Designer proteins with improved properties

Abstract

The present invention relates to proteins, as well as nucleic acids encoding such proteins, capable of forming fibres, films, nanofibres, and / or hydrogels. The present invention also relates to recombinant cells and / or organisms which synthesize these proteins. Proteins of the invention can be used for a variety of purposes such as in the production of personal care products, plastics, textiles, and biomedical products.

Description

Designer proteins with improved propertiesField of the invention

[0001] The present invention relates to proteins, as well as nucleic acids encoding such proteins, capable of forming fibres. The present invention also relates to recombinant cells and / or organisms which synthesize these proteins. Proteins of the invention can be used for a variety of purposes such as in the production of personal care products, plastics, textiles, and biomedical products.Cross reference to earlier application

[0002] This application claims priority to Australian provisional application no. 2024903913, the entire content of which is hereby incorporated by reference in their entirety.Background of the invention

[0003] Silk fibres have been used for many years as sutures for a wide variety of important surgical procedures. Silk fibres also hold great promise as materials for artificial ligaments, artificial tendons, elastic bandages for skin grafts in burn patients, and scaffolds that can provide support and, in some cases, temporary function during regeneration of bone, periodontal, and connective tissues. The development of silk fibres as materials for ligaments and tendons is expected to become increasingly important as the incidence of anterior cruciate ligament (ACL) and other joint injuries requiring surgical repairs increases in the ageing population. While a small proportion of fibres currently used as sutures is derived from natural silkworm silk, most are produced as synthetic polymers by the chemical industry. A major limitation of this approach is that it can only provide silk fibres with a narrow range of physical properties, such as diameter, strength, and elasticity.

[0004] In order for a silk fibre to be considered useful from a commercial standpoint, the fibre must possess adequate tensile (strength) and flexibility and / or elasticity characteristics and be suitable for the creation of fibres in the desired commercial application.

[0005] There is a need for new and / or improved proteins that have the capability to form a fibre.1006253961

[0006] Reference to any prior art in the specification is not an acknowledgment or suggestion that this prior art forms part of the common general knowledge in any jurisdiction or that this prior art could reasonably be expected to be understood, regarded as relevant, and / or combined with other pieces of prior art by a skilled person in the art.Summary of the invention

[0007] In one aspect, the present disclosure provides a polypeptide capable of forming a fibre, preferably a birefringent fibre, wherein when the fibre is wet-spun at a diameter of 17pm it exhibits a breaking stress of greater than 132 MPa, greater than 200 MPa, greater than or equal to 250 MPa, greater than or equal to 300 MPa, greater than or equal to 350 MPa, greater than or equal to 400 MPa, or greater than or equal to 450 MPa.

[0008] In any embodiment wherein the fibre is wet-spun at a diameter of 17pm, the fibre may exhibit a breaking stress of greater than 132 MPa and less than 532 MPa, greater than 200 MPa and less than 600 MPa, greater than 250 MPa and less than 650 MPa, greater than 350 MPa and less than 750 MPa, greater than 400 MPa and less than 800 MPa, or greater than 450 MPa and less than 850 MPa.

[0009] In any embodiment, the breaking stress may be measured by any methods known in the art, or described herein (e.g., in the Examples). For example, the breaking stress may be measured by applying masking tape at each end of a fibre, wherein weights (e.g., 2.5 g, 6 g, and 10 g) are attached to one end, and suspending the fibre from the other end to determine if it can withstand the force without breaking, thus providing a range of breaking stress (which may be referred to as “tensile strength”). The breaking stress or tensile strength may be calculated by the following equation:where a is tensile strength (MPa), m is mass (kg), a is gravitational acceleration (9.8m / s), and A is cross sectional area (m2). m ■ a may be represented as force (F in Newton).

[0010] In another aspect, the present disclosure provides a polypeptide capable of forming a fibre, preferably a birefringent fibre, wherein when the fibre is wet-spun at1006253961about 30 pm it exhibits an ultimate tensile strength that is at least or about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, or 14-fold greater than the ultimate tensile strength of a fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41.

[0011] In any embodiment, the ultimate tensile strength may be measured by any methods known in the art, or described herein (e.g., in the Examples). For example, the ultimate tensile strength may be measured using a universal testing machine, optionally quipped with a 250mN load cell. A fibre may be mounted onto frames (which may be produced by laser cutting) with a defined gauge length of 5mm, and subject to testing at a rate of 0.5 mm / min until failure. Engineering stress and strain may be calculated from the recorded force-displacement data and the fibre’s cross-sectional area. The cross- sectional area may be calculated from optical microscope images.

[0012] In any embodiment, a fibre of the present disclosure may have an ultimate tensile strength of about 55 MPa, about 70 MPa, about 85 MPa, about 90 MPa, about 95 MPa, about 100 MPa, about 105 MPa, about 110 MPa, about 115 MPa, about 130 MPa, about 135 MPa, about 140 MPa, about 145 MPa, about 150 MPa, about 155 MPa, about 160 MPa, or greater. Optionally, the ultimate tensile strength is from about 55 MPa to about 105 MPa, from about 70 MPa to about 120 MPa, from about 85 MPa to about 135 MPa, from about 90 MPa to about 140 MPa, from about 95 MPa to about 145 MPa, from about 100 MPa to about 150 MPa, from about 105 MPa to about155 MPa, from about 110 MPa to about 160 MPa, from about 115 MPa to about165 MPa, from about 130 MPa to about 180 MPa, from about 135 MPa to about185 MPa, from about 140 MPa to about 190 MPa, from about 145 MPa to about195 MPa, from about 150 MPa to about 200 MPa, from about 155 MPa to about205 MPa, or from about 160 MPa to about 210 MPa. Preferably, a fibre of the present disclosure may have an ultimate tensile strength of 55 MPa, 70 MPa, 85 MPa, 90 MPa, 95 MPa, 100 MPa, 105 MPa, 110 MPa, 115 MPa, 130 MPa, 135 MPa, 140 MPa, 145 MPa, 150 MPa, 155 MPa, 160 MPa, or greater. A fibre of the present disclosure may have an ultimate tensile strength of about 115 MPa ± 25 MPa. A polypeptide of the disclosure may be capable of forming such a fibre.

[0013] In any embodiment, the polypeptide is capable of forming a fibre, preferably a birefringent fibre, with a diameter from about 10 pm to about 50 pm, from about 15 pm1006253961to about 45 pm, from about 15 pm to about 40 pm, or from about 15 pm to about 35 pm. Preferably, the fibre has a diameter from about 15 pm to about 31 pm, from about 16 pm to about 32 pm, from about 17 pm to about 33 pm, from about 18 pm to about 34 pm, from about 19 pm to about 35 pm, from about 20 pm to about 36 pm, from about 21 pm to about 37 pm, from about 22 pm to about 38 pm, from about 23 pm to about 39 pm, from about 24 pm to about 40 pm, or from about 25 pm to about 41 pm. The fibre may have an average diameter of about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, about 41 pm, about42 pm, or about 43 pm. Preferably the fibre has an average diameter of about 17 pm or 30 pm.

[0014] In some embodiments, the fibre is un-drawn, wherein the fibre has not undergone a controlled mechanical drawing process after spinning. For example, the fibre may be prepared according to any methods described herein, including in the examples. The fibre may be prepared by extruding a solution of 5% (w / v) of the polypeptide of the disclosure from a 29-gauge needle at a constant flow rate of 150 pL min-1into a coagulation bath of 70% methanol, extracting the solidified fibre from the coagulation bath, and winding the fibre onto a spool followed by air-drying.

[0015] In any embodiment, the polypeptide is capable of self-association, preferably concentration dependent self-association. The self-association may occur at a concentration lower than the self-association of a fibroin protein from a Hymenopteran, preferably Apis mellifera. Preferably the fibroin protein from Apis mellifera is any one of SEQ ID NOs: 38 to 41 . For example, self-association may occur at a concentration equal to or less than 0.1 mg / ml, equal to or less than 0.075mg / ml, equal to or less than 0.02 mg / ml, or equal to or less than 0.01 mg / ml. Self-association may occur at a concentration from 0.1 mg / ml to 0.01 mg / ml, from 0.075 mg / ml to 0.01 mg / ml, from 0.02 mg / ml to 0.01 mg / ml, from 0.1 mg / ml to 0.02 mg / ml, from 0.1 mg / ml to 0.075 mg / ml, from 0.075 mg / ml to 0.01 mg / ml, or from 0.075 mg / ml to 0.02 mg / ml. The selfassociation can occur in any buffer described herein, including the Examples, for example 20mM Tris, pH 7.1006253961

[0016] Typically, the self-associated polypeptides form a structure that includes coiled- coil and / or [3-sheet.

[0017] In any embodiment, the polypeptide is capable of reversible thermal denaturation. For example, the polypeptide is capable of forming a structure that includes coiled-coil and / or [3-sheet after thermal denaturation. The reversal thermal denaturation may be measured by any methods known in the art, or described herein (e.g., in the Examples). The reversal thermal denaturation may be measured by circular dichroism, and the capability of reversal thermal denaturation may be determined by comparing circular dichroism spectra before thermal denaturation and after thermal denaturation when the temperature is reduced (e.g. to at, or about, 20°C). The thermal denaturation may occur at a temperature from 30°C to 94°C, for example as described in the Examples.

[0018] In any embodiment, the formation of a coiled-coil structure may be determined by any methods known in the art, or described herein (e.g., in the Examples). For example, the formation of the coiled-coil structure may be determined by circular dichroism analyses, including the assessment of the minima of the circular dichroism spectra at around 208 nm and 222 nm when the concentration of the polypeptide increases. Preferably, an increase in the ratio of the MRE value at 222 nm and the MRE value at 208 nm (MRE222 nm I MRE208nm) indicate the formation of a coiled-structure. More preferably, the formation of the coiled-coil structure is determined when the MRE222 nm I MRE208nm ratio is greater than about 0.9, greater than about 1.0, or greater than about 1.1. Optionally, the polypeptide shows a MRE222 nm I MRE208nm of about 1 .0 at a concentration of about 0.0125 mg / mL to about 0.05 mg / mL, and aMRE222 nm I MRE208nm of about 1 .2 at a concentration of about 0.1 mg / mL to about 0.4 mg / mL.

[0019] In any embodiment, the polypeptide may have an amino acid sequence where the percentage of amino acids that are alanine is equal to, or greater than, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56% or 57%.

[0020] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 1 to 36, (ii) an amino1006253961acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 1 to 36, or (iii) a biologically active fragment of (i) or (ii).

[0021] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 1 to 25, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 1 to 25, or (iii) a biologically active fragment of (i) or (ii).

[0022] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 26 to 36, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 26 to 36, or (iii) a biologically active fragment of (i) or (ii).

[0023] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5, 3, 26, 27, 29, 30, or 36, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5, 3, 26, 27, 29, 30, or 36, or (iii) a biologically active fragment of (i) or (ii).

[0024] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, 2, 31 , or 28, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, 2, 31 , or 28, or (iii) a biologically active fragment of (i) or (ii).1006253961

[0025] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 14, 16, 19, 34 or 32, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 14, 16, 19, 34 or 32, or (iii) a biologically active fragment of (i) or (ii).

[0026] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, 25, 35, or 33, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 4, 20,6, 23, 15, 25, 35, or 33, or (iii) a biologically active fragment of (i) or (ii).

[0027] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5 or 3, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5 or 3.

[0028] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10,7, 13, or 2, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, or 2, or (iii) a biologically active fragment of (i) or (ii).

[0029] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 14, 16 or 19, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 14, 16 or 19, or (iii) a biologically active fragment of (i) or (ii).1006253961

[0030] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, or 25, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, or 25, or (iii) a biologically active fragment of (i) or (ii).

[0031] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 26, 27, 29, 30, and / or 36, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 26, 27, 29, 30, or 36, or (iii) a biologically active fragment of (i) or (ii).

[0032] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 31 and / or 28, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 31 or 28, or (iii) a biologically active fragment of (i) or (ii).

[0033] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 34 or 32, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 34 or 32, or (iii) a biologically active fragment of (i) or (ii).

[0034] In one embodiment, the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 35 or 33, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 35 or 33, or (iii) a biologically active fragment of (i) or (ii).1006253961

[0035] In any embodiment, where the polypeptide comprises, consists essentially of or consists of an amino acid sequence that exhibits variation to any one of SEQ ID NO: 1 to 36, or a biologically active fragment thereof, (i.e. is not identical to an amino acid sequence of any one of SEQ ID NO: 1 to 36) the polypeptide sequence exhibits the same or substantially similar alanine percentage as any one of SEQ ID NO: 1 to 36.

[0036] In one embodiment, the polypeptide is a variant of an amino acid sequence of any one of SEQ ID NO: 1 to 36, or biologically active fragment thereof, where the variant contains the same or substantially similar alanine percentage to the corresponding amino acid sequence of any one of SEQ ID NO: 1 to 36. For example, a polypeptide of the disclosure that is a variant of SEQ ID NO: 1 , or biologically active fragment thereof, may exhibit an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO: 1 and the amino acid sequence has an alanine percentage of 50.9%, or an alanine percentage greater than or equal to 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49% or 50%. Therefore, a polypeptide of the disclosure that is a variant of any one of SEQ ID NO: 1 to 36, or biologically active fragment thereof, may exhibit an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of SEQ ID NO: 1 to 36 respectively and the amino acid sequence has an alanine percentage as shown in Table 3 for the corresponding SEQ ID NO., or an alanine percentage within, or within about, 10% of the alanine percentage shown in Table 3 for the corresponding SEQ ID NO.

[0037] In any embodiment, the polypeptide may further comprise a tag, preferably at the N-terminus. The tag may assist with purification. In one embodiment, the tag comprises, consists essentially of or consist of the amino acid sequence of SEQ ID NO: 37.

[0038] In any embodiment, the polypeptide is a soluble polypeptide. Typically, the polypeptide exhibits a greater level of solubility than a fibroin protein from a Hymenopteran, preferably Apis mellifera. The fibroin protein may have the amino acid sequence of any one of a fibroin 1 protein, a fibroin 2 protein, a fibroin 3 protein or a fibroin 4 protein. Preferably the fibroin protein from Apis mellifera is any one of SEQ ID NOs: 38 to 41.1006253961

[0039] In any aspect or embodiment, the polypeptide is expressed in a soluble form when produced by a recombinant cell. Preferably, the recombinant cell is a recombinant bacterial cell.

[0040] In another embodiment, the polypeptide is capable of being solubilised after expression as an insoluble polypeptide when produced by a recombinant cell. Preferably, the recombinant cell is a recombinant bacterial cell.

[0041] In another embodiment, the polypeptide is capable of being expressed at yields greater than a fibroin protein from a Hymenopteran, preferably Apis mellifera, typically when expressed in a recombinant bacterial cell. Preferably the fibroin protein from Apis mellifera is any one of SEQ ID NOs: 38 to 41 . Preferably, the polypeptide is capable of being expressed at about 100mg / L, about 120mg / L, about 140mg / L, about 160mg / L, about 180mg / L or about 200mg / L when expressed in a 1 L shaker flask as per the scaled up 1 L expression method described in the Examples. Preferably, the polypeptide is capable of being expressed at greater than about 100mg / L, greater than about 120mg / L, greater than about 140mg / L, greater than about 160mg / L, greater than about 180mg / L or greater than about 200mg / L when expressed in a 1 L shaker flask as per the scaled up 1 L expression method described in the Examples. Preferably, the polypeptide is capable of being expressed at about 500mg / L, about 750mg / L, about 1000mg / L, about 1250mg / L, or about 1500mg / L when expressed in a 1 L bioreactor, typically using the method described in J Biomol NMR. 2018 Aug;71 (4):247-262. Preferably, the polypeptide is capable of being expressed at greater than about 500mg / L, greater than about 750mg / L, greater than about 1000mg / L, greater than about 1250mg / L, or greater than about 1500mg / L when expressed in a 1 L bioreactor, typically using the method described in J Biomol NMR. 2018 Aug;71 (4):247-262.

[0042] In another embodiment, the polypeptide is capable of being expressed at yields greater than a fibroin protein from a Hymenopteran, preferably Apis mellifera, typically when expressed in a recombinant bacterial cell. Preferably the fibroin protein from Apis mellifera is any one of SEQ ID NOs: 38 to 41 . Preferably, the polypeptide is capable of being expressed at about 100mg / L, about 120mg / L, about 140mg / L, about 160mg / L, about 180mg / L, about 200mg / L, about 400mg / L, about 600mg / L, about 800mg / L, about 1 g / L, about 1.2g / L, about 1.4g / L, about 1.6g / L, about 1.8g / L, about 2.0g / L, about 2.2g / L, about 2.4g / L, about 2.6g / L, about 2.8g / L, or about 3.0g / L when expressed in a 1 L shaker flask as per the scaled up 1 L expression method described in the Examples.1006253961Preferably, the polypeptide is capable of being expressed at greater than about 100mg / L, greater than about 120mg / L, greater than about 140mg / L, greater than about 160mg / L, greater than about 180mg / L, greater than about 200mg / L, greater than about 400mg / L, greater than about 600mg / L, greater than about 800mg / L, greater than about 1 g / L, greater than about 1 ,2g / L, greater than about 1 ,4g / L, greater than about 1 ,6g / L, greater than about 1.8g / L, greater than about 2.0g / L, greater than about 2.2g / L, greater than about 2.4g / L, greater than about 2.6g / L, greater than about 2.8g / L, or greater than about 3.0g / L when expressed in a 1 L shaker flask as per the scaled up 1 L expression method described in the Examples. Preferably, the polypeptide is capable of being expressed at about 500mg / L, about 750mg / L, about 1000mg / L, about 1250mg / L, about 1500mg / L, about 1750mg / L, about 2000mg / L, about 2200mg / L, about 2250mg / L, about 2500mg / L, about 2750mg / L, or about 3000mg / L when expressed in a 1 L bioreactor, typically using the method described in J Biomol NMR. 2018 Aug;71 (4):247-262. Preferably, the polypeptide is capable of being expressed at greater than about 500mg / L, greater than about 750mg / L, greater than about 1000mg / L, greater than about 1250mg / L, greater than about 1500mg / L, greater than about 1750mg / L, greater than about 2000mg / L, greater than about 2200mg / L, greater than about 2250mg / L, greater than about 2500mg / L, greater than about 2750mg / L, or greater than about 3000mg / L when expressed in a 1 L bioreactor, typically using the method described in J Biomol NMR. 2018 Aug;71 (4):247-262.

[0043] In any embodiment, the polypeptide is capable of being expressed at yields greater than a fibroin protein from a Hymenopteran, preferably Apis mellifera, in the soluble fraction, typically when expressed in a recombinant bacterial cell. Preferably the fibroin protein from Apis mellifera is any one of SEQ ID NOs: 38 to 41. Preferably, the polypeptide is capable of being expressed at about 20 mg / L, about 40mg / L, about 60mg / L, about 80mg / L, about 100mg / L, about 120mg / L, about 140mg / L, about 160mg / L, about 180mg / L, about 200mg / L, about 400mg / L, about 600mg / L, about 800mg / L, about 1 g / L, about 1.2g / L, about 1.4g / L, about 1.6g / L, about 1.8g / L, about 2.0g / L, about 2.2g / L, about 2.4g / L, about 2.6g / L, about 2.8g / L, or about 3.0g / L in the soluble fraction, when expressed in a 1 L shaker flask as per the scaled up 1 L expression method described in the Examples. Preferably, the polypeptide is capable of being expressed at greater than about 20 mg / L, greater than about 40mg / L, greater than about 60mg / L, greater than about 80mg / L, greater than about 100mg / L, greater than about 120mg / L, greater than about 140mg / L, greater than about 160mg / L, greater1006253961than about 180mg / L, greater than about 200mg / L, greater than about 400mg / L, greater than about 600mg / L, greater than about 800mg / L, greater than about 1 g / L, greater than about 1 .2g / L, greater than about 1 .4g / L, greater than about 1 .6g / L, greater than about 1 .8g / L, greater than about 2.0g / L, greater than about 2.2g / L, greater than about 2.4g / L, greater than about 2.6g / L, greater than about 2.8g / L, or greater than about 3.0g / L in the soluble fraction when expressed in a 1 L shaker flask as per the scaled up 1 L expression method described in the Examples. Preferably, the polypeptide is capable of being expressed at about 100mg / L, about 200mg / L, about 300mg / L, about 400mg / L, about 500mg / L, about 750mg / L, about 1000mg / L, about 1250mg / L, about 1500mg / L, about 1750mg / L, about 2000mg / L, about 2200mg / L, about 2250mg / L, about 2500mg / L, about 2750mg / L, or about 3000mg / L in the soluble fraction when expressed in a 1 L bioreactor, typically using the method described in J Biomol NMR. 2018 Aug;71 (4):247-262. Preferably, the polypeptide is capable of being expressed at greater than about 100mg / L, greater than about 200mg / L, greater than about 300mg / L, greater than about 400mg / L, greater than about 500mg / L, greater than about 750mg / L, greater than about 1000mg / L, greater than about 1250mg / L, greater than about 1500mg / L, greater than about 1750mg / L, greater than about 2000mg / L, greater than about 2200mg / L, greater than about 2250mg / L, greater than about 2500mg / L, greater than about 2750mg / L, or greater than about 3000mg / L in the soluble fraction, when expressed in a 1 L bioreactor, typically using the method described in J Biomol NMR. 2018 Aug;71 (4):247-262.

[0044] In any embodiment, the polypeptide does not form amyloid-like [3-sheet fibrils, typically observed in assembly of lepidopteran and arachnid silk proteins, in solution.For example, the polypeptide of the disclosure is not amyloidogenic. The absence of the [3-sheet may be determined by any assays known in the art or described herein (e.g., in the Examples), e.g., a Thioflavin T (ThT) fluorescence assay wherein no increase in fluorescence over time indicates the absence of the [3-sheet. Optionally, the solution-state process of the polypeptide is driven by a-helical coiled-coil interactions, absent of [3-sheet intermediates.

[0045] In any embodiment, the polypeptide forms a single, monodisperse species, preferably a stable homotetramer, more preferably a coiled coil homotetramer, in solution. The formation of the species or the homotetramer may be determined by any assays known in the art or described herein (e.g., in the Examples), e.g., Size Exclusion Chromatography (SEC) or Dynamic Light Scattering (DLS). Optionally, the1006253961homotetramer has a particle size distribution at about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, or about 16 nm, at a concentration of about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL, about 7 mg / mL, or about 8 mg / mL as measured by Dynamic Light Scattering (DLS).Preferably, as concentration of the polypeptide increases from about 1 mg / mL to about 5 mg / mL, the particle size distribution shifts from a 7 nm species to a 13 nm species, consistent with a coiled coil homotetramer, and then to a higher order helical assembly.

[0046] In any embodiment, the polypeptide is capable of forming a transparent film. For example, a solution of polypeptide in water where the polypeptide is at a concentration of about 50mg / ml can form a transparent film when dried (e.g. dried at room temperature for at least about 16 hours). The transparent film may be flexible.

[0047] A film of the present disclosure may have reflections with d-spacings of about 8.54 A (010), about 4.34 A ((210), inter-[3-strand distance), and about 4.03 A ((020), inter-[3-sheet distance), and / or a calculated lattice parameter of a equalling to about 11 .34 A and b equalling to about 8.42 A, and / or an orientation index of about 0.77 quantified through azimuthal analysis of the prominent (020) reflection, and / or a calculated crystallite length of about 1.39 nm, as determined by synchrotron wide-angle X-ray scattering WAXS. Preferably, the film has a correlation peak at q-values corresponding to a long period (L) of about 9.0 A, and / or an observed periodicity L of about 9.0 A, as determined by small-angle X-ray scattering (SAXS).

[0048] In any embodiment, the polypeptide is capable of forming a sponge or spongelike structure. For example, lyophilized polypeptide may be treated or washed with methanol and then dried to form a solid sponge or sponge-like structure.

[0049] The transparent film and the sponge (or sponge-like material) may be waterinsoluble and may remain water-insoluble following methanol soaking.

[0050] In any embodiment, the polypeptide is capable of forming a fibre as described herein, a transparent film as described herein and a sponge as described herein.

[0051] As outlined above, the polypeptide is capable of forming a fibre. For example, the polypeptide may be capable of forming a fibre by a process whereby a 10% w / v solution of one or more polypeptides in water is extruded through a 29-gauge needle at a speed of 4 m min-1(or 150 pL / min) directly into a coagulation bath containing 100%1006253961methanol, maintained at room temperature. The polypeptide may also be capable of forming a fibre by a process whereby a 5% w / v solution of one or more polypeptides in water is extruded through a 29-gauge needle at a speed of 4 m min’1(or 150 pL / min-1) directly into a coagulation bath containing 70% methanol. The fibres may be capable of being wound onto a spool, preferably at a take-up speed of 4 m min-1.

[0052] In another aspect, the present disclosure provides a fusion protein comprising a polypeptide of the disclosure.

[0053] In another aspect, the present disclosure provides a composition comprising a polypeptide and / or fusion protein of the disclosure.

[0054] In another aspect, the present disclosure provides a fibre comprising at least one polypeptide of the disclosure. The fibre may be prepared using any process described herein, including in the Examples. The fibre may be a wet-spun fibre. The fibre may be a birefringent fibre.

[0055] A fibre of the present disclosure may have a diameter of greater than 9 pm, greater than or equal to 10 pm, greater than or equal to 11 pm, greater than or equal to 12 pm, greater than or equal to 13 pm, greater than or equal to 14 pm, greater than or equal to 15 pm , greater than or equal to 16 pm , or greater than or equal to 17 pm .

[0056] A fibre of the present disclosure may have a breaking stress of greater than 132 MPa, greater than 200 MPa, greater than or equal to 250 MPa, greater than or equal to 300 MPa, greater than or equal to 350 MPa, greater than or equal to 400 MPa, or greater than or equal to 450 MPa. A polypeptide of the disclosure may be capable of forming such a fibre.

[0057] A fibre of the present disclosure may have a breaking stress of greater than 132 MPa and less than 532 MPa, greater than 200 MPa and less than 600 MPa, greater than 250 MPa and less than 650 MPa, greater than 350 MPa and less than 750 MPa, greater than 400 MPa and less than 800 MPa, or greater than 450 MPa and less than 850 MPa. A polypeptide of the disclosure may be capable of forming such a fibre.

[0058] A fibre of the present disclosure may have a diameter of greater than or equal to 19 pm, greater than or equal to 21 pm, greater than or equal to 23 pm, greater than or equal to 25 pm, greater than or equal to 27 pm, greater than or equal to 29 pm,1006253961greater than or equal to 31 pm, greater than or equal to 33 pm, greater than or equal to 35 pm, greater than or equal to 37 pm, greater than or equal to 39 pm, greater than or equal to 41 pm, or greater than or equal to 43 pm. Preferably the fibre has a diameter of greater than or equal to 30 pm. A polypeptide of the disclosure may be capable of forming such a fibre.

[0059] A fibre of the present disclosure may have a calculated lattice parameter of a equalling to about 10.30 A and b equalling to about 8.06 A, and / or an orientation index of about 0.87 quantified through azimuthal analysis of the prominent (020) reflection, and / or a calculated crystallite length of about 1 .71 nm, as determined by synchrotron wide-angle X-ray scattering WAXS. Preferably, the fibre has a correlation peak at q- values corresponding to a long period (L) of about 9.0 A, and / or an observed periodicity L of about 9.0 A, as determined by small-angle X-ray scattering (SAXS). A polypeptide of the disclosure may be capable of forming such a fibre.

[0060] A fibre of the disclosure may have a Young’s modulus that is at least or about 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1 -fold, 2.2-fold, 2.3-fold, or 2.4-fold greater than the Young’s modulus of a fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41 . A fibre of the present disclosure may have a Young’s modulus of about 0.9 GPa, about 1.1 GPa, about 1 .3 GPa, about 1 .5 GPa, about 1 .7 GPa, about 1 .9 GPa, about 2.1 GPa, about 2.3 GPa, about 2.5 GPa, or greater.Optionally, the Young’s modulus is from about 0.9 GPa to about 1 .9 GPa, from about 1 .1 GPa to about 2.1 GPa, from about 1 .3 GPa to about 2.3 GPa, from about 1 .5 GPa to about 2.5 GPa, from about 1.7 GPa to about 2.7 GPa, from about 1 .9 GPa to about 2.9 GPa, from about 2.1 Gpa to about 3.1 GPa, from about 2.3 Gpa to about 3.3 GPa, or from about 2.5 GPa to about 3.5 GPa. Preferably the Young’s modulus is 0.9 GPa, 1.1 GPa, 1 .3 GPa, 1 .5 GPa, 1 .7 GPa, 1 .9 GPa, 2.1 GPa, 2.3 GPa, or 2.5 GPa. A fibre of the present disclosure may have a Young’s modulus of about 1.9 ± 0.5 GPa. A polypeptide of the disclosure may be capable of forming such a fibre.

[0061] A fibre of the present disclosure may have an ultimate tensile strength that is at least or about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, or 14-fold greater than the ultimate tensile strength of a fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41 . A fibre of the present disclosure may have an1006253961ultimate tensile strength of about 55 MPa, about 70 MPa, about 85 MPa, about 90 MPa, about 95 MPa, about 100 MPa, about 105 MPa, about 110 MPa, about 115 MPa, about 130 MPa, about 135 MPa, about 140 MPa, about 145 MPa, about 150 MPa, about 155 MPa, about 160 MPa, or greater. Optionally, the ultimate tensile strength is from about 55 MPa to about 105 MPa, from about 70 MPa to about 120 MPa, from about 85 MPa to about 135 MPa, from about 90 MPa to about 140 MPa, from about 95 MPa to about 145 MPa, from about 100 MPa to about 150 MPa, from about 105 MPa to about 155 MPa, from about 110 MPa to about 160 MPa, from about 115 MPa to about 165 MPa, from about 130 MPa to about 180 MPa, from about 135 MPa to about 185 MPa, from about 140 MPa to about 190 MPa, from about 145 MPa to about 195 MPa, from about 150 MPa to about 200 MPa, from about 155 MPa to about 205 MPa, or from about 160 MPa to about 210 MPa. Preferably, a fibre of the present disclosure may have an ultimate tensile strength of 55 MPa, 70 MPa, 85 MPa, 90 MPa, 95 MPa, 100 MPa, 105 MPa, 110 MPa, 115 MPa, 130 MPa, 135 MPa, 140 MPa, 145 MPa, 150 MPa, 155 MPa, 160 MPa, or greater. A fibre of the present disclosure may have an ultimate tensile strength of about 115 MPa ± 25 MPa. A polypeptide of the disclosure may be capable of forming such a fibre.

[0062] A fibre of the present disclosure may have a failure strain that is at least or about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, or 14- fold greater than the failure strain of a fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41 . A fibre of the present disclosure may have a failure strain of about 0.10 mm / mm, about 0.15 mm / mm, about 0.20 mm / mm, about 0.25 mm / mm, about 0.30 mm / mm, about 0.35 mm / mm, about 0.40 mm / mm, about0.45 mm / mm, about 0.50 mm / mm, about 0.55 mm / mm, about 0.60 mm / mm, about0.65 mm / mm, about 0.70 mm / mm, about 0.75 mm / mm, about 0.80 mm / mm, about0.85 mm / mm, or greater. Optionally, the failure strain is from about 0.10 mm / mm to about 0.50 mm / mm, from about 0.15 mm / mm to about 0.55 mm / mm, from about 0.20 mm / mm to about 0.60 mm / mm, from about 0.25 mm / mm to about 0.65 mm / mm, from about 0.30 mm / mm to about 0.70 mm / mm, from about 0.35 mm / mm to about 0.75 mm / mm, from about 0.40 mm / mm to about 0.80 mm / mm, from about 0.45 mm / mm to about 0.85 mm / mm, from about 0.50 mm / mm to about 0.90 mm / mm, from about 0.55 mm / mm to about 0.95 mm / mm, from about 0.60 mm / mm to about 1 .00 mm / mm, from about 0.65 mm / mm to about 1 .05 mm / mm, from about 0.70 mm / mm to about10062539611.10 mm / mm, from about 0.75 mm / mm to about 1.15 mm / mm, from about 0.80 mm / mm to about 1 .20 mm / mm, or from about 0.85 mm / mm to about 1 .25 mm / mm. Preferably the failure strain is 10 mm / mm, 0.15 mm / mm, 0.20 mm / mm, 0.25 mm / mm, 0.30 mm / mm, 0.35 mm / mm, 0.40 mm / mm, 0.45 mm / mm, 0.50 mm / mm, 0.55 mm / mm, 0.60 mm / mm, 0.65 mm / mm, 0.70 mm / mm, 0.75 mm / mm, 0.80 mm / mm, or 0.85 mm / mm. A fibre of the present disclosure may have a failure strain of about 0.45 ± 0.20 mm / mm. A polypeptide of the disclosure may be capable of forming such a fibre.

[0063] A fibre of the present disclosure may have a work of fracture that is at least or about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold or 13-fold greater than the work of fracture of a fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41 . A fibre of the present disclosure may have a work of fracture of about 5 MJ rrr3, about 7.5 MJ rrr3, about 10 MJ rrr3, about 12.5 MJ rrr3, about 15 MJ nr3, about 17.5 MJ rrr3, about 20 MJ rrr3, about 22.5 MJ nr3, about 25 MJ nr3, about 27.5 MJ rrr3, about 30 MJ rrr3, or greater. Optionally, the work of fracture is from about 5 MJ nr3about 15 MJ nr3, from about 7.5 MJ rrr3to about 17.5 MJ rrr3, from about 10 MJ rrr3to about 20 MJ rrr3, from about 12.5 MJ rrr3to about 22.5 MJ rrr3, from about 15 MJ rrr3to about 25 MJ rrr3, from about 17.5 MJ rrr3to about 27.5 MJ rrr3, from about 20 MJ rrr3to about 30 MJ rrr3, from about 22.5 MJ rrr3to about 32.5 MJ rrr3, from about 25 MJ rrr3to about 35 MJ rrr3, from about 27.5 MJ rrr3to about 37.5 MJ rrr3, or from about 30 MJ rrr3to about 40 MJ rrr3. Preferably, the work of fracture is 5 MJ rrr3, 7.5 MJ rrr3, 10 MJ rrr3, 12.5 MJ rrr3, 15 MJ rrr3, 17.5 MJ rrr3, 20 MJ rrr3, 22.5 MJ rrr3, 25 MJ nr3, 27.5 MJ nr3, or 30 MJ nr3A fibre of the present disclosure may have a work of fracture of about 20 ± 5 MJ nr3. A polypeptide of the disclosure may be capable of forming such a fibre.

[0064] In any embodiment, the polypeptide is capable of forming a nanofibre as described herein, preferably by electrospinning. For example, the polypeptide may be prepared in 1 ,1 ,1 ,3,3,3-hexafluoroisopropanol (HFIP) with magnetic stirring for about 15 h, preferably at a concentration of 14% (w / v), and the solution may be electrospun by feeding at about 0.3 m L h-1through a 21 -gauge needle with an applied voltage of about 15 kV and a collector distance of about 10 cm. Randomly oriented nanofibres may be collected on a rotating drum (150 rpm), dried for about 12 h, and treated with methanol.1006253961

[0065] A nanofibre of the present disclosure may be in the form of nanofibrous mats. Preferably, the nanofibrous mats exhibit a randomly oriented, non-woven architecture with smooth nanofibres, wherein the average diameter of the nanofibres are about 700 nm, 800 nm, about 900 nm, about 1 ,000 nm, about 1 ,100 nm, about 1 ,200 nm, about 1 ,300 nm, about 1 ,400 nm, about 1 ,500 nm, about 1 ,600 nm, about 1 ,700 nm, or about 1 ,800 nm. Optionally, the nanofibrous mats are suitable for cell culture, for example, by directly culturing cells on the nanofibrous mats. Optionally, the cells are fibroblasts, preferably human dermal fibroblasts. Optionally, upon culture on the nanofibrous mats, preferably after 3 days of the culture, the cells adhere on the nanofibrous mats, spread on the nanofibrous mats (e.g., exhibiting an extended cell morphology), have increased density, display flattened and elongated shape, and / or exhibit multiple filopodia anchoring to the nanofibres.

[0066] In any embodiment, the polypeptide is capable of forming a hydrogel as described herein, preferably by hydrogelation. For example, the polypeptide may be prepared in 4°C Milli-Q water, preferably at a concentration of 2% (w / v), by stirring at about 100 rpm at 4°C for about 16 h. The solution may then be heated to about 60°C for about 15 minutes to induce gelation. Optionally, the hydrogel has a porous hydrogel structure upon gelation, which may be assessed qualitatively via the inversion test, SEM and / or cryo-SEM to confirm a porous hydrogel structure.

[0067] In another aspect, the present disclosure provides a nucleic acid comprising, consisting essentially of or consisting of a nucleotide sequence encoding a polypeptide of the disclosure. Preferably, the nucleotide sequence encodes a polypeptide comprising, consisting essentially of or consisting of (i) an amino acid sequence of any one of SEQ ID NO: 1 to 36, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 1 to 36, or (iii) a biologically active fragment of (i) or (ii).

[0068] In another aspect, the present disclosure provides a nucleic acid comprising, consisting essentially of or consisting of a nucleotide sequence as set forth in any one of SEQ ID NOs: 42 to 121.

[0069] The present disclosure also encompasses variants of nucleic acids of the disclosure. These variants are each defined as having one or more substitutions,1006253961insertions and / or deletions as compared to any one of the nucleotide sequences encoding SEQ ID NO: 1 to 36, provided that said variants hybridize under moderately or highly stringent conditions to a nucleic acid which comprises a nucleotide sequence encoding any one of SEQ ID NO: 1 to 36, or provided that said variants comprise nucleic acid changes due to the degeneracy of the genetic code, which code for the same or a functionally equivalent amino acid as the nucleic acid sequence of SEQ ID NO: 1 to 36.

[0070] In one embodiment, the nucleic acid may be a vector, for example a cloning vector or an expression vector. The expression vector preferably comprises one or more regulatory sequences. For example, a regulator sequence may be a promoter and the nucleotide sequence encoding a polypeptide of the disclosure is operably linked to the promoter.

[0071] In another aspect, the present disclosure provides a cell comprising a nucleic acid or vector of the disclosure. The host cell can be any type of cell. Examples include, but are not limited to, a mammalian (preferably non-human cell, or an isolated or recombinant human cell), bacterial, yeast or plant cell.

[0072] In another aspect, the present disclosure provides a process for preparing a polypeptide, composition, fibre, transparent film or sponge of the disclosure, the process comprising culturing a host cell of the disclosure, or a vector of the disclosure, under conditions which allow expression of the polynucleotide encoding the polypeptide, and optionally recovering the expressed polypeptide.

[0073] In one embodiment, the process further comprises the set of contacting or washing the polypeptide, fibre, transparent film or sponge with an alcohol. Preferably the alcohol is methanol.

[0074] In another aspect, the present invention provides an alcohol contacted or washed polypeptide, fibre, transparent film or sponge of the disclosure. Preferably the alcohol is methanol.

[0075] In another aspect, the present disclosure provides a product comprising at least one polypeptide of the disclosure, a fibre of the disclosure, a film of the disclosure, a sponge of the disclosure, a nanofibre of the disclosure, and / or a hydrogel of the disclosure. Examples of products include, but are not limited to, personal care products,1006253961textiles, plastics, and biomedical products. Preferably, a product of the disclosure is for use in tissue engineering, soft robotics, and regenerative medicine.

[0076] In another aspect, the present disclosure provides a hydrogel comprising at least one polypeptide of the disclosure, a fibre of the disclosure, a film of the disclosure, a sponge of the disclosure, and / or a nanofibre of the disclosure.

[0077] In a further aspect, the present disclosure provides a kit comprising at least one polypeptide of the disclosure, at least one polynucleotide of the disclosure, at least one vector of the disclosure, and / or at least one silk fibre of the disclosure.

[0078] Preferably, the kit further comprises information and / or instructions for use of the kit.

[0079] As used herein, except where the context requires otherwise, the term "comprise" and variations of the term, such as "comprising", "comprises" and "comprised", are not intended to exclude further additives, components, integers or steps.

[0080] Further aspects of the present invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings.Brief description of the drawings

[0081] Figure 1 : SDS-PAGE analysis of lysates from various expression cultures. Lane 1 : Low molecular weight protein marker; Lanes 2 and 3: Lysates demonstrating overexpression of two designer silk proteins; Lane 4: Lysate showing no soluble expression of the natural silk protein.

[0082] Figure 2: SDS-PAGE analysis showing the secretion of secretory signal peptide tagged designer silk protein. Lane 1 : Low molecular weight marker, Lane 2: Supernatant showing secretion of designer silk protein (~35 kDa), Lane 3: Supernatant of secretory signal peptide tagged natural silk fibroin protein, indicating no secretion.

[0083] Figure 3: SDS-PAGE analysis of immobilized metal affinity chromatography (IMAC) Ni-NTA purified His-tagged designer silk proteins from the lysate, showing1006253961relative yields and soluble expression. "M" indicates the molecular weight protein marker, while lanes labelled A1 to G7 represent various purified designer silk proteins.

[0084] Figure 4: Ratio between CD minima at 222 nm and 208 nm of varying silk fibroin proteins (0.5 mg / mL protein in 20 mM Tris, pH 7). A ratio >1.0 indicates the formation of a coiled-coil structure, suggesting self-assembly. "SF" denotes the silk fibroin proteins from Apis mellifera. These results demonstrate that designer silk proteins self-associate at lower concentrations compared to individual natural silk proteins and a combination of four natural silk fibroin proteins.

[0085] Figure 5: Example of the concentration dependent self-association observed in designer silk proteins. Increasing concentration correlates with a deeper minimum at 222 nm, indicating the formation of a coiled-coil structure, suggesting the self-assembly of these proteins. Similar concentration dependent self-association was observed in natural silk proteins but only at much higher concentrations. (A) Lines from high MRE to low MRE at approximately 220 nm: 0.1 mg / mL, 0.075 mg / mL, 0.02 mg / mL, and0.01 mg / mL. (B) Lines from high MRE to low MRE at approximately 220 nm: 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, 0.05 mg / mL, 0.025 mg / mL, and 0.0125 mg / mL. (C) Ratio of 222 nm and 208 nm of A6 (denoted Protosilk) and A. mellifera silk fibroin proteins 1-4 (F1-4) at varied concentrations, measured using circular dichroism. A ratio of >1.0 suggests coiled coil formation. A6 is shown to form a coiled coil at a lower concentration than natural silk proteins. Dots around and above the 1 .0 ratio: Protosilk; dots below the 0.9 ratio: Combined F1-4.

[0086] Figure 6: Example of the reversible thermal denaturation observed in designer silk proteins. (A) displays the circular dichroism (CD) spectrum of the protein in solution during heating up to 94°C. (B) shows the CD spectrum before and after heating, demonstrating minimal loss of secondary structure following refolding.

[0087] Figure 7: (A) FTIR results of the freeze dried A6 before and after MeOH treatment. Labelled peaks show increase in [3-sheet content following MeOH. Lines from high %T to low %T at approximately 1 ,300 cm’1: After MeOH Wash, and Before MeOH Wash. (B) Fourier Transform Infrared (FTIR) spectroscopy analysis of various concentrations (1 .0-100 mg / mL, D2O). Lines from high absorbance to low absorbance at approximately 1 ,640 cm’1: 50 mg / mL, 25 mg / mL, 10 mg / mL, 5 mg / mL, and 1 mg / mL. (C) Results from a second-derivative analysis. Lines from high absorbance to low1006253961absorbance at approximately 1 ,640 cm-1: 1 mg / mL, 5 mg / mL, 10 mg / mL, 25 mg / mL, and 50 mg / mL.

[0088] Figure 8: Results of a Thioflavin T (ThT) fluorescence assay. Lines from high absorbance to low absorbance: lysozyme 10 mg / mL, lysozyme 1.0 mg / mL, silk 10 mg / mL, silk 1.0 mg / mL, and blank.

[0089] Figure 9: (A) UV-Trace of Analytical Size Exclusion Chromatography (SEC) of the designer silk proteins. Apparent mass calculated as 126 kDa, representative AF2 homotetramer structure shown. (B) Natural heterotetramer (left) vs designed homotetramer (right).

[0090] Figure 10: (A) Number distribution as calculated by Dynamic Light Scattering (DLS) at varied concentrations (0.1 - 10 mg / mL), and proposed monomer to tetramer transition. (B) Alternative graphical representation of the data.

[0091] Figure 11 : (A) Clear air-dried film formed from 5% designer silk protein. (B) and (C) display SEM images of this film, revealing a smooth surface. (D) Lyophilized, MeOH-washed designer silk protein forming a sponge. (E) and (F) show SEM images of the surface of this sponge. Both materials are water insoluble following MeOH soaking.

[0092] Figure 12: Optical microscopy of a wet-spun silk fibre (10% silk protein spun into 100% MeOH) showing an even, birefringent fibre with a diameter of 17 pm. The scale bar represents 100 pm.

[0093] Figure 13: Lyophilised designer silk proteins (middle) and materials they can be processed to (fibre, top left; film, top right; nanofibers, bottom left; and hydrogel, bottom right).

[0094] Figure 14: SAXS / WAXS analysis showing structural transitions and ordered packing in ancestral silk materials, (a) Extracted scattering intensity as a function of scattering vector corresponding to the A6 fibre and along the equatorial and azimuthal direction. Lines from high to low scattering intensity at q = approximately 0.8 A’1: fibre equatorial (blue), fibre (red) and fibre meridoional (purple), (b) Comparison of the scattering intensity as a function of scattering vector between the designer silk fibre and film. Lines from high to low scattering intensity at q = approximately 0.8 A’1: fibre (red) and film (green), (c) Comparison of the scattering intensity as a function of scattering1006253961vector between the film and the scattering intensity of the silk fibre along the meridional direction. Lines from high to low scattering intensity at q = approximately 0.8 A’1: film (green) and fibre meridional (purple), (d) WAXS (top) and SAXS (bottom) 2D patterns of designer silk fibre, (e) WAXS (top) and SAXS (bottom) 2D patterns of designer silk film.

[0095] Figure 15: Mechanical testing of designer protein and A. mellifera silk fibroin fibres, (a) Normalised stress-strain curves from designer protein (synthetic) and A. mellifera silk fibroins (F1-4) (natural), spun from an aqueous solution of lyophilised protein into methanol. Line from high o to low o: Synthetic, and Natural, (b) Ultimate tensile strength, (c) Youngs modulus, (d) Failure strain, (e) Toughness.

[0096] Figure 16: Characterisation of electrospun designer silk nanofibrous membrane and cell interactions. (A) Scanning electron microscopy (SEM) images of designer silk nanofibres (low and high magnification), showing a uniform, bead-free and randomly oriented submicron / nanofibrous distribution. (B) LIVE / DEAD fluorescence staining of hDF cells on Day 1 , and Day 3, Live cells (green, first column), and Dead cells (red, second column). The third column is the merged image, with fibres showing autofluorescence. Scale bar indicates 200 pm. (c) SEM images of hDF cells cultured on nanofibres at low and high magnifications.Sequence information

[0097] Table 1. Protein sequences100625396110062539611006253961100625396110062539611006253961

[0098] Table 2. Nucleotide sequences10062539611006253961100625396110062539611006253961100625396110062539611006253961100625396110062539611006253961100625396110062539611006253961100625396110062539611006253961100625396110062539611006253961100625396110062539611006253961100625396110062539611006253961Detailed description of the embodiments

[0099] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0100] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0101] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0102] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.Polypeptide production and purification

[0103] The terms “polypeptide” and “protein” are generally used interchangeably and refer to a single polypeptide chain. The polypeptide may or may not be modified by addition of non-amino acid groups.

[0104] The terms “isolated" or “purified” when used to describe the various polypeptides disclosed herein, mean the polypeptide that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with1006253961diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1 ) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. Isolated protein includes polypeptide in situ within recombinant cells, since at least one component of the polypeptide natural environment will not be present. Ordinarily, however, isolated polypeptide will be prepared by at least one purification step.

[0105] A "fragment" is a portion of a polypeptide of the present invention that retains substantially similar functional activity or substantially the same biological function or activity as the polypeptide, which can be determined using assays described herein.

[0106] As used herein a "biologically active" fragment is a portion of a polypeptide of the invention which maintains a defined activity of the full-length polypeptide, namely the ability to be used to produce a fibre. Biologically active fragments can be any size as long as they maintain the defined activity.

[0107] The term “recombinant” in the context of a polypeptide refers to the polypeptide when produced by a cell, or in a cell-free expression system, in an altered amount or at an altered rate compared to its native state. In one embodiment the cell is a cell that does not naturally produce the polypeptide. However, the cell may be a cell which comprises a non-endogenous gene that causes an altered, preferably increased, amount of the polypeptide to be produced. A recombinant polypeptide of the disclosure includes polypeptides which have not been separated from other components of the transgenic (recombinant) cell, or cell-free expression system, in which it is produced, and polypeptides produced in such cells or cell-free systems which are subsequently purified away from at least some other components.

[0108] “Percent (%) amino acid sequence identity” or “percent (%) identical” with respect to a polypeptide sequence, i.e. a polypeptide of the invention defined herein, is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific polypeptide of the disclosure, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum1006253961percent sequence identity, and not considering any conservative substitutions as part of the sequence identity.

[0109] Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms (non-limiting examples described below) needed to achieve maximal alignment over the full-length of the sequences being compared. When amino acid sequences are aligned, the percent amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain percent amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as: percent amino acid sequence identity = X / Y100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not equal the percent amino acid sequence identity of B to A.

[0110] In calculating percent identity, typically exact matches are counted. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. A nonlimiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTN and BLASTX programs of Altschul et al. (1990) J. Mol. Biol. 215:403. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., BLASTX and BLASTN) can be used. Alignment may also be performed manually by inspection. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the ClustalW algorithm (Higgins et al. (1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences and aligns the entirety of the amino acid or DNA sequence, and thus can provide data about the sequence conservation of the entire amino acid sequence. The ClustalW algorithm is used in several commercially1006253961available DNA / amino acid analysis software packages, such as the ALIGNX module of the Vector NTI Program Suite (Invitrogen Corporation, Carlsbad, CA). After alignment of amino acid sequences with ClustalW, the percent amino acid identity can be assessed. A non-limiting example of a software program useful for analysis of ClustalW alignments is GENEDOC™ or JalView (http: / / www.jalview.org / ). GENEDOC™ allows assessment of amino acid (or DNA) similarity and identity between multiple proteins. Another non- limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4:11 -17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG Wisconsin Genetics Software Package, Version 10 (available from Accelrys, Inc., 9685 Scranton Rd., San Diego, CA, USA). When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0111] The polypeptide desirably comprises an amino end and a carboxyl end. The polypeptide can comprise D-amino acids, L-amino acids or a mixture of D- and L-amino acids. The D-form of the amino acids, however, is particularly preferred since a polypeptide comprised of D-amino acids is expected to have a greater retention of its biological activity in vivo.

[0112] The polypeptide can be prepared by any of a number of conventional techniques. The polypeptide can be isolated or purified from a naturally occurring source or from a recombinant source. Recombinant production is preferred. For instance, in the case of recombinant polypeptides, a DNA fragment encoding a desired peptide can be subcloned into an appropriate vector using well-known molecular genetic techniques (see, e.g., Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1982); Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd ed. (Cold Spring Harbor Laboratory, 1989). The fragment can be transcribed and the polypeptide subsequently translated in vitro. Commercially available kits also can be employed (e.g., such as manufactured by Clontech, Palo Alto, Calif.; Amersham Pharmacia Biotech Inc., Piscataway, N.J.; InVitrogen, Carlsbad, Calif., and the like). The polymerase chain reaction optionally can be employed in the manipulation of nucleic acids.

[0113] The term "conservative substitution" as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-1006253961naturally occurring amino acid or a peptidomimetic having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non- naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).

[0114] Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art. The following six groups are examples of amino acids that may be considered to be conservative substitutions for one another:1 ) Alanine (A), Serine (S), Threonine (T);2) Aspartic acid (D), Glutamic acid (E);3) Asparagine (N), Glutamine (Q);4) Arginine (R), Lysine (K);5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0115] As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be determined bearing in mind the fact that replacement of charged amino acids by sterical ly similar non-charged amino acids are considered as conservative substitutions. For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled person and non-natural or unnatural amino acids are described further below. When affecting conservative substitutions, the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

[0116] In any embodiment, a variant of a polypeptide of the disclosure, e.g. a variant of a polypeptide comprising, consisting essentially of or consisting of an amino acid1006253961sequence of any one of SEQ ID NO: 1 to 36, includes conservative substitutions. Preferably, the conservative substitutions do not significantly reduce the alanine percentage of the variant compared to the “parental” polypeptide.

[0117] Table 3: Alanine percentage

[0118] The phrase "non-conservative substitution" or a “non-conservative residue” as used herein refers to replacement of the amino acid as present in the parent sequence1006253961by another naturally or non-naturally occurring amino acid, having different electrochemical and / or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and / or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid. Non-conservative substitution includes any mutation that is not considered conservative.

[0119] A non-conservative amino acid substitution can result from changes in: (a) the structure of the amino acid backbone in the area of the substitution; (b) the charge or hydrophobicity of the amino acid; or (c) the bulk of an amino acid side chain. Substitutions generally expected to produce the greatest changes in protein properties are those in which: (a) a hydrophilic residue is substituted for (or by) a hydrophobic residue; (b) a proline is substituted for (or by) any other residue; (c) a residue having a bulky side chain, e.g., phenylalanine, is substituted for (or by) one not having a side chain, e.g., glycine; or (d) a residue having an electropositive side chain, e.g., lysyl, arginyl, or histadyl, is substituted for (or by) an electronegative residue, e.g., glutamyl or aspartyl.

[0120] Alterations of the native amino acid sequence to produce mutant polypeptides, such as by insertion, deletion and / or substitution, can be done by a variety of means known to those skilled in the art. For instance, site-specific mutations can be introduced by ligating into an expression vector a synthesized oligonucleotide comprising the modified site. Alternately, oligonucleotide-directed site-specific mutagenesis procedures can be used, such as disclosed in Walder et al., Gene 42: 133 (1986); Bauer et al., Gene 37: 73 (1985); Craik, Biotechniques, 12-19 (January 1995); and U.S. Pat. Nos. 4,518,584 and 4,737,462. A preferred means for introducing mutations is the QuikChange Site-Directed Mutagenesis Kit (Stratagene, LaJolla, Calif.).

[0121] In designing amino acid sequence mutants or variants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1 ) substituting first with conservative amino acid choices and then with more radical1006253961selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

[0122] Amino acid sequence deletions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.

[0123] Substitution mutants have at least one amino acid residue in the polypeptide molecule removed and a different residue inserted in its place. Sites of interest for substitutional mutagenesis include sites that do not contain an alanine.

[0124] The terms "N-terminal" and "C-terminal" are used herein to designate the relative position of any amino acid sequence or polypeptide domain or structure to which they are applied. The relative positioning will be apparent from the context. That is, an "N-terminal" feature will be located at least closer to the N-terminus of the polypeptide molecule than another feature discussed in the same context (the other feature possible referred to as "C-terminal" to the first feature). Similarly, the terms "5'-" and "3'-" can be used herein to designate relative positions of features of polynucleotides.

[0125] A recombinant polypeptide made in accordance with the methods of the present invention may also be modified by, conjugated or fused to another moiety to facilitate purification of the polypeptides, or for use in enzymatic assays using methods known in the art. For example, a polypeptide of the invention may be modified by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage, etc.

[0126] Modifications contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and / or their derivatives during polypeptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on the polypeptides of the disclosure.

[0127] Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and / or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 4.1006253961

[0128] Table 4Non-conventional Code Non-conventional Code amino acid amino acid a-aminobutyric acid Abu L-N-methylalanine Nmala a-amino-a-m ethyl butyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine NmileD-alanine Dal L-N-methylleucine NmleuD-arginine Darg L-N-methyllysine NmlysD-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Deys L-N-methylnorleucine NmnleD-glutamine Dgln L-N-methylnorvaline NmnvaD-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine NmpheD-isoleucine Dile L-N-methylproline NmproD-leucine Dleu L-N-methylserine NmserD-lysine Dlys L-N-methylthreonine NmthrD-methionine Dmet L-N-methyltryptophan NmtrpD-ornithine Dorn L-N-methyltyrosine NmtyrD-phenylalanine Dphe L-N-methylvaline NmvalD-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine NmtbugD-threonine Dthr L-norleucine NleD-tryptophan Dtrp L-norvaline NvaD-tyrosine Dtyr a-methyl-aminoisobutyrate MaibD-valine Dval a-methyl-y-aminobutyrate MgabuD-a-methylalanine Dmala a-methylcyclohexylalanine Mchexa1006253961D-a-methylarginine Dmarg a-methylcylcopentylalanine McpenD-a-methylasparagine Dmasn a-methyl-a-napthylalanine ManapD-a-methylaspartate Dmasp a-methylpenicillamine MpenD-a-methylcysteine Dmcys N-(4-aminobutyl)glycine NgluD-a-methylglutamine Dmgln N-(2-aminoethyl)glycine NaegD-a-methylhistidine Dmhis N-(3-aminopropyl)glycine NornD-a-methylisoleucine Dmile N-amino-a-methylbutyrateNmaabuD-a-methylleucine Dmleu a-napthylalanine AnapD-a-methyllysine Dmlys N-benzylglycine NpheD-a-methylmethionine Dmmet N-(2-carbamylethyl)glycine NglnD-a-methylornithine Dmorn N-(carbamylmethyl)glycine NasnD-a-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine NgluD-a-methylproline Dm pro N-(carboxymethyl)glycine NaspD-a-methylserine Dmser N-cyclobutylglycine NcbutD-a-methylthreonine Dmthr N-cycloheptylglycine NchepD-a-methyltryptophan Dmtrp N-cyclohexylglycine NchexD-a-methyltyrosine Dmty N-cyclodecylglycine NcdecD-a-methylvaline Dmval N-cylcododecylglycine NcdodD-N-methylalanine Dnmala N-cyclooctylglycine NcoctD-N-methylarginine Dnmarg N-cyclopropylglycine NcproD-N-methylasparagine Dnmasn N-cycloundecylglycine NcundD-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-y-aminobutyrateNmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionineDnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine1006253961NmcpenN-methylglycine Nala D-N-methylphenylalanineDnmpheN-methylaminoisobutyrate Nmaib D-N-methylprolineDnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanineNmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen y-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine NhtyrL-f-butylglycine Tbug N-(thiomethyl)glycine NcysL-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-a-methylalanine MalaL-a-methylarginine Marg L-a-methylasparagine MasnL-a-methylaspartate Masp L-a-methyl-f-butylglycine MtbugL-a-methylcysteine Mcys L-methylethylglycine MetgL-a-methylglutamine Mgln L-a-methylglutamate MgluL-a-methylhistidine Mhis L-a-methylhomophenylalanine MhpheL-a-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-a-methylleucine Mleu L-a-methyllysine MlysL-a-methylmethionine Mmet L-a-methylnorleucine MnleL-a-methylnorvaline Mnva L-a-methylornithine MornL-a-methylphenylalanine Mphe L-a-methylproline MproL-a-methylserine Msec L-a-methylthreonine MthrL-a-methyltryptophan Mtrp L-a-methyltyrosine MtyrL-a-methylvaline Mval L-N-methylhomophenylalanineNmhpheN-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine1 -carboxy-1 -(2,2-diphenyl-Nmbc ethylamino)cyclopropane1006253961Mechanical testing

[0129] The term “strength” refers to the maximum stress a material can withstand while being stretched or pulled before breaking (measured from tensile test, where material stretched until failure):Maximum ForceStren = - ; -C ross — sections area

[0130] The term “elasticity” describes the ability of a material to return to their original shape after being deformed (stretched or compressed). This property is often quantified in terms of the material's Young's modulus, which measures the stiffness of a solid material. I is determined from the slope of the initial, linear part of a stressstrain curve during tensile testing:Stress CT

[0131] The term “toughness” is a measure of a material's ability to absorb energy and plastically deform without fracturing. It is calculated as the area under the stressstrain curve up to the point of failure:

[0132] The term “Young’s modulus” measures the tensile or compressive stiffness when the force is applied lengthwise. It is the elastic modulus for tension or axial compression. Young's modulus (E) is defined as the ratio of the stress (force per unit area; o) applied to the object and the resulting axial strain (a dimensionless quantity that quantifies relative deformation; s) in the linear elastic region of the material. As such, Young's modulus is similar to and proportional to the spring constant in Hooke's law, but with dimensions of pressure instead of force per distance.1006253961

[0133] The term “ultimate tensile strength” refers to the maximum stress value recorded in a stress-strain curve during a tensile test. It typically represents the limit of the material's ability to resist tension and is typically expressed in Megapascals (MPa) or Gigapascals (GPa).

[0134] The term “failure strain” refers to the ratio of the change in length of a specimen at the point of fracture to its original length. It typically indicates the ductility of the material and is expressed as a percentage (%) or a dimensionless ratio (mm / mm).

[0135] The term “work of fracture” is defined as the total energy absorbed by the material per unit volume or area until rupture occurs. Mathematically, it typically corresponds to the area under the stress-strain curve and is typically expressed in Joules per square meter (J / m2) or Megajoules per cubic meter (MJ / m3).Physical characteristics

[0136] As used herein, the term “[3-sheet” preferably refers to a protein secondary structure consisting of beta-strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet. These structures may be parallel (strands running in the same direction) or anti-parallel (strands running in opposite directions) and are typically associated with mechanical strength and rigidity in proteinaceous materials. Amyloid-like [3-sheet fibrils are typically observed in assembly of lepidopteran and arachnid silk proteins.

[0137] As used herein, the term “amyloidogenic” describes a polypeptide sequence or region capable of self-assembling into amyloid fibrils. These fibrils are typically characterised by a cross-diffraction pattern, where [3-strands run perpendicular to the fibril axis and hydrogen bonds run parallel to the axis.

[0138] The presence or absence of [3-sheets, and / or whether a polypeptide is amyloidogenic, may be determined by any assays known in the art or described herein (e.g., in the Examples), e.g., a Thioflavin T (ThT) fluorescence assay.

[0139] As used herein, the term “coiled-coil” refers to a structural motif in proteins in which typically two to seven a-helices are coiled together like the strands of a rope. The structure may be stabilised by hydrophobic interactions between the helices, typically following a “knobs-into-holes” packing arrangement, and is often associated with1006253961structural elasticity or oligomerization. A coiled coil homotetramer therefore refers to a protein complex or assembly comprised of four identical polypeptide chains (monomers) that associate to form a coiled bundle. The formation of the species or the homotetramer may be determined by any assays known in the art or described herein (e.g., in the Examples), e.g., Size Exclusion Chromatography (SEC) or Dynamic Light Scattering (DLS).

[0140] As used herein, the term “reflection” refers to a discrete maximum of scattered X-ray intensity resulting from constructive interference of X-rays scattered by periodic electron density fluctuations within the material. In Wide-Angle X-ray Scattering (WAXS), reflections typically correspond to specific crystallographic planes (hkl) governed by Bragg's Law. In Small-Angle X-ray Scattering (SAXS), reflections typically indicate larger-scale periodic order, such as lamellar stacking or fibril packing.

[0141] As used herein, the term “d-spacing” in relation to reflections refers to the perpendicular distance between adjacent parallel planes of atoms or scattering centres in a crystal lattice. It is typically calculated from the angular position of a reflection using Bragg's Law: nA = 2d sin(0), wherein A is the X-ray wavelength, 6 is the scattering angle, and n is an integer representing the order of reflection.

[0142] As used herein, the term “a” in relation to lattice parameters refers to the magnitude of the unit cell vector a (or the linear dimension of the unit cell along the a- axis), and the term “b” in relation to lattice parameters refers to the magnitude of the unit cell vector b (or the linear dimension of the crystallographic unit cell along the b-axis).

[0143] As used herein, the term “orientation index” or “Ol” refers to a dimensionless value representing the degree of molecular or crystal alignment relative to a reference axis, calculated according to the equation OI =180~FWHMThe orientation index may be determined by azimuthal analysis, which involves integrating the X-ray scattering intensity of a specific reflection (such as the (020) reflection) as a function of the azimuthal angle, fitting the resulting distribution (e.g., using a Gaussian or Lorentzian function) to determine the Full Width at Half Maximum (FWHM) in degrees.

[0144] As used herein, the term “correlation peak” refers to a maximum observed in the one-dimensional electron density correlation function derived from X-ray scattering data, representing the most probably distance between scattering heterogeneities.1006253961Accordingly, the term “q-value” refers to the magnitude of the scattering vector, defined by the equation q =471 S1.n(e)(where 20 is the scattering angle and is the wavelength of the incident radiation), and the term “long period” refers to the average repeating distance of the supramolecular nanostructure, calculated from the position of the primary scattering peak (qmax) of the correlation peak using the relationship L = y.

[0145] Any physical characteristics described herein may be assessed by any assays known in the art or described herein (e.g., in the Examples), e.g., synchrotron small- and wide-angle X-ray scattering (SAXS / WAXS).Nucleic acids

[0146] As used herein an "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the polypeptide encoding nucleic acid. An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature. Isolated nucleic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells. However, an isolated nucleic acid molecule includes nucleic acid molecules contained in cells that ordinarily express the nucleic acid where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.

[0147] The terms “nucleic acid molecule” and “polynucleotide” may be used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid sequence which “encodes” a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.1006253961

[0148] Polynucleotides of the disclosure can be synthesised according to methods well known in the art, as described by way of example in Sambrook et al (1989, Molecular Cloning — a laboratory manual; Cold Spring Harbor Press).

[0149] As used herein, “codon optimised” refers to optimisation of the DNA sequence to resemble the codon usage of genes in host microorganism. In preferred embodiments, the codon usage in the sequence is optimised to resemble that of highly expressed E. coli genes.

[0150] The polynucleotide molecules of the present disclosure may be provided in the form of an expression cassette which includes control sequences operably linked to the inserted sequence, thus allowing for expression of the polypeptide. These expression cassettes, in turn, are typically provided within vectors (e.g., plasmids or recombinant vectors). A suitable vector may be any vector which is capable of carrying a sufficient amount of genetic information, and allowing expression of a polypeptide of the invention.

[0151] The present disclosure thus includes expression vectors that comprise such polynucleotide sequences. Expression vectors are routinely constructed in the art of molecular biology and may for example involve the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements which may be necessary, and which are positioned in the correct orientation, in order to allow for expression of a desired polypeptide. Other suitable vectors would be apparent to persons skilled in the art. By way of further example in this regard Sambrook et al is referred to.

[0152] Thus, a polypeptide of the disclosure may be provided by delivering such a vector to a cell and allowing transcription from the vector to occur. The skilled person will be familiar with standard techniques for delivery such expression vectors to a cell, including transformation techniques and the like.

[0153] The vector may be a plasmid. In certain embodiments, the plasmid is a high copy number plasmid or a low copy number plasmid. Vectors are well known in the art and may include cloning vectors, expression vectors, etc. A cloning vector is a recombinant nucleic acid construct which is able to replicate autonomously or integrated in the genome in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant1006253961vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is a recombinant nucleic acid construct into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode polypeptides or enzymes whose activities are detectable by standard assays known in the art (e.g., [3-galactosidase, luciferase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., fluorescent proteins such as green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.

[0154] As used herein, a coding sequence and regulatory sequences are said to be "operably" joined or linked when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined or linked if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1 ) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined or linked to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.

[0155] The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of1006253961transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. In particular, such 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.

[0156] Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.

[0157] A “promoter” is a nucleotide sequence which initiates and regulates transcription of a polypeptide-encoding polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. It is intended that the term “promoter” or “control element” includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions.

[0158] The nucleic acids of the present invention are preferably operably linked to promoters such that the subject enzymes are expressed in the cell when cultured under suitable conditions for enabling consumption of hydrogen, as described herein. The promoters may be specific for individual bacterial cell species. The promoter may be a heterologous promoter which increases the expression of the gene above the typical expression level observed in the cell. The promoter may be an inducible promoter.

[0159] A polynucleotide, expression cassette or vector according to the present invention may additionally comprise a signal peptide sequence. The signal peptide sequence is generally inserted in operable linkage with the promoter such that the signal peptide is expressed and facilitates secretion of a polypeptide encoded by coding sequence also in operable linkage with the promoter. It may further be understood that in any embodiment, any of the exemplary expression cassettes, vectors or sequences described herein may be further modified so as to not include a signal peptide sequence.1006253961

[0160] Any appropriate expression vector (e.g., as described in Pouwels et al., Cloning Vectors: A Laboratory Manual (Elsevier, N.Y.: 1985)) and corresponding suitable host can be employed for production of recombinant polypeptides. Expression hosts include, but are not limited to, bacterial species within the genera Escherichia, Bacillus, Pseudomonas, Salmonella, host cell systems and the like. The skilled person is aware that the choice of expression host has ramifications for the type of polypeptide produced.Host cells

[0161] Another embodiment of the present disclosure includes a host cell, preferably a recombinant host cell transformed with one or more nucleic acids or vectors of the present disclosure, or progeny cells thereof Transformation of a nucleic acid into a cell can be accomplished by any method by which a nucleic acid can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed nucleic acids of the present disclpsure can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e. , recombinant) cell in such a manner that their ability to be expressed is retained.

[0162] Suitable host cells to transform include any cell that can be transformed with a nucleic acid or vector of the present disclosure. Host cells of the present disclosure either can be endogenously (i.e., naturally) capable of producing polypeptides of the present disclosure or can be capable of producing such polypeptides after being transformed with at least one nucleic acid or vector of the present disclosure. Host cells of the present invention can be any cell capable of producing at least one polypeptide of the present disclosure, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells. Examples of host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells, and Vero cells. Further examples of host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia nr', and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine1006253961or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH / 3T3 cells, LMTK cells and / or HeLa cells. Particularly preferred host cells are plant cells such as those available from Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH (German Collection of Microorganisms and Cell Cultures).

[0163] Methods of transforming microorganisms are well known in the art, and can include such non-limiting examples as electroporation, calcium chloride-, or lithium acetate-based methods.

[0164] The skilled person will be familiar with methods for confirming successful transformation of relevant constructs.

[0165] Successful transformation can also be determined by the inclusion of selection marker genes in the plasmid of vector to be transformed into the cell. As used herein, the term "selection marker genes" refer to genetic material that encodes a protein necessary for the survival and / or growth of a host cell grown in a selective culture medium. Typical selection marker genes for use in microorganisms, including in E. coli are well known to the skilled person.

[0166] Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules of the present invention include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules of the present invention to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts.1006253961

[0167] In particularly preferred embodiments, culturing of the microorganisms or host cells, as described herein, may be performed under aerobic conditions.

[0168] The skilled person will appreciate that culturing of recombinant host cells for production of recombinant proteins will be carried out at a temperature that is optimal for the growth and expression of proteins in the organism. For example, the optimum temperature for growth of E. coli and related bacterial organisms is about 37 °C and the temperature for growth of yeasts for producing recombinant proteins is about 30-32°C.Recovery methods and production of polypeptides and fibres

[0169] A polypeptide may be extracted and purified from recombinant cells, such as plant, bacteria or yeast cells, producing said polypeptide by a variety of methods. In one embodiment, the method involves removal of native cell proteins from homogenized cells / tissues / plants etc. by lowering pH and heating, followed by ammonium sulfate fractionation. Briefly, total soluble proteins are extracted by homogenizing cells / tissues / plants. Native proteins are removed by precipitation at pH 4.7 and then at 60° C. The resulting supernatant is then fractionated with ammonium sulfate at 40% saturation. The resulting protein will be of the order of 95% pure.Additional purification may be achieved with conventional gel or affinity chromatography.

[0170] In another example, cell lysates are treated with high concentrations of acid e.g. HCI or propionic acid to reduce pH to about 1 -2 for 1 hour or more which will solubilise the polypeptides but precipitate other proteins.

[0171] In another example, a polypeptide of the disclosure may be purified by a method described in the Examples.

[0172] Fibrillar aggregates will form from solutions by spontaneous self-assembly of polypeptides of the disclosure when the polypeptide concentration exceeds a critical value. The aggregates may be gathered and mechanically spun into macroscopic fibres according to the method of O'Brien et al. (I. O'Brien et al., “Design, Synthesis and Fabrication of Novel Self-Assembling Fibrillar Proteins”, in Silk Polymers: Materials Science and Biotechnology, pp. 104-117, Kaplan, Adams, Farmer and Viney, eds., c. 1994 by American Chemical Society, Washington, D.C.) or as described herein, including the Examples.1006253961

[0173] Fibres of the disclosure have a low processing requirement. The polypeptides of the disclosure require minimal processing e.g. spinning to form a strong fibre as they spontaneously forms strong coiled coils. This contrasts with B. mori and spider recombinant silk polypeptides which require sophisticated spinning techniques in order to obtain the secondary structure ([3-sheet) and strength of the fibre.

[0174] However, fibres may be spun from solutions having properties characteristic of a liquid crystal phase. The fibre concentration at which phase transition can occur is dependent on the composition of a polypeptide or combination of polypeptides present in the solution. Phase transition, however, can be detected by monitoring the clarity and birefringence of the solution. Onset of a liquid crystal phase can be detected when the solution acquires a translucent appearance and registers birefringence when viewed through crossed polarizing filters.

[0175] In one fibre-forming technique, fibres can first be extruded from the protein solution through an orifice into methanol, until a length sufficient to be picked up by a mechanical means is produced. Then a fibre can be pulled by such mechanical means through a methanol solution, collected, and dried. Methods for drawing fibres are considered well-known in the art.

[0176] Further examples of methods which may be used for producing fibres of the disclosure are described in US 2004 / 0170827 and US 2005 / 0054830.Uses

[0177] Polypeptides of the disclosure are useful for the creation of new biomaterials because of their exceptional toughness and strength.

[0178] The polypeptides, fibres, films, sponge, nanofibres, or hydrogel of the disclosure can be used for a broad and diverse array of medical, military, industrial and commercial applications. The fibres can be used in the manufacture of medical devices such as sutures, skin grafts, cellular growth matrices, replacement ligaments, and surgical mesh, and in a wide range of industrial and commercial products, such as, for example, cable, rope, netting, fishing line, clothing fabric, bullet-proof vest lining, container fabric, backpacks, knapsacks, bag or purse straps, adhesive binding material, non-adhesive binding material, strapping material, tent fabric, tarpaulins, pool1006253961covers, vehicle covers, fencing material, sealant, construction material, weatherproofing material, flexible partition material, sports equipment; and, in fact, in nearly any use of fibre or fabric for which high tensile strength and elasticity are desired characteristics. The polypeptides, fibres, films, sponge, nanofibres, or hydrogel of the disclosure also have applications in compositions for personal care products such as cosmetics, skin care, hair care and hair colouring, and a film forming agent; and in coating of particles, such as pigments. The polypeptides, fibres, films, sponge, nanofibres, or hydrogel of the disclosure further have applications in tissue engineering, soft robotics and regenerative medicine.

[0179] As used herein, a “film forming agent” refers to compositions that leave a pliable, cohesive, and continuous covering over the hair or skin when applied to their surface. This film typically has strong hydrophilic properties and leaves a smooth feel on skin. A film forming agent may be used as ingredients of cosmetics, particular hair care products, and moisturizers or other skin-care products. Existing film forming agents are typically silicon based. In contrast, the inventors anticipates that the polypeptides of the disclosure may be used to prepare a film forming agent that is not silicon based.

[0180] Polypeptides of the disclosure can be spun together and / or bundled or braided with other fibre types. Examples include, but are not limited to, polymeric fibres (e.g., polypropylene, nylon, polyester), fibres and silks of other plant and animal sources (e.g., cotton, wool, Bombyx mori or spider silk), and glass fibres. A preferred embodiment is silk fibre braided with 10% polypropylene fibre. The present invention contemplates that the production of such combinations of fibres can be readily practiced to enhance any desired characteristics, e.g., appearance, softness, weight, durability, water-repellent properties, improved cost-of-manufacture, that may be generally sought in the manufacture and production of fibres for medical, industrial, or commercial applications.

[0181] Compositions of the present invention may include an “acceptable carrier”. Examples of such acceptable carriers include water, saline, Ringer's solution, dextrose solution, Hank's solution, and other aqueous physiologically balanced salt solutions. Nonaqueous vehicles, such as fixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.1006253961Personal Care Products

[0182] Cosmetic and skin care compositions may be anhydrous compositions comprising an effective amount of polypeptide in a cosmetically acceptable medium. The uses of these compositions include, but are not limited to, skin care, skin cleansing, make-up, and anti-wrinkle products. An effective amount of a polypeptide in cosmetic and skin care compositions is herein defined as a proportion of from about 10"4to about 30% by weight, but preferably from about 10"3to 15% by weight, relative to the total weight of the composition. This proportion may vary as a function of the type of cosmetic or skin care composition. Suitable compositions for a cosmetically acceptable medium are described in U.S. Pat. No. 6,280,747. For example, the cosmetically acceptable medium may contain a fatty substance in a proportion generally of from about to about 90% by weight relative to the total weight of the composition, where the fatty phase containing at least one liquid, solid or semi-solid fatty substance. The fatty substance includes, but is not limited to, oils, waxes, gums, and so-called pasty fatty substances. Alternatively, the compositions may be in the form of a stable dispersion such as a water-in-oil or oil-in-water emulsion. Additionally, the compositions may contain one or more conventional cosmetic or dermatological additives or adjuvants, including but not limited to, antioxidants, preserving agents, fillers, surfactants, UVA and / or UVB sunscreens, fragrances, thickeners, wetting agents and anionic, nonionic or amphoteric polymers, and dyes or pigments.

[0183] Emulsified cosmetics and quasi drugs which are producible with the use of emulsified materials comprising at least one polypeptide of the disclosure include, for example, cleansing cosmetics (beauty soap, facial wash, shampoo, rinse, and the like), hair care products (hair dye, hair cosmetics, and the like), basic cosmetics (general cream, emulsion, shaving cream, conditioner, cologne, shaving lotion, cosmetic oil, facial mask, and the like), make-up cosmetics (foundation, eyebrow pencil, eye cream, eye shadow, mascara, and the like), aromatic cosmetics (perfume and the like), tanning and sunscreen cosmetics (tanning and sunscreen cream, tanning and sunscreen lotion, tanning and sunscreen oil, and the like), nail cosmetics (nail cream and the like), eyeliner cosmetics (eyeliner and the like), lip cosmetics (lipstick, lip cream, and the like), oral care products (tooth paste and the like) bath cosmetics (bath products and the like), and the like.1006253961

[0184] The cosmetic composition may also be in the form of products for nail care, such as a nail varnish. Nail varnishes are herein defined as compositions for the treatment and colouring of nails, comprising an effective amount of polypeptide in a cosmetically acceptable medium. An effective amount of a polypeptide for use in a nail varnish composition is herein defined as a proportion of from about 10’4to about 30% by weight relative to the total weight of the varnish. Components of a cosmetically acceptable medium for nail varnishes are described in U.S. Pat. No. 6,280,747. The nail varnish typically contains a solvent and a film forming substance, such as cellulose derivatives, polyvinyl derivatives, acrylic polymers or copolymers, vinyl copolymers and polyester polymers. The composition may also contain an organic or inorganic pigment.

[0185] Hair care compositions are herein defined as compositions for the treatment of hair, including but not limited to shampoos, conditioners, lotions, aerosols, gels, and mousses, comprising an effective amount of polypeptide in a cosmetically acceptable medium. An effective amount of a polypeptide for use in a hair care composition is herein defined as a proportion of from about 10"2to about 90% by weight relative to the total weight of the composition. Components of a cosmetically acceptable medium for hair care compositions are described in US 2004 / 0170590, U.S. Pat. No.6,280,747, U.S. Pat. No. 6,139,851 , and U.S. Pat. No. 6,013,250. For example, these hair care compositions can be aqueous, alcoholic or aqueous-alcoholic solutions, the alcohol preferably being ethanol or isopropanol, in a proportion of from about 1 to about 75% by weight relative to the total weight, for the aqueous-alcoholic solutions. Additionally, the hair care compositions may contain one or more conventional cosmetic or dermatological additives or adjuvants, as given above.

[0186] Hair colouring compositions are herein defined as compositions for the colouring, dyeing, or bleaching of hair, comprising an effective amount of polypeptide a cosmetically acceptable medium. An effective amount of a polypeptide for use in a hair colouring composition is herein defined as a proportion of from about 10"4to about 60% by weight relative to the total weight of the composition. Components of a cosmetically acceptable medium for hair colouring compositions are described in US 2004 / 0170590, U.S. Pat. No. 6,398,821 and U.S. Pat. No. 6,129,770. For example, hair colouring compositions generally contain a mixture of inorganic peroxygen-based dye oxidizing agent and an oxidizable coloring agent. The peroxygen-based dye1006253961oxidizing agent is most commonly hydrogen peroxide. The oxidative hair coloring agents are formed by oxidative coupling of primary intermediates (for example p- phenylenediamines, p-aminophenols, p-diaminopyridines, hydroxyindoles, aminoindoles, aminothymidines, or cyanophenols) with secondary intermediates (for example phenols, resorcinols, m-aminophenols, m-phenylenediamines, naphthols, pyrazolones, hydroxyindoles, catechols or pyrazoles). Additionally, hair colouring compositions may contain oxidizing acids, sequestrants, stabilizers, thickeners, buffers carriers, surfactants, solvents, antioxidants, polymers, non-oxidative dyes and conditioners.

[0187] The polypeptides can also be used to coat pigments and cosmetic particles in order to improve dispersibility of the particles for use in cosmetics and coating compositions. Cosmetic particles are herein defined as particulate materials such as pigments or inert particles that are used in cosmetic compositions. Suitable pigments and cosmetic particles, include, but are not limited to, inorganic color pigments, organic pigments, and inert particles. The inorganic color pigments include, but are not limited to, titanium dioxide, zinc oxide, and oxides of iron, magnesium, cobalt, and aluminium. Organic pigments include, but are not limited to, D&C Red No. 36, D&C Orange No. 17, the calcium lakes of D&C Red Nos. 7, 11 , 31 and 34, the barium lake of D&C Red No. 12, the strontium lake D&C Red No. 13, the aluminium lake of FD&C Yellow No. 5 and carbon black particles. Inert particles include, but are not limited to, calcium carbonate, aluminium silicate, calcium silicate, magnesium silicate, mica, talc, barium sulfate, calcium sulfate, powdered Nylon™, perfluorinated alkanes, and other inert plastics.

[0188] The polypeptides may also be used in dental floss (see, for example, US 2005 / 0161058). The floss may be monofilament yam or multifilament yam, and the fibres may or may not be twisted. The dental floss may be packaged as individual pieces or in a roll with a cutter for cutting pieces to any desired length. The dental floss may be provided in a variety of shapes other than filaments, such as but not limited to, strips and sheets and the like. The floss may be coated with different materials, such as but not limited to, wax, polytetrafluoroethylene monofilament yam for floss.

[0189] The polypeptides may also be used in soap (see, for example, US 2005 / 0130857).1006253961Pigment and Cosmetic Particle Coating

[0190] The effective amount of a polypeptide for use in pigment and cosmetic particle coating is herein defined as a proportion of from about 10"4to about 50%, but preferably from about 0.25 to about 15% by weight relative to the dry weight of particle. The optimum amount of the polypeptide to be used depends on the type of pigment or cosmetic particle being coated. For example, the amount of polypeptide used with inorganic color pigments is preferably between about 0.01 % and 20% by weight. In the case of organic pigments, the preferred amount of polypeptide is between about 1 % to about 15% by weight, while for inert particles, the preferred amount is between about 0.25% to about 3% by weight. Methods for the preparation of coated pigments and particles are described in U.S. Pat. No. 5,643,672. These methods include: adding an aqueous solution of the polypeptide to the particles while tumbling or mixing, forming a slurry of the polypeptide and the particles and drying, spray drying a solution of the polypeptide onto the particles or lyophilizing a slurry of the polypeptide and the particles. These coated pigments and cosmetic particles may be used in cosmetic formulations, paints, inks and the like.Biomedical

[0191] The polypeptides may be used as a coating on a bandage to promote wound healing. For this application, the bandage material is coated with an effective amount of the polypeptide. For the purpose of a wound-healing bandage, an effective amount of polypeptide is herein defined as a proportion of from about 10"4to about 30% by weight relative to the weight of the bandage material. The material to be coated may be any soft, biologically inert, porous cloth or fibre. Examples include, but are not limited to, cotton, silk, rayon, acetate, acrylic, polyethylene, polyester, and combinations thereof. The coating of the cloth or fibre may be accomplished by a number of methods known in the art. For example, the material to be coated may be dipped into an aqueous solution containing the polypeptide. Alternatively, the solution containing the polypeptide may be sprayed onto the surface of the material to be coated using a spray gun. Additionally, the solution containing the polypeptide may be coated onto the surface using a roller coat printing process. The wound bandage may include other additives including, but not limited to, disinfectants such as iodine, potassium iodide, povidon iodine, acrinol, hydrogen peroxide, benzalkonium chloride, and chlorohexidine; cure accelerating agents such as allantoin, dibucaine1006253961hydrochloride, and chlorophenylamine malate; vasoconstrictor agents such as naphazoline hydrochloride; astringent agents such as zinc oxide; and crust generating agents such as boric acid.

[0192] The polypeptides of the present disclosure may also be used in the form of a film as a wound dressing material. The use of polypeptides, in the form of an amorphous film, as a wound dressing material is described in U.S. Pat. No. 6,175,053. The amorphous film comprises a dense and nonporous film of a crystallinity below 10% which contains an effective amount of polypeptide. For a film for wound care, an effective amount of polypeptide is herein defined as between about 1 to 99% by weight. The film may also contain other components including but not limited to other proteins such as sericin, and disinfectants, cure accelerating agents, vasoconstrictor agents, astringent agents, and crust generating agents, as described above. Other proteins such as sericin may comprise 1 to 99% by weight of the composition. The amount of the other ingredients listed is preferably below a total of about 30% by weight, more preferably between about 0.5 to 20% by weight of the composition. The wound dressing film may be prepared by dissolving the above mentioned materials in an aqueous solution, removing insolubles by filtration or centrifugation, and casting the solution on a smooth solid surface such as an acrylic plate, followed by drying.

[0193] The polypeptides of the present disclosure may also be used in sutures (see, for example, US 2005 / 0055051 ). Such sutures can feature a braided jacket made of ultrahigh molecular weight fibres and silk fibres. The polyethylene provides strength. Polyester fibres may be woven with the high molecular weight polyethylene to provide improved tie down properties. The silk may be provided in a contrasting color to provide a trace for improved suture recognition and identification. Silk also is more tissue compliant than other fibres, allowing the ends to be cut close to the knot without concern for deleterious interaction between the ends of the suture and surrounding tissue. Handling properties of the high strength suture also can be enhanced using various materials to coat the suture. The suture advantageously has the strength of Ethibond No. 5 suture, yet has the diameter, feel and tie-ability of No. 2 suture. As a result, the suture is ideal for most orthopedic procedures such as rotator cuff repair, Achilles tendon repair, patellar tendon repair, ACL / PCL reconstruction, hip and shoulder reconstruction procedures, and replacement for suture used in or with suture anchors. The suture can be uncoated, or coated with wax (beeswax, petroleum wax,1006253961polyethylene wax, or others), silicone (Dow Corning silicone fluid 202A or others), silicone rubbers, PBA (polybutylate acid), ethyl cellulose (Filodel) or other coatings, to improve lubricity of the braid, knot security, or abrasion resistance, for example.

[0194] The polypeptides of the present disclosure may also be used in stents (see, for example, US 2004 / 0199241 ). For example, a stent graft is provided that includes an endoluminal stent and a graft, wherein the stent graft includes fibre. The fibre induces a response in a host who receives the stent graft, where the response can lead to enhanced adhesion between the fibre stent graft and the host's tissue that is adjacent to the fibre of the fibre stent graft. The fibre may be attached to the graft by any of various means, e.g., by interweaving the fibre into the graft or by adhering the fibre to the graft (e.g., by means of an adhesive or by means of suture). The polypeptide may be in the form of a thread, a braid, a sheet, powder, etc. As for the location of the fibre on the stent graft, the fibre may be attached only the exterior of the stent, and / or the fibre may be attached to distal regions of the stent graft, in order to assist in securing those distal regions to neighbouring tissue in the host. A wide variety of stent grafts may be utilized within the context of the present invention, depending on the site and nature of treatment desired. Stent grafts may be, for example, bifurcated or tube grafts, cylindrical or tapered, self-expandable or balloon-expandable, unibody or, modular, etc.

[0195] The polypeptides of the present disclosure may also be used in a matrix for producing ligaments and tendons ex vivo (see, for example, US 2005 / 0089552). A silk- fibre-based matrix can be seeded with pluripotent cells, such as bone marrow stromal cells (BMSCs). The bioengineered ligament or tendon is advantageously characterized by a cellular orientation and / or matrix crimp pattern in the direction of applied mechanical forces, and also by the production of ligament and tendon specific markers including collagen type I, collagen type III, and fibronectin proteins along the axis of mechanical load produced by the mechanical forces or stimulation, if such forces are applied. In a preferred embodiment, the ligament or tendon is characterized by the presence of fibre bundles which are arranged into a helical organization. Some examples of ligaments or tendons that can be produced include anterior cruciate ligament, posterior cruciate ligament, rotator cuff tendons, medial collateral ligament of the elbow and knee, flexor tendons of the hand, lateral ligaments of the ankle and tendons and ligaments of the jaw or temporomandibular joint. Other tissues that may1006253961be produced by methods of the present invention include cartilage (both articular and meniscal), bone, muscle, skin and blood vessels.

[0196] The polypeptides of the present disclosure may also be used in hydrogels (see, for example, US 2005 / 0266992). Silk fibroin hydrogels can be characterized by an open pore structure which allows their use as tissue engineering scaffolds, substrate for cell culture, wound and bum dressing, soft tissue substitutes, bone filler, and as well as support for pharmaceutical or biologically active compounds.

[0197] The polypeptides may also be used in dermatological compositions (see, for example, US 2005 / 0019297). Furthermore, the polypeptides of the invention and derivatives thereof may also be used in sustained release compositions (see, for example, US 2004 / 0005363).Textiles

[0198] The polypeptides of the present disclosure may also be applied to the surface of fibres for subsequent use in textiles. This provides a monolayer of the protein film on the fibre, resulting in a smooth finish. U.S. Pat. No. 6,416,558 and U.S. Pat. No. 5,232,611 describe the addition of a finishing coat to fibres. The methods described in these disclosures provide examples of the versatility of finishing the fibre to provide a good feel and a smooth surface. For this application, the fibre is coated with an effective amount of the polypeptide. For the purpose of fibre coating for use in textiles, an effective amount of polypeptide is herein defined as a proportion of from about 1 to about 99% by weight relative to the weight of the fibre material. The fibre materials include, but are not limited to textile fibres of cotton, polyesters such as rayon and Lycra™, nylon, wool, and other natural fibres including native silk. Compositions suitable for applying the polypeptide onto the fibre may include cosolvents such as ethanol, isopropanol, hexafluoranols, isothiocyanouranates, and other polar solvents that can be mixed with water to form solutions or microemulsions. The polypeptide-containing solution may be sprayed onto the fibre or the fibre may be dipped into the solution. While not necessary, flash drying of the coated material is preferred. An alternative protocol is to apply the polypeptide composition onto woven fibres. An ideal embodiment of this application is the use of polypeptides to coat stretchable weaves such as used for stockings.1006253961Composite Materials

[0199] Fibres can be added to polyurethane, other resins or thermoplastic fillers to prepare panel boards and other construction material or as moulded furniture and benchtops that replace wood and particle board. The composites can be also be used in building and automotive construction especially rooftops and door panels. The fibres re-enforce the resin making the material much stronger and allowing lighter weight construction which is of equal or superior strength to other particle boards and composite materials. Fibres may be isolated and added to a synthetic compositeforming resin or be used in combination with plant-derived proteins, starch and oils to produce a biologically-based composite materials. Processes for the production of such materials are described in JP 2004284246, US 2005175825, U.S. Pat. No.4,515,737, JP 47020312 and WO 2005 / 017004.Paper Additives

[0200] The fibre properties of the polypeptides of the disclosure can add strength and quality texture to paper making. Silk papers are made by mottling silk threads in cotton pulp to prepare extra smooth handmade papers is used for gift wrapping, notebook covers, carry bags. Processes for production of paper products which can include polypeptides of the invention are generally described in JP 2000139755.Advanced Materials

[0201] Areas of substantial growth in the clothing textile industry are the technical and intelligent textiles. There is a rising demand for healthy, high value functional, environmentally friendly and personalized textile products. Fibres, such as those of the invention, that do not change properties when wet and in particular maintain their strength and extensibility are useful for functional clothing for sports and leisure wear as well as work wear and protective clothing.

[0202] Developments in the weapons and surveillance technologies are prompting innovations in individual protection equipments and battle-field related systems and structures. Besides conventional requirements such as material durability to prolonged exposure, heavy wear and protection from external environment, textiles of the disclosure including polypeptides of fibres of the disclosure can be processed to resist1006253961ballistic projectiles, fire and chemicals. Processes for the production of such materials are described in WO 2005 / 045122 and US 2005268443.

[0203] It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.Examples

[0204] Example 1 : Design processAncestral sequence reconstruction

[0205] A library of similar and related protein sequences was compiled using the 4 silk fibroins from Apis mellifera as the starting point (SEQ ID NOs: 38-41 ). All sequences in this library originated from hymenopteran insect species, and include the 4-5 silk fibroin protein sequences from each species included. From this a phylogenetic tree was created, which was used to reconstruct probable ancestral protein sequences. Meaning whilst these ancestral sequences are novel, they will have some similarities to the hymenopteran proteins used to create the tree. These protein sequences are the basis for this invention.

[0206] In particular, in order to compile a sequence library the NCBI BLAST-P server was initially utilised. Each of the 4 silk fibroins from Apis mellifera were used as the query with default parameters and all results with a sequence identity >30% were appended to the library. This gave a library of around 50 sequences. Each of these 50 proteins were processed through AlphaFold2 (AF2) to predict their structures and to fetch the multiple sequence alignments (MSA) which is created in the process of structural prediction. The full sequences from these alignments were programmatically fetched from NCBI and UniProt sequence databases, redundant sequences removed along with those outside a determined sequence cut off (200-500). This gave a preliminary library of ~200 sequences. In order to align these sequences the following approaches were sampled: CLUSTAL W (default), MUSCLE (default and profileprofile), MAFFT (default, profile-profile, LINSI, EINSI, GINSI, BLOSUM45, and BLOSUM30), MUSTANG (default), and PROMALS3D (default).1006253961

[0207] These MSAs were assessed visually and phylogenetically to determine an optimal method, by examining the alignment and placement of residues and creating phylogenetic trees respectively. It was determined that MAFTT-GINSI generated the most well-aligned MSA. This MSA was trimmed using BMGE and manual trimming based on IQ-TREE generated test trees to remove long branch length causing sequences. This MSA was used as input for IQ-TREE, executed with the following parameters: model selection with ModelFinder Plus, ultrafast bootstrap approximation with 10,000 replicates, SH-like approximate likelihood ratio test, and ancestral state reconstruction. Over 100 replicate trees gave surprisingly conserved topologies suggesting a strong phylogenetic signal. These trees were combined to a single file and the Approximately Unbiased Test, Kishino-Hasegawa Test, Shimodaira- Hasegawa Test, Bootstrap Proportional Resampling using the Expected Log- Likelihood, and Corrected Expected Likelihood Weight were utilised to select the best candidate. In addition to the reconstructions from the IQ-TREE state file, this tree was used as input for PAML to provide alternate reconstructions in order to sample more sample space. The probability of insertions and deletions in these reconstructed sequences was estimated.

[0208] This chosen tree matched the taxonomic tree as expected and showed clades belonging to each of the 4 fibroin proteins, with outgroups selected based on related species. From this tree 20 nodes of interest were determined along the phylogeny relating ancestral sequences at varying points along the tree were selected. These nodes were reconstructed with both PAML and IQTree, using both reconstruction methods to sample more sequence space. Removal of the signal peptides was undertaken with SignalP 5.0 alongside manual data analysis. Ambiguous sites with low posterior probabilities may be substituted with the next-most-likely residue, enabling the creation of variants with minimal deviation from the ML sequence.

[0209] This approach enabled the successful synthesis of 36 designer silk proteins (SEQ ID NOs: 1-36), which may be encoded by the codon-optimized nucleotides set forth in any one of SEQ ID NOs: 42-121 This high-throughput strategy proved highly successful. Expression screening revealed remarkably high yields, with most of the ancestors expressing readily (see Example 2).1006253961

[0210] Example 2: Screening of sequences and characterization of proteins96 deep well expression culture screen

[0211] DNA sequences encoding an N-terminal His6 tag and TEV protease cleavage site were codon-optimized for expression in Escherichia coli, synthesized, and cloned between the Ndel and Xhol sites in pET-28b(+) vectors. The plasmids were transformed into chemically competent E. coli BL21(DE3) (NEB) using the recommended heat-shock protocol and plated onto LB Miller agar supplemented with 50 mg mL'1kanamycin before being incubated overnight at 37°C. A single colony for each sequence was picked and used to inoculate a 96-deep-well plate containing 1 .5 mL aliquots of simple autoinduction media (5 g L’1yeast extract, 20 g L'1tryptone, 5 g L‘1NaCI, 6 g L’1Na2HPO4-7H2O, 3 g L’1KH2PO4, 0.19 g L’1MgCI2, 6 g L’1glycerol, 0.5 g L'1glucose, 2 g L"1d-lactose) supplemented with 0.5 mg mL-1or 50 pg mL-1kanamycin. These cultures were incubated at 18°C, 24°C, and 30°C for 24 h with shaking at 1 ,050 rpm. For example, an expression strategy involved growing cultures at 37 °C to an OD600of 0.6, followed by reducing the temperature to 18 °C for 24 h of expression with shaking at 1050 rpm.

[0212] Many candidates showed expression in the soluble fraction at 18°C, so this condition was further explored by incubating cultures at 18°C for 48, 60, and 72 hours. Additionally, a further growth strategy was tested where cells were grown to OD 0.6 at 37°C and then transferred to 18°C for 24 h. The approaches led to sufficient yields, with the further growth strategy leading to the best yields for the largest number of candidates.

[0213] Cell pellets were harvested by centrifugation at 4,730 ref for 15 min at 20°C. Cells were lysed using 1X BugBuster extraction reagent, followed by the addition of 0.5 pL of Turbonuclease (Serratia marcescens, Sigma-Aldrich) and centrifugation at 2,250 ref for 20 min at 4°C to remove cell debris. The supernatant was loaded into HisPur™ Ni-NTA Spin Plates, followed by washing with equilibration buffer (20 mM Tris, 500 mM NaCI, 20 mM imidazole, pH 8.0). Target proteins were eluted with 200 pL of elution buffer (20 mM Tris, 500 mM NaCI, 500 mM imidazole, pH 8.0).

[0214] An exemplary SDS-page analysis of lysates from various expression cultures is shown in Figure 1. Furthermore, as shown in Figure 2, the addition of a secretory1006253961signal peptide to the designer silk protein enabled its secretion, whereas the addition of the secretory signal peptide to the natural silk fibroin protein did not lead to secretion (Figure 2).

[0215] An exemplary SDS-PAGE analysis of immobilized metal affinity chromatography (IMAC) Ni-NTA purified His-tagged designer silk proteins from the lysates is shown in Figure 3. Crucially, over 30% showed significant accumulation in the soluble fraction, even under standard autoinduction conditions. This contrasts sharply with extant hymenopteran silk proteins, including the comparatively well- studied fibroins from A. mellifera, which typically express predominantly as insoluble inclusion bodies in E. coli, requiring harsh denaturation and refolding protocols for purification.Scaled up 1L expression

[0216] Transformations were carried out as described above. A single colony for each sequence was picked and used to inoculate a 10 mL LB starter culture supplemented with 0.5 mg mL'1kanamycin and incubated at 37°C for 6-8 hours, shaking at 200 rpm. This was used to inoculate a 1 L shaker flask containing simple autoinduction media (5 g L'1yeast extract, 20 g L’1tryptone, 5 g L'1NaCI, 6 g L’1Na2HPO4'7H2O, 3 g L’1KH2PO4, 0.19 g L’1MgCI2, 6 g L’1glycerol, 0.5 g L’1glucose, 2 g L"1d-lactose) supplemented with 0.5 mg mL-1kanamycin, at a 1 :100 ratio.

[0217] The cultures were incubated at 37°C for 3 h, or until an OD 0.6 was reached, with shaking at 250 rpm, then incubated at 18°C for 18 h or 24 h. Cell pellets were harvested by centrifugation at 5,000 ref for 15 min at 20°C. Cells were resuspended in lysis buffer (20 mM Tris, 500 mM NaCI, pH 7.0; or 20 mM Tris, 500 mM NaCI, 20 mM imidazole, pH 8.0), lysed using sonication (2 x 50% power, 50% pulse, 5 min, rest on ice for 5 min), followed by the addition of 0.5 pL of Turbonuclease and centrifugation at 1000 ref for 75 min at 4°C to remove cell debris. The supernatant was filtered and loaded into a HisTrap™ Ni-NTA column, followed by washing with 10 CV equilibration buffer (20 mM Tris, 500 mM NaCI, 20 mM imidazole, pH 8.0). Target proteins were eluted using a gradient method on an AKTA start, from 0 to 100% elution buffer (20 mM Tris, 500 mM NaCI, 500 mM imidazole, pH 8.0). Fractions containing target protein were assessed by SDS-PAGE analysis, combined, and dialyzed into 100x Milli-Q water for 12 h twice or thrice at 4°C. The samples were snap-frozen using liquid1006253961nitrogen, then lyophilized using a LabConco FreeZone Benchtop Freeze Dry system to form a spongy white solid, yielding up to 200 mg / L, or yielding 2.2 g / L. These samples were stored in airtight containers at room temperature.Circular dichroism

[0218] To elucidate the structural basis for the designer silk protein’s material properties, the inventors first investigated its solution-state behaviour.

[0219] Samples were prepared at varying concentrations ranging from 0.01 to 2 mg mL"1in 20 mM Tris buffer, pH 7. Circular dichroism (CD) spectrum measurements were acquired using an Applied Photophysics CD spectrometer at 20°C, using a Hellma Analytics quartz SUPRASIL 10x4 mm high-precision cell. Measurements were taken in triplicate in millidegrees (m°) at wavelengths 200 to 260 nm, with 1 second time-per-point and a 0.5 nm or 1 nm step size. Replicates were averaged and adjusted from millidegrees to mean residue ellipticity (MRE) using the following equation: m°xMRWMRE (deg ■ cm2■ dmol1Ixc where m° = CD data (millidegrees), MRW = molecular weight (Daltons) divided by the number of backbone amides, I = light path length (mm), I = concentration (mg mL-1). CD spectra were smoothed using the 2nd order smoothing algorithm (4 neighbours).

[0220] Circular dichroism thermal denaturation curves were measured at wavelengths 208 and 222 nm, with 1 second time-per-point, a 1 °C step, and a 1 °C min-1temperature ramp, with a 0.2°C tolerance, from 20°C to 94°C or from 20°C to 95°C. Full spectrum measurements were taken before and after thermal denaturation as described above to monitor refolding. Spectra may be baseline-corrected and converted to mean residue ellipticity (MRE) in deg cm2dmol-1.

[0221] Exemplary circular dichroism analyses of the designer silk proteins as compared to the natural silk fibroin proteins are shown in Figures 4 to 6.

[0222] The protein's structure was dominated by a concentration-dependent, coupled folding-and-assembly mechanism. At dilute concentrations, the silk proteins exhibited CD spectra characteristic of a-helical structures, with distinct minima around 208 nm and 222 nm (Figure 5). As concentration increased, significant changes in the CD1006253961spectra were observed, specifically an increase in the 222 / 208 nm ratio which is indicative of coiled-coil formation and inter-chain association. This concentrationdependent transition mirrors the expected self-assembly pathway of hymenopteran silks.

[0223] The assembly of A. mellifera has been most extensively studied, with heterotetramer assembly reportedly driven by initial association of F2 and F4, and subsequently F1 and F3. It is proposed that a similar mechanism of assembly occurs for the designer silk protein homotetramer formation.

[0224] These results demonstrate that:- designer silk proteins self-associated / self-assembled at significantly lower concentrations as compared to individual natural silk proteins and a combination of four natural silk fibroin proteins (Figure 4), potentially facilitating material formation under milder conditions- designer silk protein showed concentration dependent self-association at much lower concentrations as compared to natural silk proteins- designer silk proteins showed minimal loss of secondary structure following refolding.Fourier transform infrared spectroscopy (FTIR)

[0225] The concentration-structure relationship observed in the above experiments was probed further using Fourier Transform Infrared (FTIR) spectroscopy, to study the secondary structure of solution state designer silk proteins at higher concentrations.

[0226] Infrared spectroscopy was performed on lyophilized silk before and after MeOH treatment using a PerkinElmer Spectrum 2 FT-IR spectrometer (equipped with an attenuated total reflectance accessory) at a resolution of 1 cm-1. For each sample, 32 scans may be co-added over a range of 4000 to 650 cm-1at a resolution of 4 cm-1. Amide I, II, and III regions were assigned between 1 ,700 and 1 ,600 cm-1, 1 ,600 to 1 ,500 cm-1, and 1 ,350 to 1 ,200 cm-1, respectively. Transition from coiled coil to increased [3-sheet content following MeOH treatment was inferred based on absorbance band shifts in each of the amide regions. Amide I peak shifted from 1649.80 cm-1to 1645.09 cm-1, amide II peak shifted from 1546.67 cm-1to 1531.96 crrr10062539611, and amide III peak shifted from 1257.20 cm-1to 1240.68 cm-1(Figure 7A). Absorbance bands before MeOH treatment are assigned as random coil or a-helix, bands following treatment assigned as [3-sheet or [3-tum.

[0227] In all concentrations studied the Amide I band appeared as a broad peak typically centred around 1640 cm-1, characteristic of a folded, helical protein (Figure 7B). To further confirm these assignments a second-derivative analysis was performed (Figure 7C), which revealed a strong peak at 1650 cm-1and a distinct "triplet" signature (1650, 1640, and 1625 cm-1), characteristic of an a-helical coiled- coil.

[0228] In order to further validate interpretation of this complex signal, a Thioflavin T (ThT) fluorescence assay was performed. This showed no significant increase in fluorescence over time (Figure 8), indicating that there was no solution-state formation of amyloid-like [3-sheet fibrils typically observed in assembly of lepidopteran and arachnid silk proteins. Taken in context with the CD data, this further validates the formation of stable coiled coil structures in solution.

[0229] To further understand these observations, the inventors used analytical Size Exclusion Chromatography (SEC). The analysis revealed that the designer silk proteins existed predominantly as a single, monodisperse species with a molecular weight consistent with a stable homotetramer (Figure 9A). The experimental result provides direct validation for the in silico AlphaFold2 model, which predicted a high-confidence tetrameric coiled-coil assembly (Figure 9B). This is a critical finding, as it confirms that the designer protein correctly recapitulates the tetrameric architecture widely observed in extant hymenopteran silks (Figure 9B), validating its use as a single-component, selfassembling material.

[0230] The hypothesised oligomeric state was then explored using Dynamic Light Scattering (DLS). As concentration increased, the particle size distribution shifted from a 7 nm species, consistent with the calculated hydrodynamic diameter of an a-helical monomer, to a 13 nm species, consistent with a coiled coil homotetramer, and then to a higher order helical assembly (Figure 10). This taken in context with the rest of the results, paint a consistent picture of a concentration dependant driven association of the designer silk monomers, and most interestingly highlight the atypical assembly1006253961mechanism of hymenopteran silk proteins where there is no presence of [3-sheet in solution.

[0231] Collectively, the biophysical data reveals a complete, concentrationdependent assembly pathway for the designer silk proteins that clarifies the atypical mechanism of hymenopteran silks. The inventors understand that this entire solutionstate process may be driven by a-helical coiled-coil interactions, fundamentally distinguished by the complete absence of the [3-sheet intermediates characteristic of other silk systems. Dynamic Light Scattering (DLS) provided the definitive physical validation of this mechanism (Figure 10). At low concentrations, the protein exists as a ~7 nm species, consistent with the hydrodynamic diameter of the disordered monomer. As concentration increases, the population shifts to a ~13 nm species, confirming a coupled folding-and-assembly into the homotetrameric coiled-coil predicted by the AlphaFold2 model. This establishes the designer silk protein precursor as a defined, helical, self-assembling system, poised for triggered, solid- state fabrication.Air-dried protein film and lyophilized sponge

[0232] A protein solution was buffer-exchanged into Milli-Q water using a centrifugal concentrator and concentrated to approximately 50 mg / mL. Volumes ranging from 10 pL to 100 pL were pipetted onto the surface of a petri dish and dried at room temperature for 16 hours, until a transparent film was formed. Films were stored in methanol until analysis. Lyophilized pure protein was treated with methanol and dried to a white solid, which was also stored in methanol until analysis. Both materials were water-insoluble and remained resistant following methanol soaking.

[0233] An exemplary image of a clear air-dried film formed from 5% designer silk protein is shown in Figure 11 A. An exemplary image of a lyophilized, methanol- washed designer silk protein forming a sponge is shown in Figure 11 D.Wet Spinning

[0234] A wet-spinning apparatus was designed to extrude designer silk protein fibres in a continuous spinning process. A 10% (w / v) protein solution in Milli-Q water was prepared by dissolving lyophilized protein. This solution was loaded into a syringe fitted with a 29-gauge needle and mounted on a syringe pump. The protein solution1006253961was extruded at a speed of 4 m mirr1(or 150 pL / min) directly into a coagulation bath containing 100% methanol, maintained at room temperature. The fibres remained in the methanol bath for approximately 30 minutes to complete solidification. After spinning, fibres were carefully drawn, wound onto a spool (e.g., at a take-up speed of 4 m min-1), and allowed to air-dry under ambient conditions.

[0235] The inventors have also produced fibres with a diameter range from 20 pm to 36 pm, with an average diameter of approximately 30 pm. These fibres were undrawn, wherein they did not undergo a controlled mechanical drawing process following spinning. A solution of 5% (w / v) lyophilised protein in Milli-Q water was prepared by stirring at 100 rpm at 4°C for 16h. The solution was loaded into a 1 mL syringe fitted with a 29-gauge needle. The solution was extruded at a constant flow rate of 150 pL min-1into a coagulation bath of 70% methanol. The solidified fibre was extracted from the bath with tweezers and manually wound onto a spool, then allowed to air-dry under ambient conditions.Microscopy

[0236] Fiber and film morphology was examined using a Leica M205 C polarized light optical microscope.

[0237] Silk film and sponge were analysed by scanning electron microscopy (SEM) to explore their microstructures. Samples were stored in methanol, dried onto aluminium sheets, and mounted using carbon tape. Samples were sputter-coated with platinum (Emitech K550X) at 20 mA for 2 minutes using an Emitech K550X Sputter Coater and examined using a Zeiss UltraPlus field emission scanning electron microscope (FESEM) with the following settings: Electron High Tension (EHT): 5 kV, Aperture: 30 pM, Signal: InLens, Working Distance: 2-4 mm, and Stage Distance: 40 mm. Exemplary SEM images of a clear air-dried film formed from 5% designer silk protein are shown in Figure 11 B-C. Exemplary SEM images of a lyophilized, methanol- washed designer silk protein formed sponge is shown in Figure 11 E-F.

[0238] Wet-spun fibres were assessed for birefringence using polarized light optical microscopy. Five fibres were mounted on microscope slides and viewed under crosspolarized light using a Leica M205 C microscope equipped with a Flexacam C3 camera. The polarizer was adjusted to visually assess birefringence through the1006253961characteristic bright and dark patterns. An exemplary image of a wet-spun designer silk fibre is shown in Figure 12.Electrospinning

[0239] A 14% (w / v) solution of the designer silk was prepared in 1 ,1 ,1 ,3,3,3- hexafluoroisopropanol (HFIP) with magnetic stirring for 15 h. The solution was electrospun using a NanoNC ESR100D system. The solution was fed at 0.3 mL h-1through a 21 -gauge needle with an applied voltage of 15 kV and a collector distance of 10 cm. Randomly oriented nanofibres were collected on a rotating drum (150 rpm), dried overnight, and treated with methanol.Hydrogelation

[0240] A 2% (w / v) solution of the designer silk was prepared in 4°C Milli-Q water by stirring at 100 rpm at 4°C for 16h. The sample was then heated to 60°C for 15 minutes to induce gelation. Gelation was assessed qualitatively via the inversion test, followed by SEM and cryo-SEM to confirm a porous hydrogel structure.Characterisation of designer silk materials

[0241] The favourable solution properties of the designer silk proteins translated into versatile, single protein subunit material formation capabilities (Figure 13).Concentrated aqueous solutions could be readily cast into optically transparent films, wet-spun into continuous fibres, electrospun into nanofibres, or self-assembled into hydrogels.

[0242] To resolve the multiscale structure of the silk materials, the inventors employed synchrotron small- and wide-angle X-ray scattering (SAXS / WAXS). SAXS and WAXS data were collected at the SAXS / WAXS beamline of the Australian Synchrotron (ANSTO). Fibres were mounted horizontally and films were mounted in transmission mode. Data was acquired with a photon energy of 18.1 keV and an exposure time of 1 s. The sample-to-detector distance was 435 mm, covering a scattering vector (q) range from 0.08 to 4.3 A-1.

[0243] The data reveals a detailed picture of a hierarchical architecture that is directly controlled by the material's processing route. The wide-angle X-ray scattering (WAXS) patterns identified the presence of semi-crystalline [3-sheet domains organized within an1006253961orthorhombic crystal lattice (Figure 14d-e). In the designer silk fibre, prominent reflections were identified with d-spacings of 8.54 A (010), 4.34 A ((210), inter-[3-strand distance), and 4.03 A ((020), inter-[3-sheet distance) (Figure 14a). The film exhibited analogous reflections at slightly larger spacings, and its calculated lattice parameters (a = 11 .34 A, b = 8.42 A) indicated a less compact unit cell compared to the fibre (a = 10.30 A, b = 8.06 A) (Figure 14d). This demonstrates that the mechanical drawing inherent in wet-spinning forces the protein chains into a more tightly packed conformation.

[0244] The processing route's most significant impact is the induction of molecular alignment. The 1 D equatorial scattering profile of the fibre (Figure 14c, red line) shows key reflections shifted to higher q-values compared to the film (Figure 14c, green line). This was quantified through azimuthal analysis of the prominent (020) reflection, which yielded a considerably higher orientation index (Ol) for the fibre (Ol = 0.87) compared to the nearly isotropic film (Ol = 0.77). This confirms that wet-spinning created a high degree of uniaxial alignment of the [3-sheet crystallites along the fibre axis. Furthermore, the calculated crystallite length was larger in the fibre (1 .71 nm) than in the film (1.39 nm), suggesting the spinning process also promoted the formation of more extensive ordered domains.

[0245] Complementary SAXS analysis revealed features at larger length scales. Both samples displayed a broad correlation peak at lower q-values (Fig. 3d), corresponding to a long period (L) of approximately 9.0 A. This feature is often associated with the periodic packing of crystalline and amorphous domains. Remarkably, this observed periodicity (L « 9.0 A) is only slightly larger than the crystallographic d010 spacing (8.54 A). This strongly suggests a highly compact and integrated nanostructure where the amorphous regions separating the [3-sheet crystallites are extremely thin, likely consisting of just a few residues in tight molecular turns, aligning with predicted coiled coil tetramers.

[0246] These structural analyses, combined with AlphaFold2 predictions supporting stable coiled-coil formation by the designer silk protein monomers and potential oligomers, paint a consistent picture of silk assembly. The proteins exist as soluble a-helical monomers, which assemble upon concentration increase to homotetrameric coiled coils, or transition to include intermolecular [3-sheets upon exposure to hydrophobic triggers or processing stresses like shear via association of the disorder1006253961terminal regions. This transition locks the structure into an insoluble, semi-crystalline material containing oriented domains, consistent with the known behaviour of extant multi-component hymenopteran silks but achieved here within a single-component system.Mechanical testing

[0247] Fibres were wet-spun as described above. Fibres were cut to 5 cm in length, and their diameters were measured using a Leica M205 C microscope equipped with a Flexacam C3 camera and Leica software with built-in measurement tools. Fibers were prepared for testing by applying masking tape at each end. Weights of 2.5 g, 6 g, and 10 g were attached to one end, and the fibre was suspended from the other end to determine if it could withstand the force without breaking, thus providing a range of tensile strength. Tensile strength was calculated using the following equations:F = m - a, where F = force (N), m = mass (kg), and a = gravitational acceleration (9.8m / s), a = where a = tensile strength (MPa), F = force (N), A = cross sectional area (m2).

[0248] Exemplary results of the mechanical testing are shown in Table 5. The designer silk proteins showed a higher tensile strength than the natural silk proteins.

[0249] Table 5: Breaking stress of natural and wet-spun Apis mellifera silk fibroin proteins compared to wet-spun designer silk protein. A range is provided for A6 as this is based on preliminary data, measured using incremental tensile load testingTensile Testing

[0250] The inventors recombinantly produced the designer silk proteins and natural silk proteins (A. mellifera, fibroins 1-4), resuspended in Milli-Q water to form a 5% (w / v)1006253961dope, and wetspun both into a 70% methanol coagulation bath to form continuous monofilament fibres. The inventors then performed uniaxial tensile testing to characterise the mechanical properties of the resulting fibres (n=7 for synthetic, n=5 for natural).

[0251] The mechanical properties of wet-spun fibres were characterized using a universal testing machine equipped with a 250mN load cell. Individual fibres were mounted onto laser cut frames with a defined gauge length of 5mm. Testing was performed at a rate of 0.5mm / min until failure. Engineering stress and strain were calculated from the recorded force-displacement data and the fibre’s cross-sectional area, which was calculated from optical microscope images. Three measurements along the mounted fibre were taken from above and side on views, these values were used to calculate the cross-sectional area.

[0252] The representative stress-strain curves immediately revealed a profound difference in mechanical behaviour between the two groups (Figure 15a). The curve for the synthetic fibres exhibited a steeper initial slope, a substantially higher ultimate tensile strength (UTS), and a much greater area under the curve compared to the natural silk, indicating simultaneous improvements in stiffness, strength, and toughness.

[0253] Quantitatively, the synthetic fibres demonstrated a marked enhancement across all key mechanical metrics. The Young’s modulus of the synthetic fibres was 1 .9 ± 0.5 GPa, a 1 .9-fold increase over the natural control (1 .0 ± 0.3 GPa; p < 0.05, two-tailed Student's t-test), confirming a significant increase in material stiffness (Figure 15c). This was accompanied by an even more dramatic improvement in strength. The synthetic fibres achieved a UTS of 115 ± 25 MPa, nearly three-fold greater than the natural fibres (40 ± 15 MPa; p < 0.001) (Figure 15b).

[0254] Crucially, this substantial increase in strength and stiffness did not come at the expense of extensibility; in fact, ductility was radically improved. The failure strain of the synthetic fibres was 0.45 ± 0.20 mm / mm, a 9-fold improvement upon the 0.05 ± 0.02 mm / mm recorded for the natural silk (p < 0.05) (Figure 15d). The simultaneous enhancement of both strength and ductility culminated in a remarkable 8-fold increase in toughness. The work of fracture for the synthetic fibres was calculated to be 20 ± 5 MJ rrr3, far exceeding the 2.5 ± 1.0 MJ rrr3of the control fibres (p < 0.0001) (Figure 15e).1006253961

[0255] Taken together, these results demonstrate that the designer silk proteins, when spun into fibre, overcome the typical trade-off between strength and toughness, yielding a material superior to their natural counterpart in every measured mechanical property.Biocompatibility of designer silk protein scaffolds

[0256] Given the potential utility of these novel silk materials in biomedical contexts, preliminary assessments of their biocompatibility were undertaken. The ability to process designer silk proteins into various formats, including electrospun nanofibrous mats, provides suitable substrates for evaluating cell-material interactions. The inventors anticipated that human dermal fibroblasts (hDFs) could be cultured directly on these electrospun silk mats.

[0257] Human dermal fibroblasts (hDFs; Cascade Biologies) were cultured in human fibroblast expansion medium (Gibco) supplemented with Low Serum Growth Supplement (LSGS). Electrospun membranes were sterilized, punched into 8 mm discs, and seeded with 1 x 104cells / well in a 48-well plate. Cell viability at 1 and 3 days was assessed using a LIVE / DEAD™ imaging kit (ThermoFisher Scientific), where Calcein AM stains live cells (green) and ethidium homodimer-1 stains dead cells (red). Images were acquired with a Leica Stellaris 8 confocal laser scanning microscope.

[0258] For cell adhesion analysis, cell morphology on the fibre mats was examined after fixing the samples in 4% paraformaldehyde, dehydrating through a graded ethanol series, and drying with hexamethyldisilazane. Dried samples were platinum-coated and imaged via FESEM.

[0259] The surface morphology of the electrospun designer silk nanofibres was investigated via FE-SEM, and observed to exhibit randomly oriented, non-woven architecture with smooth nanofibres with average diameter of 1237 ± 487 nm (Figure 16a). The uniformity and continuity of the fibres suggest optimized electrospun parameters. The fibres diameters distribution histogram confirms the predominantly submicron to nanometer. Additionally, produced fibres were continuous surface with uniform and porous structures with interconnecting nanofibres, which are favourable for mimicking natural extracellular matrix, and promoting cellular interactions.

[0260] The LIVE / DEAD staining images at day 1 and day 3 exhibited high cell viability of hDF cells, with abundant green fluorescence (live) cells and very few red1006253961fluorescence (dead) cells (Figure 16b). The merged images of live / dead indicate excellent cell adhesion and spreading on designer silk nanofibrous membrane. The increased cell density and extended cell morphologies at day 3 supports the biocompatibility and suitability for various biomedical applications.

[0261] The SEM images of cell on designer silk nanofibres further confirm the cellular adhesion and morphology. Fibroblasts displayed characteristic flattened, elongated shapes with multiple filopodia anchoring to the nanofibres (Figure 16c). Cells appeared well spread across the nanofibrous membrane, forming strong interactions with the fibrous matrix. The high-resolution images demonstrate close contact between cells and nanofibres, highlighting the topographical suitability for cell attachments and proliferation.

[0262] Overall, the combined morphological and cytocompatibility results confirm that the designer silk electrospun nanofibrous membrane supports cell adhesion, viability, and ECM-like architecture, making it a promising candidate for tissue engineering applications.Discussion

[0263] Protein-based materials such as silk are prized for their exceptional mechanical properties and biocompatibility, driving their exploration for applications from textiles to advanced composites. However, traditional silk harvesting is resource-intensive, and the production of recombinant B. mori silk fibroin faces challenges due to its large size. Spider dragline silk offers superior strength and toughness, but large-scale production is hampered by difficulties in recombinant expression. From these limitations arise a need for alternative protein sources and innovative production strategies.

[0264] Here, the inventors developed a panel of silk fibroins with inherently improved handling and production characteristics, bypassing the complexities of multi-component systems or the intractability of large fibroins. The inventors showed that this approach solved the primary limitations of both natural and synthetic silk proteins: the designer silk not only was produced in high, soluble yields, but also, as a single component, self-assembled into a stable, tetrameric coiled-coil architecture.

[0265] Simple processing, either solvent exchange with methanol (proven by FTIR) or the shear and dehydration of wet-spinning (proven by WAXS), drives an irreversible1006253961structural transition. This "structural lock" involves the formation of [3-sheet crystallites, creating a composite material. The SAXS / WAXS analysis confirms this is not a random aggregation but a highly ordered event, resulting in a semi-crystalline orthorhombic lattice. The spinning process may direct this transition, creating a high degree of uniaxial alignment of the [3-sheet crystallites along the fibre axis, which may contribute to high-performance mechanical properties. The inventors proposed that the extended plastic region observed in stress-strain curves may correspond to substantial molecular rearrangement during the drawing process, and this energy-dissipating mechanism may involve the further alignment of the [3-sheet nanostructures, a process that enhances the initial uniaxial orientation confirmed by the SAXS / WAXS analysis.

[0266] This triggered transition places the designer silk proteins in a unique position among self-assembling biomaterials. While many engineered peptides self-assemble into [3-sheet fibrils, these are almost universally amyloidogenic, which the negative ThT assay confirms the designer silk proteins are not. Furthermore, compared to spider silk, whose complex pH / ion-triggered spinning is difficult to replicate in vitro, the present system's [3-sheet transition is triggered by a simple and highly tunable change in the solvent dielectric. This provides a robust and scalable processing handle for fabricating diverse material formats, from the aligned fibre to films, hydrogels, and nanofibres.

[0267] The inventors demonstrated this assembly could be triggered into diverse, biocompatible material formats that exhibited superior mechanical toughness and elasticity to their natural, multi-protein counterparts, and exhibited biocompatibility. This work thus enables the production of a new class of high-performance, single-component, structural protein based biomaterials.

[0268] Beyond its structural performance, this designer silk material is also biologically functional. The inventors demonstrated that electrospun nanofibres, a material format composed entirely of the triggered, [3-sheet-locked protein, are highly biocompatible. The robust growth of hDfs on these scaffolds confirms that the final material is non-toxic and suitable for human-interfacing applications. This combination of tunable mechanics and biocompatibility opens a clear pathway for its use in tissue engineering, soft robotics, and regenerative medicine.1006253961

Claims

CLAIMS1 . A polypeptide capable of forming a fibre, preferably a birefringent fibre, wherein when the fibre is wet-spun at a diameter of 17pm it exhibits a breaking stress of greater than 132 MPa, greater than 200 MPa, greater than or equal to 250 MPa, greater than or equal to 300 MPa, greater than or equal to 350 MPa, greater than or equal to 400 MPa, or greater than or equal to 450 MPa.

2. A polypeptide capable of forming a fibre, preferably a birefringent fibre, wherein when the fibre is wet-spun at 30 pm it exhibits an ultimate tensile strength that is at least or about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold, 13-fold, or 14-fold greater than the ultimate tensile strength of a fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41 .

3. The polypeptide of claim 1 or 2, wherein the polypeptide is capable of forming a fibre, preferably a birefringent fibre, with a diameter from about 10 pm to about 50 pm, from about 15 pm to about 45 pm, from about 15 pm to about 40 pm, or from about 15 pm to about 35 pm; preferably the fibre has a diameter from about 15 pm to about 31 pm, from about 16 pm to about 32 pm, from about 17 pm to about 33 pm, from about 18 pm to about 34 pm, from about 19 pm to about 35 pm, from about 20 pm to about 36 pm, from about 21 pm to about 37 pm, from about 22 pm to about 38 pm, from about 23 pm to about 39 pm, from about 24 pm to about 40 pm, or from about 25 pm to about 41 pm; optionally the fibre has an average diameter of about 15 pm, about 16 pm, about 17 pm, about 18 pm, about 19 pm, about 20 pm, about 21 pm, about 22 pm, about 23 pm, about 24 pm, about 25 pm, about 26 pm, about 27 pm, about 28 pm, about 29 pm, about 30 pm, about 31 pm, about 32 pm, about 33 pm, about 34 pm, about 35 pm, about 36 pm, about 37 pm, about 38 pm, about 39 pm, about 40 pm, about 41 pm, about 42 pm, or about 43 pm; preferably the fibre has an average diameter of about 17 pm or 30 pm; optionally, the fibre is prepared by a process whereby a 10% w / v solution of one or more polypeptides in water is extruded through a 29-gauge needle at a speed1006253961of 4 m min-1(or 150 pL / min) directly into a coagulation bath containing 100% methanol, maintained at room temperature; or the fibre is prepared by extruding a solution of 5% (w / v) of the polypeptide of the disclosure from a 29-gauge needle at a constant flow rate of 150 pL min-1into a coagulation bath of 70% methanol, extracting the solidified fibre from the coagulation bath, and winding the fibre onto a spool followed by air-drying; optionally, the fibre is un-drawn, wherein the fibre has not undergone a controlled mechanical drawing process after spinning.

4. The polypeptide of any one of claims 1 to 3, wherein the fibre has: a Young’s modulus that is at least or about 1 .4-fold, 1 .5-fold, 1.6-fold, 1 .7- fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1 -fold, 2.2-fold, 2.3-fold, or 2.4-fold greater than the Young’s modulus of a fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41 ; optionally the Young’s modulus is about 0.9 GPa, about 1.1 GPa, about 1.3 GPa, about 1.5 GPa, about 1.7 GPa, about 1.9 GPa, about 2.1 GPa, about 2.3 GPa, about 2.5 GPa, or greater; an ultimate tensile strength of about 55 MPa, about 70 MPa, about 85 MPa, about 90 MPa, about 95 MPa, about 100 MPa, about 105 MPa, about 110 MPa, about 115 MPa, about 130 MPa, about 135 MPa, about 140 MPa, about 145 MPa, about 150 MPa, about 155 MPa, about 160 MPa, or greater; a failure strain that is at least or about 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9- fold, 10-fold, 11 -fold, 12-fold, 13-fold, or 14-fold greater than the failure strain of a fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41 ; optionally the failure strain is about 0.10 mm / mm, about 0.15 mm / mm, about 0.20 mm / mm, about 0.25 mm / mm, about 0.30 mm / mm, about 0.35 mm / mm, about 0.40 mm / mm, about0.45 mm / mm, about 0.50 mm / mm, about 0.55 mm / mm, about 0.60 mm / mm, about0.65 mm / mm, about 0.70 mm / mm, about 0.75 mm / mm, about 0.80 mm / mm, about0.85 mm / mm, or greater; and / or a work of fracture that is at least or about 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11 -fold, 12-fold or 13-fold greater than the work of fracture of a1006253961fibre formed by a fibroin protein from a Hymenopteran, preferably Apis mellifera, more preferably a fibroin protein set forth in any one of SEQ ID NOs: 38 to 41 ; optionally the work of fracture of about 5 MJ nr3, about 7.5 MJ nr3, about 10 MJ rrr3, about 12.5 MJ rrr3, about 15 MJ rrr3, about 17.5 MJ rrr3, about 20 MJ rrr3, about 22.5 MJ nr3, about 25 MJ nr3, about 27.5 MJ rrr3, about 30 MJ rrr3, or greater.

5. The polypeptide of any one of claims 1 to 4, wherein the fibre has a calculated lattice parameter of a equalling to about 10.30 A and b equalling to about 8.06 A, and / or an orientation index of about 0.87 quantified through azimuthal analysis of the prominent (020) reflection, and / or a calculated crystallite length of about 1.71 nm, as determined by synchrotron wide-angle X-ray scattering WAXS; preferably the fibre has a correlation peak at q-values corresponding to a long period (L) of about 9.0 A, and / or an observed periodicity L of about 9.0 A.

6. The polypeptide of any one of claims 1 to 5, wherein the polypeptide is capable of self-association, preferably concentration dependent self-association.

7. The polypeptide of claim 6, wherein the self-association occurs at a concentration lower than the self-association of a fibroin protein from a Hymenopteran, preferably Apis mellifera, preferably the fibroin protein from Apis mellifera is any one of SEQ ID NOs: 38 to 41.

8. The polypeptide of claim 6 or 7, wherein the self-association occurs at a concentration equal to or less than 0.1 mg / ml, equal to or less than 0.075mg / ml, equal to or less than 0.02mg / ml, equal to or less than 0.01 mg / ml.

9. The polypeptide of any one of claims 6 to 8, wherein the self-association occurs at a concentration from 0.1 mg / ml to 0.01 mg / ml, from 0.075 mg / ml to 0.01 mg / ml, from 0.02 mg / ml to 0.01 mg / ml, from 0.1 mg / ml to 0.02 mg / ml, from 0.1 mg / ml to 0.075 mg / ml, from 0.075 mg / ml to 0.01 mg / ml, or from 0.075 mg / ml to 0.02 mg / ml.

10. The polypeptide of any one of claims 1 to 9, wherein the polypeptide is capable of reversible thermal denaturation.11 . The polypeptide of any one of claims 1 to 10, wherein the polypeptide: does not form amyloid-like [3-sheet fibrils in solution;1006253961is not amyloidogenic; and / or forms a single, monodisperse species, preferably a stable homotetramer, more preferably a coiled coil homotetramer in solution.

12. The polypeptide of any one of claims 1 to 11 , wherein the polypeptide comprises, consists essentially of or consist of an amino acid sequence where the percentage of amino acids that are alanine is equal to, or greater than, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56% or 57%.

13. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 1 to 36, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 1 to 36, or (iii) a biologically active fragment of (i) or (ii).

14. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises an amino acid sequence of any one of SEQ ID NO: 1 to 36.

15. The polypeptide of any one of claims 1 to 12, wherein polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 1 to 25, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 1 to 25, or (iii) a biologically active fragment of (i) or (ii).

16. The polypeptide of any one of claims 1 to 12, wherein polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 1 to 25.

17. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 26 to 36, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%,100625396194%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 26 to 36, or (iii) a biologically active fragment of (i) or (ii).

18. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 26 to 36.

19. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5, 3, 26, 27, 29, 30, or 36, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5, 3, 26, 27, 29, 30, or 36, or (iii) a biologically active fragment of (i) or (ii).

20. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5, 3, 26, 27, 29, 30, or 36.21 . The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, 2, 31 , or 28, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, 2, 31 , or 28, or (iii) a biologically active fragment of (i) or (ii).

22. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, 2, 31 , or 28.

23. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 14, 16, 19, 34 or 32, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 14, 16, 19, 34 and 32, or (iii) a biologically active fragment of (i) or (ii).100625396124. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 14, 16, 19, 34 or 32.

25. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, 25, 35, or 33, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, 25, 35, or 33, or (iii) a biologically active fragment of (i) or (ii).

26. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, 25, 35, or 33.

27. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5 or 3, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5 or 3, or (iii) a biologically active fragment of (i) or (ii).

28. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 11 , 17, 8, 12, 5 or 3.

29. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, or 2, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, or 2, or (iii) a biologically active fragment of (i) or (ii).

30. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 21 , 24, 9, 18, 22, 10, 7, 13, or 2.100625396131 . The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 14, 16 or 19, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 14, 16 or 19, or (iii) a biologically active fragment of (i) or (ii).

32. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 14, 16 or 19.

33. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, or 25, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, or 25, or (iii) a biologically active fragment of (i) or (ii).

34. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 4, 20, 6, 23, 15, or 25.

35. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 26, 27, 29, 30, or 36, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 26, 27, 29, 30, or 36, or (iii) a biologically active fragment of (i) or (ii).

36. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 26, 27, 29, 30, or 36.

37. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 31 or 28, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%,100625396194%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 31 or 28, or (iii) a biologically active fragment of (i) or (ii).

38. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 31 or 28.

39. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 34, or 32, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 34, or 32, or (iii) a biologically active fragment of (i) or (ii).

40. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 34, or 32.41 . The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises, consists essentially of or consists of (i) an amino acid sequence of any one of SEQ ID NO: 35 or 33, (ii) an amino acid sequence having at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to an amino acid sequence of any one of SEQ ID NO: 35 or 33, or (iii) a biologically active fragment of (i) or (ii).

42. The polypeptide of any one of claims 1 to 12, wherein the polypeptide comprises (i) an amino acid sequence of any one of SEQ ID NO: 35 or 33.

43. The polypeptide of any one of claims 1 to 42, wherein the polypeptide is a soluble polypeptide.

44. The polypeptide of any one of claims 1 to 43, wherein the polypeptide exhibits a greater level of solubility that a fibroin protein from a Hymenopteran, preferably Apis mellifera.

45. The polypeptide of any one of claims 1 to 44, wherein the polypeptide is capable of being expressed at yields greater than a fibroin protein from a Hymenopteran, preferably Apis mellifera, preferably when expressed in a recombinant bacterial cell.100625396146. The polypeptide of any one of claims 1 to 45, wherein the polypeptide is capable of forming a transparent film, preferably a flexible transparent film.

47. The polypeptide of any one of claims 1 to 46, wherein the polypeptide is capable of forming a sponge or sponge-like structure.

48. The polypeptide of any one of claims 1 to 47, wherein the polypeptide is capable of forming a nanofibre, optionally nanofibrous mats.

49. The polypeptide of any one of claims 1 to 48, wherein the polypeptide is capable of forming a hydrogel.

50. A fibre comprising at least one polypeptide of the any one of claims 1 to 49.51 . The fibre of claim 50, wherein the fibre is a wet-spun fibre.

52. The fibre of claim 50 or 51 , wherein the fibre is a birefringent fibre.

53. The fibre of any one of claims 50 to 52, wherein the fibre has a diameter of greater than 9pm, greater than or equal to 10 pm, greater than or equal to 11 pm, greater than or equal to 12 pm, greater than or equal to 13 pm, greater than or equal to 14 pm, greater than or equal to 15 pm, greater than or equal to 16 pm, or greater than or equal to 17 pm.

54. The fibre of any one of claims 50 to 53, wherein the fibre has a breaking stress of greater than 132 Mpa, greater than 200 Mpa, greater than or equal to 250 Mpa, greater than or equal to 300 MPa, greater than or equal to 350 MPa, greater than or equal to 400 MPa, or greater than or equal to 450 MPa.

55. The fibre of any one of claims 50 to 54, wherein the fibre has a diameter of greater than or equal to 19 pm, greater than or equal to 21 pm, greater than or equal to 23 pm, greater than or equal to 25 pm, greater than or equal to 27 pm, greater than or equal to 29 pm, greater than or equal to 31 pm, greater than or equal to 33 pm, greater than or equal to 35 pm, greater than or equal to 37 pm, greater than or equal to 39 pm, greater than or equal to 41 pm, or greater than or equal to 43 pm; preferably the fibre has a diameter of greater than or equal to 30 pm.

56. The fibre of any one of claims 50 to 55, wherein the fibre has:1006253961a Young’s modulus of about 0.9 GPa, about 1 .1 GPa, about 1 .3 GPa, about1 .5 GPa, about 1 .7 GPa, about 1 .9 GPa, about 2.1 GPa, about 2.3 GPa, about2.5 GPa, or greater; an ultimate tensile strength of about 55 MPa, about 70 MPa, about 85 MPa, about 100 MPa, about 115 MPa, about 130 MPa, about 145 MPa, about 160 MPa, or greater; a failure strain of about 0.10 mm / mm, about 0.15 mm / mm, about0.20 mm / mm, about 0.25 mm / mm, about 0.30 mm / mm, about 0.35 mm / mm, about0.40 mm / mm, about 0.45 mm / mm, about 0.50 mm / mm, about 0.55 mm / mm, about0.60 mm / mm, about 0.65 mm / mm, about 0.70 mm / mm, about 0.75 mm / mm, about0.80 mm / mm, about 0.85 mm / mm, or greater; and / or a work of fracture of about 5 MJ nr3, about 7.5 MJ nr3, about 10 MJ nr3, about 12.5 MJ rrr3, about 15 MJ rrr3, about 17.5 MJ rrr3, about 20 MJ rrr3, about22.5 MJ rrr3, about 25 MJ rrr3, about 27.5 MJ nr3, about 30 MJ nr3, or greater.

57. A film comprising at least one polypeptide of the any one of claims 1 to 49, preferably the film has, as determined by synchrotron wide-angle X-ray scattering WAXS: reflections with d-spacings of about 8.54 A (010), about 4.34 A ((210), inter- [3-strand distance), and about 4.03 A ((020), inter-[3-sheet distance); a calculated lattice parameter of a equalling to about 11 .34 A and b equalling to about 8.42 A; an orientation index of about 0.77 quantified through azimuthal analysis of the prominent (020) reflection; and / or a calculated crystallite length of about 1 .39 nm; preferably, the film has a correlation peak at q-values corresponding to a long period (L) of about 9.0 A, and / or an observed periodicity L of about 9.0 A, as determined by small-angle X-ray scattering (SAXS).

58. A nanofibre comprising at least one polypeptide of the any one of claims 1 to 49.100625396159. A hydrogel comprising at least one polypeptide of the any one of claims 1 to 49.

60. A nucleic acid comprising, consisting essentially of or consisting of a nucleotide sequence encoding a polypeptide of any one of claims 1 to 49.61 . The nucleic acid of claim 60, wherein the nucleic acid comprises, consists essentially of or consists of a nucleotide sequence as set forth in any one of SEQ ID NO: 42-121.

62. A vector comprising a nucleic acid of claim 60 or 61 .

63. A cell comprising a nucleic acid of claim 60 or 61 , or the vector of claim 62.

64. A product comprising at least one polypeptide of any one of claims 1 to 49, a fibre of any one of claims 50 to 56, a film of claim 57, a nanofibre of claim 58, or a hydrogel of claim 59.

65. The product of claim 64, wherein the product is a personal care product such as cosmetics, skin care, hair care and hair colouring, and a film forming agent; a military product; a textile; a plastic; or a biomedical product.1006253961

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