursor solution of a film formed by evaporation of said precursor solution may be used to carry out step (iii) of the method of the third aspect of the invention.

[0129]

[0106] In preferred embodiments, the method of the third aspect of the invention further comprises the step of (iv) submitting the product of (iii) to a step of cross-linking the Tetratricopeptide Repeat (TPR) polypeptide. As mentioned above, any cross-linking method known in the art for the cross-linking of peptides, in particular via amino groups comprised in the amino acid side chains of said peptide, may be used to carry out step (iv) of the method of the third aspect of the invention. Preferably, step (iv) is carried out using vapour diffusion through the product of step (iii) of a cross-linking agent such as glutaraldehyde. As will be obvious to the skilled person having regard of common general knowledge, the conditions of step (iv) may have to be adapted depending on the chemical nature of the cross-linking agent.

[0130]

[0107] When the composition of the second aspect further comprises a salt, the aqueous solution of step (i) of the method of the third aspect of the invention further comprises said salt. Said salt and its amount may be as defined above in the embodiments of the second aspect of the invention defining these features.

[0131]

[0108] As mentioned above, the fourth aspect of the invention relates to a product obtainable according to the method of the third aspect of the invention.

[0132]

[0109] In preferred embodiments, the product of the fourth aspect of the invention is obtainable by the method as defined in any of the particular and preferred embodiments of the third aspect of the invention defined above.

[0133]

[0110] The fifth aspect of the invention relates to the use of the composition according to the second or the fourth aspect of the invention as a separator for a battery or for a supercapacitor.

[0134]

[0111] The inventors have found that the composition according to the second or the fourth aspect of the invention provides surprisingly improved wettability to the separator, which reduces manufacturing and assembly times of the capacitator significantly. In addition, the composition of the invention is electrochemically stable and exhibits suitable ion conductivity for acting as a separator.

[0135]

[0112] In preferred embodiments, the fifth aspect of the invention relates to the use of the composition as defined in any of the embodiments described above defining the composition of the second or the fourth aspect of the invention as a separator for a battery or for a supercapacitor.

[0136]

[0113] As mentioned above, the sixth aspect of the invention relates to a device, such as a battery or a supercapacitor, comprising the composition according to the second or the fourth aspect of the invention.

[0137]

[0114] In preferred embodiments, the sixth aspect of the invention relates to a device, such as a battery or a supercapacitor, comprising the composition as defined in any of the embodiments described above defining the composition of the second or the fourth aspect of the invention. The device of the sixth aspect of the invention is preferably an electrochemical device, such as a battery or a supercapacitor, whereby the composition of the second or the fourth aspect of the invention is arranged for being in electrochemical contact with the cathode and the anode of said device.

[0138]

[0115] Throughout the description and claims the word “comprises" and variations of the word, are not intended to exclude other technical features, additives, components or steps. Furthermore, the word “comprise” encompasses the cases of “consist of” and “consists essentially of”. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples are provided by way of illustration, and they are not intended to be limiting of the present invention.

[0139] The invention is further defined by the following aspects:

[0140] 1. A Tetratricopeptide Repeat (TPR) polypeptide having the sequence according to [Xi X2 X3 W X5 Xe X7 G X9 X10Y X12 X13 X14 X15X16Y Xis X19A X21 X22 X23Y X25 X26A X28 X29 X30 X31 P X33 X34]n (SEQ ID NO: 1), wherein a glutamic acid (E) or aspartic acid (D) is present in between 9 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, and wherein n equals to or is more than 1; preferably n equals to or is more than 4.

[0141] 2. The Tetratricopeptide Repeat (TPR) polypeptide according to aspect 1 wherein a glutamic acid or an aspartic acid amino acid is present in at least positions 2, 5, 6, 16, 18, 19, 22, 29 and 31.

[0142] 3. The Tetratricopeptide Repeat (TPR) polypeptide according to aspect 2 having the sequence according to [Xi E X3WEE X7G X9X Y X12 X13 X14 X15 DYDEA X21E X23Y X25X26A X28E X30 DP X33 X34]n (SEQ ID NO: 2) wherein n > 1.

[0143] 4. The Tetratricopeptide Repeat (TPR) polypeptide according to aspect 3 having the sequence according to SEQ ID NO: 3.

[0144] 5. The Tetratricopeptide Repeat (TPR) polypeptide according to aspect 2 or 3 further comprising a glutamic acid or an aspartic acid amino in at least one of positions 9, 12, 25 and 26.

[0145] 6. The Tetratricopeptide Repeat (TPR) polypeptide according to aspect 5 having the sequence according to [Xi E X3WEE X7G E X Y E X13 X14 X15 DYDEA X21E X23Y X25X26A X28E X30 DP X33 X34]n (SEQ ID NO: 4) wherein n > 1.

[0146] 7. The Tetratricopeptide Repeat (TPR) polypeptide according to aspect 5 or 6 having the sequence according to [Xi E X3WEE X7G E X10Y E X13 X14 X15 DYDEA X21E X23Y E E A X28E X30DP X33 X34]n (SEQ ID NO: 5) wherein n > 1.

[0147] 8. The Tetratricopeptide Repeat (TPR) polypeptide according to any of aspects 5 to 7 having a sequence according to SEQ ID NO: 6 or SEQ ID NO: 7.

[0148] 9. The Tetratricopeptide Repeat (TPR) polypeptide according to any of aspects 1 to 8 wherein n= 1. 10. A composition comprising a Tetratricopeptide Repeat (TPR) polypeptide and a plasticizer that is a polymer having a molecular weight of ate least 1 ,000 g / mol; preferably comprised between 1 ,000 g / mol and 10,000 g / mol, wherein the Tetratricopeptide Repeat (TPR) polypeptide has the sequence [Xi X2 X3 W X5 Xe X7G X9 X10Y X12 X13 X14 X15X16 Y Xis X19 A X21 X22 X23Y X25X26A X28X29X30X31 P X33 X34]n (SEQ ID NO: 1), wherein a glutamic acid (E) or aspartic acid (D) is present in between 7 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, and wherein n equals to or is more than 1.

[0149] 11. The composition according to aspect 10 wherein the Tetratricopeptide Repeat (TPR) polypeptide has a sequence according to SEQ ID NO: 8.

[0150] 12. Composition according to aspect 10 or 11 wherein the Tetratricopeptide Repeat (TPR) polypeptide is cross-linked.

[0151] 13. Composition according to any one of aspects 10 to 12 wherein the plasticizer is poly(ethylene)glycol.

[0152] 14. Composition according to aspect 13 wherein the molecular weight of poly(ethylene)glycol is of about 4,000 g / mol.

[0153] 15. Composition according to any one of aspects 10 to 14 wherein the amount of the Tetratricopeptide Repeat (TPR) polypeptide is of between 5 and 50 .mol of TPR polypeptide per each gram of plasticizer in the composition; preferably, the amount of the TPR polypeptide is of between 5 and 20 .mol of TPR per each gram of plasticizer in the composition; more preferably of about 12 .mol of TPR polypeptide per each gram of plasticizer in the composition.

[0154] 16. Composition according to any one of aspects 10 to 15 that is provided in the form of a film or a membrane.

[0155] 17. Composition according to aspect 16 wherein the thickness of the film or membrane is comprised between 10 and 100 .m; preferably between 30 and 50 .m.

[0156] 18. A method for the preparation of a composition according to any of aspects 16 to 17 comprising the steps of:

[0157] (i) providing an aqueous solution of the Tetratricopeptide Repeat (TPR) polypeptide and the plasticizer wherein the plasticizer and the amount of TPR polypeptide are preferably as defined in any of aspects 9 to 14;

[0158] (ii) depositing the solution provided in (i) on a surface; and

[0159] (iii) drying the product of step (ii).

[0160] 19. The method according to aspect 18 wherein the concentration of Tetratricopeptide Repeat (TPR) polypeptide in the solution of step (i) is of between 100 iM and 1,000 .M; preferably of between 400 iM and 800 .M; more preferably of 600 .M. 20. The method according to any of aspects 18 to 19 further comprising the step of (iv) submitting the product of (iii) to a step of cross-linking the Tetratricopeptide Repeat (TPR); preferably using vapour diffusion of a cross-linking agent such as glutaraldehyde.

[0161] 21. Product obtainable by the method of any of aspects 18 to 20.

[0162] 22. Use of the composition according to any of aspects 10 to 17 or according to aspect 21 as a separator for a battery or for a supercapacitor.

[0163] 23. Device, such as a battery or a supercapacitor, comprising the composition according to any of aspects 10 to 17 or according to aspect 21.

[0164]

[0116] The project leading to this application has received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 964593.

[0165] EXAMPLES

[0166] Materials

[0167]

[0117] Polyethylene glycol (PEG) with average Mw of 400 g mol’1and 4,000 g mol’1were purchased from Scharlab and Sigma Aldrich, respectively. Commercial activated carbon (YP-80F) purchased from Kuraray Corporation was utilized as active material and C-NERGY™ Super C45 as conductive carbon additive from Imerys. As binder, polytetrafluoroethylene (PTFE) was employed. The electrolyte (3 M KCI in water) and glass fiber (GF / D) used as separator were purchased from Merck KGaA.

[0168] Preparation of TPR proteins

[0169]

[0118] Tetratricopeptide Repeats (TPR) proteins containing different amounts of acidic residues, in particular glutamic acid (Glu, E), were constructed. In this way, four distinct TPR variants were engineered including the wild type CTPR protein which inherently presents 7 acidic residues (aspartic and glutamic acids) per repeat, CTPR_2E (SEQ ID NO: 3), CTPR_4E (SEQ ID NO: 6), and CTPR_6E (SEQ ID NO: 7) units integrating 2, 4, and 6 additional Glu residues per repeat, respectively. Specifically, mutations were introduced at non-conserved positions within the CTPR sequence, thereby preserving the CTPR fold. The resulting mutant sequences CTPR_2E (Y5E_N6E); CTPR_4E (Y5E_N6E_N9E_Y12E), and CTPR_6E (Y5E_N6E_ N9E_Y12E_Q25E_K26E) were simulated through molecular modeling, protein minimization, and structure validation with Molprobity (Ian W. Davis, Andrew Leaver-Fay, Vincent B. Chen, Jeremy N. Block, Gary J. Kapral, Xueyi Wang, Laura W. Murray, W. Bryan Arendall, Jack Snoeyink, Jane S. Richardson, David C. Richardson, MolProbity: all-atom contacts and structure validation for proteins and nucleic acids, Nucleic Acids Research, Volume 35, Issue suppl_2, 1 July 2007, Pages W375-W383, incorporated herein by reference), as shown on Figure 1. Furthermore, AlphaFold predictions based on the sequences corroborated that the proposed sequences are compatible with the preservation of the CTPR structure. TPR proteins comprised of 4 identical repeats of these modified modules were built resulting in the CTPR4 E-series (CTPR4_WT (SEQ ID NO: 8), CTPR4_2E, CTPR4_4E, and CTPR4_6E). These protein variants were expressed as His-tagged fusions and purified through standard metal affinity chromatography. Following purification, their purity and molecular weight (MW) were validated through gel electrophoresis and mass spectrometry.

[0170] Preparation of self-standing CTPR membranes

[0171]

[0119] To prepare Tetratricopeptide Repeats (TPR) based self-standing protein films, 150 pL of a protein solution containing TPR protein (600 pM) and 5 wt.% of PEG-4000 were mixed and subsequently drop cast onto a Teflon surface. Once dried, the film was crosslinked through vapor diffusion with a glutaraldehyde solution 0.5%v for 24 hours. After that, the film was gently removed from the teflon surface. The final thickness of the membranes was 45, 35, 42 and 40 pm for CTPR4_WT, CTPR4_2E, CTPR4_4E and CTPR4_6E, respectively, as measured using a standard micrometer device. Figure 2 shows (a) a scheme of the process of preparation of the membrane and (b) a sample of a membrane obtained according to this process.

[0172]

[0120] The inventors found that this procedure allows preparing membranes that are robust and flexible enough to be handled easily. This is attributed to the ability of the TPR to self-assemble in nanofibers and thin films. The inventors indeed found that when TPR is replaced by Bovine Serum Albumin (BSA) or Lysozyme (LYS) (which are suitable for cross-linking with glutaraldehyde) in this procedure, the method provides brittle films. No modification of the above procedure, for instance by modifying the drying method or modifying the molecular weight of PEG allowed overcoming the formation of brittle films. This is illustrated in Figure 3 which shows the product of cross-linking of a composition prepared according to the present procedure replacing Consensus Tetratricopeptide Repeats (CTPR) by BSA (top) or LYS (bottom).

[0173] Electrode fabrication

[0174]

[0121] Standard self-standing electrodes were prepared using YP-80F as the activated carbon, C65 as the conductive additive and PTFE as binder in a ratio of 90:5:5 (YP-80F:C65:PTFE). The fabrication procedure consisted of the mixing of the different elements using ethanol as dispersant, until a paste consistency was achieved. Then, a glass rod was used to manually smash the mixture until a compact film was obtained. The electrodes were dried at room temperature overnight and then subjected to a post drying step at 120 °C under vacuum at least during 24 h. To homogenize the dried electrodes, a final step of calendering was done at 80 °C. The measured thickness of the calendered electrodes was around 100 m. The final self-standing electrodes were punched into circular electrodes with 4 mm of diameter.

[0175] Supercapacitor assembly

[0176]

[0122] The supercapacitor was assembled using the previously described electrodes as positive and negative electrodes with a 1:1 mass ratio. The full cells were assembled in open air using a 2-electrode Swagelok® configuration with the TPR film sandwiched between the two self-standing electrodes. 3M KCI (50 pL) in water was added as electrolyte before closing the cell. The cell was closed and measured without any extra pressure. As a reference, similar Swagelok® cells with a commercial glass fiber separator of 1 mm thickness were also prepared and tested.

[0177] Electrochemical characterization

[0178]

[0123] Electrochemical characterization was conducted using a VMP3 Biologic potentiostat. This involved cyclic voltammetry (CV) at various sweep rates and galvanostatic charge / discharge (GPCL) at different current densities. Moreover, cell cyclability was also performed at constant current density of 1 A g-1which is equivalent to a discharging time of around 20 seconds in these systems. The specific capacitance (Equation I), gravimetric energy density (Equation II), and power density (Equation III) values obtained from electrochemical measurements are calculated using the following equations:

[0179] Cceii (F / g)= 1 / 2 (I At) / (meAV) Equation I E (Wh / kg)= 1 / 2 Cceii(V2max-V2min) Equation 11 P(W / kg)=E / At Equation III where C is capacitance, I is the current, At is discharge time, methe is mass per electrode, Vmax is the maximum cell voltage and Vmin is the minimum cell voltage, E is energy, and P is power.

[0180] Electrochemical performance in aqueous electrolytes

[0181]

[0124] The capacitive behavior of the previously assembled supercapacitors employing the CTPR4_E series as a membrane were tested with aqueous electrolyte such as 3M KCI. Figure 4a shows the electrochemical impedance spectroscopy (EIS) curves of the different devices before cycling them. Comparison with glass fiber separator is incorporated. The Figure 4b shows the high frequency amplification region of the plots in which the Z' axis intercept of the plots in the high frequency region refers to the equivalent series resistance (Rs), the size of the semicircular plots between the high frequency and intermediate frequency region reflects the interfacial charge transfer resistance (Ret), the length of the straight line in the intermediate frequency region determines the ion transfer between the electrolyte and porous electrode surface, and the slope of the plots in the low frequency region determines the ion diffusion through electrode microporosity. The smaller Ret is the one measured for the wild-type unmodified Consensus Tetratricopeptide Repeats (CTPR) variant. The results show in particular that low slope is larger for the glass fiber-based device, which indicates that the membranes comprising the TPR membranes exhibit a better wettability than the glass fiber separator. Table 1 below summarizes some data extracted from the EIS curves of Figure 4.

[0182] Table 1

[0183] Separator Rs Ret

[0184] type (Ohm cm2) (Ohm cm2)

[0185] CTPR4_WT 0.045 0.88

[0186] CTPR4_2E 0.015 1.33

[0187] CTPR4_4E 0.007 1.57

[0188] CTPR4_6E -0.151 3.33

[0189] Glass fiber 0.16 0.35

[0190]

[0191]

[0125] Fig. 5 shows cyclic voltammetry curves of all tested materials. All tested materials show the typical rectangle of a physical adsorption / desorption charge storage mechanism of an electrochemical double-layer capacitor. The CV curves of all the membranes exhibit a regular rectangle with a little difference in the integrated area of the curves, indicating their good double layer capacitance behavior and the similar specific capacitance. No redox peak is also observed, indicating the electrochemical stability of the membrane in the tested conditions.

[0192]

[0126] Fig. 6 shows constant current charge / discharge cycling experiments performed at current densities in the 0.1-20 A g-1range with the prepared devices. At low current densities, the cell voltage profiles are symmetrical for proteins CTPR4_WT, CTPR4_2E, CTPR4_4E and present an isosceles triangle shape, which is indicative of the good reversibility of all the TPR membranes. On the contrary, when Glass fiber is employed as a membrane, the GCPL curve deviates significantly from this shape. This is clearly proven by the coulombic efficiencies (CE). At 0.1 A g-1the CE is greater than 90% for the membranes based on CTPR4_WT, CTPR4_2E and CTPR4_4E. This is indicative of the absence of secondary reactions, such as electrolyte decomposition or partial carbon oxidation. On the other hand, the observed CE for CTPR4_6E suggests that secondary reactions or decomposition might occur. The discharge profiles of all the CTPR4_WT (SEQ ID NO: 8) membranes exhibit a very small voltage drop compared to the CTPR4_E series (SEQ ID NO: 3, SEQ ID NO: 6 and SEQ ID NO: 7), indicating a lower internal resistance and excellent double-layer capacitance behavior in all the cases with very similar discharge times when compared to the glass fiber. In contrast, the higher the current density applied, the more the profiles deviate from the isosceles triangle and consequently a higher voltage drop is observed. This is indicative of a higher internal resistance, which is consistent with the previous analysis. However, the GCPL profiles still maintain a regular shape with the operating voltage range of 0 - 0.8 V, proving that the protein modification can improve the electrochemical performance of the device.

[0193]

[0127] The influence of current density on the specific capacitance of the prepared devices was then investigated, as shown in Fig. 7. With the increase of current density, the specific capacitance of the analyzed materials shows a decreasing trend which is parallel in all the cases except for CTPR4_6E. This decrease is caused by the much larger internal resistance of the supercapacitor. Under the same working conditions, the specific capacitance of CTPR4_6E is larger at low current densities, while CTPR4_WT, CTPR4_2E and 4E are larger at high current densities, with a less dramatic capacitance loss when the current density is increased. This is indicative of the importance of the protein structure for the separator property. The ones that present less glutamic acid modifications in their structure, are able to maintain their structural properties and elevate the transport ability of the electrolyte ions, reduce the internal resistance of the membranes, and improve the electrochemical performance. At an operating voltage of 0-0.8 V, the cell capacitance of CTPR4_4E can reach 25.8 F g-1at a current density of 0.25 A g-1, which is higher than that obtained for CTPR4_WT and CTPR4_2E (24.6 and 24.4 F g-1, respectively). Even at a high current density of 20.0 A g-1, CTPR4_4E can still exhibita high cell capacitance of 21.8 F g-1, indicating that the introduction of the glutamic residues can significantly improve the electrochemical performance of supercapacitors. In the special case of the CTPR4_6E, which has a completely different unfolded structure, the cell capacitance can reach values as high as 28.8 F g-1at a current density of 0.1 A g-1(but with an extremely low CE <70%), whereas a much lower value of 16.1 F g-1is measured at 20 A g-1. When compared to the commercial separator, it is proven the better wettability of the TPR membranes, since for the same resting time the capacities obtained for the CTPR4_4E are comparable to the ones measured with the commercial separator.

[0194]

[0128] Equation II and III are utilized to obtain from the cell capacitance values in Figure 6a, the energy-to-power values represented in a Ragone plot in Figure 7b. The plots for TPRs experience a large drop in energy densities when the power densities are increased, a drop especially drastic in the case of protein 6E, which is directly related to the higher resistance of these separators at high current densities. In the case of other TPR-based systems, capacitance values (see Figure 7a) and iR drop (see Figure 6) are very similar, thus, the difference in energy and power output is not significantly different among them.

[0195]

[0129] To investigate the cycling stability of the device CTPR4_4E, the device was cycled 10k times at a constant current density of 1 A g-1, within a discharge time of approximately 20 s. Figure 8 shows the cycle retention and CE of the CTPR4_4E with the cycle number. Although the curves show slight fluctuations at the beginning of the cycle, the cycle retention can maintain at ca. 95% and the CE can maintain at 98.8% after 10k cycles, which indicates that CTPR4_4E has good cycling stability and reversibility after charging and discharging. CTPR4_4E cyclability study demonstrates a better capacitance retention of the protein-based cell compared with the one fabricated with commercial glass fiber as separator, which ended the test with only over 90% of the initial capacitance (CE >99%). The results demonstrate that the prepared CTPR4_4E can provide excellent electrochemical performance as well as good cycling stability for the supercapacitor and show potential application prospects.

[0196] Comparison with compositions disclosed in the art

[0197]

[0130] For the purpose of comparing the performance of a film according to the invention with a film as disclosed in Sanchez-de Alcazar et al., a self-standing CTPR membrane was prepared following the procedure described above using 5 wt% of PEG-400 (poly(ethylene glycol) of molecular weight 400 g / mol) as plasticizer and the CTPR with SEQ ID NO: 8. The resulting membrane was used for the preparation of an electrode and a supercapacitator following the respective procedures described above.

[0198]

[0131] This comparative system is compared with the membrane obtained following the procedure described above using 5 wt% of PEG-4000 (poly(ethylene glycol) of molecular weight 4000 g / mol) as plasticizer and the CTPR with SEQ ID NO: 8.

[0199]

[0132] The electrochemical properties of the resulting devices were evaluated by Electrochemical Impedance Spectroscopy and Cyclic Voltammetry following the procedures described above for electrochemical characterization and performance measurement. Figure 9 shows the obtained results.

[0200]

[0133] Figure 9 shows that the membrane according to the invention presents a higher ionic conductivity (i.e. lower Rsvalue, as measured by EIS) than the membrane disclosed in the prior art. Accordingly, the membrane according to the invention is more suitable as separator in batteries or supercapacitators than the membrane according to the prior art. In addition, Figure 9 shows that the capacitance of the cell comprising the membrane according to the invention is higher than the capacitance of the cell comprising a membrane as disclosed in the prior art. Accordingly, the membrane according to the invention represents an improvement over the membranes disclosed in the art when considering applying these membranes as separators in electrochemical devices, such as batteries or supercapacitators. Example of separator composition comprising a salt

[0201] NaCI-doped CTPR4-4E films were tested as electrolytes in electrochemical double-layer capacitor (EDLC) devices under humid conditions. For this purpose, CTPR4-4E processed protein films were doped with varying NaCI concentrations (1, 10, and 100 mM) during preparation. The resulting self-standing solid electrolyte film was sandwiched between two self-standing activated carbon according to the following method:

[0202] 7.1 Materials

[0203]

[0134] Polyethylene glycol (PEG) with average Mw 4,000 g mol-1was purchased from Sigma Aldrich. Commercial activated carbon (YP-80F) purchased from Kuraray Corporation was utilized as active material and C-NERGY™ Super C45 as conductive carbon additive from Imerys. As binder, PTFE was employed. The NaCI and the glass fiber (GF / D) used as separator were purchased from Merck KGaA.

[0204] Preparation of the self-standing CTPR membrane for supercapacitors

[0205]

[0135] To fabricate self-standing CTPR-based protein films, a solution containing 600 pM CTPR protein, NaCI (1-100 mM), and 5% (w / w) PEG-4000 is drop-casted onto a hydrophobic surface (PTFE / Teflon) and left to dry overnight. Once dried, the film is crosslinked via vapor diffusion using a 0.5% (v / v) glutaraldehyde solution for 24 hours. The resulting self-standing CTPR-4E film, doped with different NaCI concentrations, can then be carefully removed from the surface.

[0206] Electrode fabrication

[0207]

[0136] Standard self-standing electrodes were prepared using YP-80F as the activated carbon, C65 as the conductive additive and PTFE as binder in a ratio of 90:5:5 (YP-80F:C65: PTFE). The fabrication procedure consists of the mixing of the different elements using ethanol as dispersant, until a paste consistency is achieved. Then, a glass rod is used to manually smash the mixture until a compact film is obtained. To homogenize the dried film, a final step of calendering is done at 80 °C and with an aperture between rolls of 100 pm. The measured thickness of the calendered electrodes is 86±10 pm. To obtain the final self-standing electrodes, the film is punched into circular electrodes with 4 mm of diameter (3.75 mg cm-2). The electrodes were dried at 200 °C overnight before being used as part of the final devices.

[0208] Electrochemical double layer capacitor assembly

[0209]

[0137] The supercapacitor was assembled using symmetrical self-standing activated carbon electrodes (YP-80F:C65:PTFE, 90:5:5 ratio) in an electrochemical double-layer capacitor (EDLC) configuration. Before assembly, 50 mL of deionized water was drop-casted onto the protein-salt film to mimic a high humidity environment. The cell was left to rest for 6 hours to ensure the complete wettability of all components. The wet CTPR4-4E protein film was sandwiched between the two electrodes as a solid-state electrolyte, and 50 pL of distilled water was added. Before assembly, all electrodes were dried at 200 °C to remove residual water and humidity from the activated carbon’s porous structure. The full cells were assembled in open air using a 2-electrode Swagelok® configuration, and as a reference, similar Swagelok® cells were prepared with a 1 mm thick commercial glass fiber separator for comparison. Different sets of CTPR-based protein membranes were fabricated to evaluate the effect of salt concentration on device performance. The fabrication process is schematized in Figure 4a.

[0210] Electrochemical characterization

[0211]

[0138] Electrochemical characterization was conducted using a VMP3 Biologic potentiostat. This involved cyclic voltammetry (CV) at various scan rates and galvanostatic charge / discharge (GPCL) at different current densities. The specific capacitance (Equation IV), energy density (Equation V), and power density (Equation VI) values obtained from electrochemical measurements are calculated using the following equations:

[0212] Ccceeini = ~ 2 m1eM-AV Equation IV

[0213] E = | Ccell• AV Equation V

[0214] P = — Equation VI

[0215]

[0216] where Cce / / is specific capacitance in F / g, I is current in A, At is discharge time, methe is the electrode mass, A Vis cell voltage, E is energy density (Wh / g), and Pis power density (W / g), both normalized to electrode mass.

[0217]

[0139] As shown in Figure 10b, the EIS Nyquist plots for the CTPR4-4E devices with varying NaCI concentrations reveal significant differences in diffusion processes at the electrode-electrolyte interface. The Nyquist plots present two distinct regimes: a region with ~45-degree slope, typically associated with open or semi-infinite diffusion, followed by a steeper, near-vertical incline, characteristic of restricted (finite-length) diffusion. This behavior is typical of systems with activated carbon (AC) electrodes, where ion transport and charge storage are strongly influenced by porosity and ion confinement effects. Notably, protein EDLC devices doped with 100 mM NaCI exhibit overall lower resistance in the open diffusion regime and show an earlier transition to the restricted diffusion regime, which appears at higher frequencies. These features suggest that increased ionic strength facilitates faster ion motion and a more rapid buildup of the electric double layer (EDL) at the electrode / electrolyte interface, a key factor to improve charge storage efficiency.

[0140] Remarkably, the cyclic voltammograms (CV) of the symmetric EDLCs (Figure 10c) show a more rectangular shape for the device containing 100 mM of NaCI than for the ones with lower concentrations. The rectangular shape indicates excellent capacitive behavior resulting from the accumulation of ions at the electrode-electrolyte interface and facilitating efficient charge storage. It is important to note that none of the CV recorded at different scan rates showed evidence of redox reactions, confirming the electrochemical stability of the CTPR4-4E films.

[0218]

[0141] Galvanostatic charge / discharge curves at a low current density (0.1 A / g) are presented in Figure 10d for the three CTPR4-4E EDCL devices doped with varying NaCI concentrations. Devices containing 100 mM NaCI showed voltage-time profiles closer to the ideal EDLC response, with a more symmetric charge-discharge behavior and a lower ohmic drop. This indicates more efficient charge transport, minimal energy loss and higher power performance. Specifically, at low current densities (0.1 A / g), the devices containing 100 mM of NaCI exhibited a significantly superior specific capacitance (23.7 F / g) — which quantifies how much charge can be accumulated per gram of activated carbon electrode — and coulombic efficiency (CE) — which measures how reversible the charge-discharge process is — , reaching up to 98.7%. In contrast, devices with lower salt concentrations presented a clear bending in the voltage-time profiles (Figure 10d) during both charging and discharging. This deviation from ideality may be linked to the same mechanisms responsible for the asymmetrical CVs. Similarly, when the current density was increased to 3 A / g, devices with lower salt doping experienced a higher ohmic loss and a reduction in specific capacitance for all cases (Figure 10e), demonstrating the direct correlation between higher salt doping and improved device performance. The device with 100 mM retains 80% of its capacitance even at density currents as high as 5 A / g, as can be seen in Figure 10d.

[0219]

[0142] Table 2. Comparison of the electrochemical performance of the EDLCs doped with various NaCI salt concentrations. Coulombic Efficiency, Ohmic Drop at 0.1 A / g and Capacitance Retention calculated at the maximum current density (5 A / g) from Figure 10e.

[0220] [NaCI] Ohmic Coulombic Capacitance (mM) Drop efficiency Retention

[0221] (mV) (%) (%)

[0222] 0.1 A / g 3 A / g 0.1 A / g 3 A / g

[0223] 1 26.3 380 95.8 98.1 6.7 10 10.7 230 96.3 99.4 8.8

[0224] 100 1.9 52 98.7 99.9 80.0

[0225]

[0226]

[0143] aCoulombic Efficiency (%) = (CspDischarge / Cspcharge)x100;bCapacitance retention (%)—(CsP@5A / g I CsP@0.1A / g)x100.

[0227]

[0144] The specific cell capacitance for CTPR4-4E EDLCs, calculated from the chargedischarge profiles as a function of applied current is presented in Figure 10e. The device prepared with 100 mM NaCI presents the maximum specific capacitance of 23.7 F / g at a current density of 0.1 A / g, retaining 80% of this capacitance when the current density is increased to 5 A / g. Finally, the overall energy storage performance of doped CTPR4-4E EDLCs is compared in a Ragone plot (Figure 10f), which establish how much energy can the device store (energy density) and how fast can it deliver it (power density). The incorporation of NaCI ions into the CTPR4-4E protein film results in a maximum energy density of 2.1 Wh / kg and a maximum power density of 4639 W / kg. Specifically, the maximum cell-specific capacitance obtained for all NaCI-doped CTPR4-4E EDLCs exceeds the values observed for those prepared with liquid NaCI at similar concentrations (1, 10, and 100 mM) demonstrating the critical role of CTPR4-4E in enhancing cell performance. This improvement translates into higher energy and power densities for doped CTPR4-4E EDCLs, highlighting the effectiveness of engineered proteins as non-liquid electrolytes.

Claims

CLAIMS1. A Tetratricopeptide Repeat (TPR) polypeptide having the sequence according to [Xi X2 X3 W X5 Xe X7 G X9 X10Y X12 X13 X14 X15X16Y Xis X19A X21 X22 X23Y X25 X26A X28 X29 X30X31 P X33 Xs4]n (SEQ ID NO: 1), wherein 9 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, are glutamic acid (E) or aspartic acid (D), and wherein n equals to or is more than 1; preferably, n equals or is larger than 4.

2. The Tetratricopeptide Repeat (TPR) polypeptide according to claim 1 having the sequence according to SEQ ID NO: 3.

3. The Tetratricopeptide Repeat (TPR) polypeptide according to any of claims 1 to 2 having a sequence according to SEQ ID NO: 6 or SEQ ID NO: 7.

4. A composition comprising a Tetratricopeptide Repeat (TPR) polypeptide and a plasticizer that is a polymer having a molecular weight of at least 1 ,000 g / mol; preferably comprised between 1,000 g / mol and 10,000 g / mol wherein the Tetratricopeptide Repeat (TPR) polypeptide has the sequence [Xi X2 X3 W X5 Xe X7G X9 X10Y X12 X13 X14 X15X16 Y Xis X19 A X21 X22 X23Y X25X26A X28X29X30X31 P X33 X34]n (SEQ ID NO: 1), wherein a glutamic acid (E) or aspartic acid (D) is present in between 7 to 20 of the amino acid positions, wherein i indicates the amino acid position in the polypeptide sequence, and wherein n equals to or is more than 1.

5. The composition according to claim 4 wherein the Tetratricopeptide Repeat (TPR) polypeptide has a sequence according to any one of SEQ ID NO: 3, SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 8.

6. The composition according to claim 4 or 5 wherein the Tetratricopeptide Repeat (TPR) polypeptide is cross-linked.

7. The composition according to any one of claims 4 to 6 wherein the plasticizer is poly(ethylene)glycol; preferably, wherein the molecular weight of poly(ethylene)glycol is of about 4,000 g / mol.

8. The composition according to any one of claims 4 to 7 wherein the amount of the Tetratricopeptide Repeat (TPR) polypeptide is of between 5 and 50 .mol of TPR polypeptide per each gram of plasticizer in the composition; preferably, the amount ofthe TPR polypeptide is of between 5 and 20 .mol of TPR per each gram of plasticizer in the composition; more preferably of about 12 .mol of TPR polypeptide per each gram of plasticizer in the composition.

9. The composition according to any one of claims 4 to 8 that is provided in the form of a film or a membrane, wherein the thickness of the film or membrane is preferably comprised between 10 and 100 .m; preferably between 30 and 50 .m.

10. A method for the preparation of a composition according to any of claims 4 to 9 comprising the steps of:(i) providing an aqueous solution of the Tetratricopeptide Repeat (TPR) polypeptide and the plasticizer wherein the plasticizer and the amount of Tetratricopeptide Repeat (TPR) polypeptide are preferably as defined in any of claims 4 to 9;(ii) depositing the solution provided in (i) on a surface; and(iii) drying the product of step (ii).

11. The method according to claim 10 wherein the concentration of Tetratricopeptide Repeat (TPR) polypeptide in the solution of step (i) is of between 100 iM and 1,000 .M; preferably of between 400 iM and 800 .M; more preferably of 600 .M.

12. The method according to any of claims 10 to 11 further comprising the step of (iv) submitting the product of (iii) to a step of cross-linking the Tetratricopeptide Repeat (TPR); preferably using vapour diffusion of a cross-linking agent such as glutaraldehyde.

13. A product obtainable by the method of any of claims 10 to 12.

14. Use of the composition according to any of claims 4 to 9 or according to claim 13 as a separator for a battery or for a supercapacitor.

15. A device, such as a battery or a supercapacitor, comprising the composition according to any of claims 4 to 9 or according to claim 13.

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