BETA-SOLENOID PROTEINS AND THEIR APPLICATION IN TEXTILE PRODUCTION
20210015962 ยท 2021-01-21
Inventors
- Milo Shaffer (London, GB)
- James T. Macdonald (London, GB)
- Paul S. Freemont (London, GB)
- Hannah Leese (London, GB)
Cpc classification
A61L27/22
HUMAN NECESSITIES
A61L31/047
HUMAN NECESSITIES
Y02P70/62
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
A61L17/00
HUMAN NECESSITIES
D02G3/02
TEXTILES; PAPER
A61L15/32
HUMAN NECESSITIES
International classification
A61L15/32
HUMAN NECESSITIES
A61L17/00
HUMAN NECESSITIES
A61L27/22
HUMAN NECESSITIES
C07K14/00
CHEMISTRY; METALLURGY
Abstract
The present invention provides synthetic fibres with advantageous properties such as high tensile strength and which can be produced by recombinant biotechnological means. The invention also provides the constituent components from which the fibres are assembled, along with fabrics formed from the synthetic fibres. Items comprising the fibres and fabrics of the invention are also included.
Claims
1. A concatemer of beta solenoid domains, wherein the concatemer comprises at least two beta solenoid domains, wherein the at least two beta solenoid domains are linked by a linker, and wherein each of the at least two beta solenoid domains comprise an amino acid sequence that comprises at least 4 units of a consensus motif.
2. The concatemer of claim 1 wherein the at least two beta solenoid domains are rod-shaped, optionally wherein one of the at least two beta solenoid domains has a longitudinal axis and a transverse axis and the ratio of longitudinal length to transverse length is at least 2:1, optionally at least 3:1, optionally at least 4:1, optionally at least 5:1, optionally at least 6:1, optionally at least 7:1, optionally at least 8:1, optionally at least 9:1, optionally 10:1; optionally wherein both of the at least two beta solenoid domains have a longitudinal axis and a transverse axis and the ratio of longitudinal length to transverse length is at least 2:1, optionally at least 3:1, optionally at least 4:1, optionally at least 5:1, optionally at least 6:1, optionally at least 7:1, optionally at least 8:1, optionally at least 9:1, optionally 10:1; optionally wherein all of the beta solenoid domains have a longitudinal axis and a transverse axis and the ratio of longitudinal length to transverse length is at least 2:1, optionally at least 3:1, optionally at least 4:1, optionally at least 5:1, optionally at least 6:1, optionally at least 7:1, optionally at least 8:1, optionally at least 9:1, optionally 10:1.
3. The concatemer of claim 1 wherein one or both of the at least two beta solenoid domains has an aspect ratio of 10:1 or less, optionally wherein all of the beta solenoid domains have an aspect ratio of 10:1 or less.
4. The concatemer of any of claims 1-3 wherein: at least one of the at least two beta solenoid domains comprises between 4 and 3000 units of the consensus motif, optionally between 5 and 2800, optionally between 6 and 2600, optionally between 7 and 2400, optionally between 8 and 2200, optionally between 9 and 2000, optionally between 10 and 1800, optionally between 11 and 1600, optionally between 12 and 1400, optionally between 13 and 1200, optionally between 14 and 1000, optionally between 15 and 800, optionally between 16 and 600, optionally between 17 and 500, optionally between 18 and 400, optionally between 19 and 300, optionally between 21 and 200, optionally between 22 and 150, optionally between 23 and 100, optionally between 24 and 90, optionally between 25 and 85, optionally between 26 and 80, optionally between 27 and 75, optionally between 28 and 70, optionally between 29 and 65, optionally between 30 and 60, optionally between 35 and 55, optionally between 40 and 50, optionally 45 units of the consensus motif; at least two of the beta solenoid domains comprise between 4 and 3000 units of the consensus motif, optionally between 5 and 2800, optionally between 6 and 2600, optionally between 7 and 2400, optionally between 8 and 2200, optionally between 9 and 2000, optionally between 10 and 1800, optionally between 11 and 1600, optionally between 12 and 1400, optionally between 13 and 1200, optionally between 14 and 1000, optionally between 15 and 800, optionally between 16 and 600, optionally between 17 and 500, optionally between 18 and 400, optionally between 19 and 300, optionally between 21 and 200, optionally between 22 and 150, optionally between 23 and 100, optionally between 24 and 90, optionally between 25 and 85, optionally between 26 and 80, optionally between 27 and 75, optionally between 28 and 70, optionally between 29 and 65, optionally between 30 and 60, optionally between 35 and 55, optionally between 40 and 50, optionally 45 units of the consensus motif; and/or all of the beta solenoid domains comprise between 4 and 3000 units of the consensus motif, optionally between 5 and 2800, optionally between 6 and 2600, optionally between 7 and 2400, optionally between 8 and 2200, optionally between 9 and 2000, optionally between 10 and 1800, optionally between 11 and 1600, optionally between 12 and 1400, optionally between 13 and 1200, optionally between 14 and 1000, optionally between 15 and 800, optionally between 16 and 600, optionally between 17 and 500, optionally between 18 and 400, optionally between 19 and 300, optionally between 21 and 200, optionally between 22 and 150, optionally between 23 and 100, optionally between 24 and 90, optionally between 25 and 85, optionally between 26 and 80, optionally between 27 and 75, optionally between 28 and 70, optionally between 29 and 65, optionally between 30 and 60, optionally between 35 and 55, optionally between 40 and 50, optionally 45 units of the consensus motif.
5. The concatemer of any of claims 1-4 wherein: at least one of the at least two beta solenoid domains comprises at least 5 units of the consensus motif, optionally at least 6 units, optionally at least 7 units, optionally at least 8 units, optionally at least 9 units, optionally at least 10 units, optionally at least 11 units, optionally at least 12 units, optionally at least 13 units, optionally at least 14 units, optionally at least 15 units, optionally at least 16 units, optionally at least 17 units, optionally at least 18 units, optionally at least 19 units, optionally at least 20 units, optionally at least 21 units, optionally at least 24 units, optionally at least 25 units, optionally at least 26 units, optionally at least 27 units, optionally at least 28 units, optionally at least 29 units, optionally at least 30 units, optionally at least 40 units, optionally at least 50 units, optionally at least 60 units, optionally at least 70 units, optionally at least 80 units, optionally at least 90 units, optionally at least 100 units, optionally at least 200 units of the consensus motif; at least two of the beta solenoid domains comprise at least 5 units of the consensus motif, optionally at least 6 units, optionally at least 7 units, optionally at least 8 units, optionally at least 9 units, optionally at least 10 units, optionally at least 11 units, optionally at least 12 units, optionally at least 13 units, optionally at least 14 units, optionally at least 15 units, optionally at least 16 units, optionally at least 17 units, optionally at least 18 units, optionally at least 19 units, optionally at least 20 units, optionally at least 21 units, optionally at least 24 units, optionally at least 25 units, optionally at least 26 units, optionally at least 27 units, optionally at least 28 units, optionally at least 29 units, optionally at least 30 units, optionally at least 40 units, optionally at least 50 units, optionally at least 60 units, optionally at least 70 units, optionally at least 80 units, optionally at least 90 units, optionally at least 100 units, optionally at least 200 units of the consensus motif; all of the beta solenoid domains comprise at least 5 units of the consensus motif, optionally at least 6 units, optionally at least 7 units, optionally at least 8 units, optionally at least 9 units, optionally at least 10 units, optionally at least 11 units, optionally at least 12 units, optionally at least 13 units, optionally at least 14 units, optionally at least 15 units, optionally at least 16 units, optionally at least 17 units, optionally at least 18 units, optionally at least 19 units, optionally at least 20 units, optionally at least 21 units, optionally at least 24 units, optionally at least 25 units, optionally at least 26 units, optionally at least 27 units, optionally at least 28 units, optionally at least 29 units, optionally at least 30 units, optionally at least 40 units, optionally at least 50 units, optionally at least 60 units, optionally at least 70 units, optionally at least 80 units, optionally at least 90 units, optionally at least 100 units, optionally at least 200 units of the consensus motif; optionally wherein the units of the consensus motif are arranged consecutively.
6. The concatemer of any of claims 1-5 wherein: at least one of the at least two beta solenoid domains comprises less than 3000 units of the consensus motif, optionally less than 2000 units, optionally less than 1000 units, optionally less than 500 units, optionally less than 400 units, optionally less than 300 units, optionally less than 200 units, optionally less than 100 units, optionally less than 90 units, optionally less than 80 units, optionally less than 70 units, optionally less than 60 units, optionally less than 50 units, optionally less than 40 units, optionally less than 30 units, optionally less than 29 units, optionally less than 28 units, optionally less than 27 units, optionally less than 26 units, optionally less than 25 units, optionally less than 24 units, optionally less than 23 units, optionally less than 22 units, optionally less than 21 units, optionally less than 20 units, optionally less than 19 units, optionally less than 18 units, optionally less than 17 units, optionally less than 16 units, optionally less than 15 units, optionally less than 14 units, optionally less than 13 units, optionally less than 12 units, optionally less than 11 units, optionally less than 10 units, optionally less than 9 units, optionally less than 8 units, optionally less than 7 units, optionally 6 units of the consensus motif; at least two of the at least two beta solenoid domains comprise less than 3000 units of the consensus motif, optionally less than 2000 units, optionally less than 1000 units, optionally less than 500 units, optionally less than 400 units, optionally less than 300 units, optionally less than 200 units, optionally less than 100 units, optionally less than 90 units, optionally less than 80 units, optionally less than 70 units, optionally less than 60 units, optionally less than 50 units, optionally less than 40 units, optionally less than 30 units, optionally less than 29 units, optionally less than 28 units, optionally less than 27 units, optionally less than 26 units, optionally less than 25 units, optionally less than 24 units, optionally less than 23 units, optionally less than 22 units, optionally less than 21 units, optionally less than 20 units, optionally less than 19 units, optionally less than 18 units, optionally less than 17 units, optionally less than 16 units, optionally less than 15 units, optionally less than 14 units, optionally less than 13 units, optionally less than 12 units, optionally less than 11 units, optionally less than 10 units, optionally less than 9 units, optionally less than 8 units, optionally less than 7 units, optionally 6 units of the consensus motif; and/or all of the beta solenoid domains comprise less than 3000 units of the consensus motif, optionally less than 2000 units, optionally less than 1000 units, optionally less than 500 units, optionally less than 400 units, optionally less than 300 units, optionally less than 200 units, optionally less than 100 units, optionally less than 90 units, optionally less than 80 units, optionally less than 70 units, optionally less than 60 units, optionally less than 50 units, optionally less than 40 units, optionally less than 30 units, optionally less than 29 units, optionally less than 28 units, optionally less than 27 units, optionally less than 26 units, optionally less than 25 units, optionally less than 24 units, optionally less than 23 units, optionally less than 22 units, optionally less than 21 units, optionally less than 20 units, optionally less than 19 units, optionally less than 18 units, optionally less than 17 units, optionally less than 16 units, optionally less than 15 units, optionally less than 14 units, optionally less than 13 units, optionally less than 12 units, optionally less than 11 units, optionally less than 10 units, optionally less than 9 units, optionally less than 8 units, optionally less than 7 units, optionally 6 units of the consensus motif; optionally wherein the units of the consensus motif are arranged consecutively.
7. The concatemer of any of claims 1-6 wherein each beta solenoid domain comprises the same number of units of the consensus motif.
8. The concatemer of any of claims 1-7 wherein at least one of the beta solenoid domains comprises at least two identical units of the consensus motif, optionally wherein all of the units of the consensus motif are identical within a given beta solenoid domain.
9. The concatemer of any of claims 1-8 wherein all of the beta solenoid domains comprise at least two identical units of the consensus motif, optionally wherein all of the units of the consensus motif are identical within a given beta solenoid domain, optionally wherein all of the units of the consensus motif are identical within the concatemer.
10. The concatemer of any of claims 1-9 wherein at least one of the beta solenoid domains comprises non-identical units of the consensus motif, optionally wherein all of the units of the consensus motif are different to one another within the beta solenoid domain.
11. The concatemer according to any of claims 1-10 wherein all of the beta solenoid domains comprise non-identical units of the consensus motif, optionally wherein all of the units of the consensus motif are different to one another within a given beta solenoid domain.
12. The concatemer according to any of claims 1-11 wherein at least two of the at least two beta solenoid domains have an identical amino acid sequence, optionally wherein all of the beta solenoid domains have an identical amino acid sequence.
13. The concatemer according to any of claims 1-12 wherein at least two of the at least two beta solenoid domains have a non-identical amino acid sequence, optionally wherein all of the beta solenoid domains have non-identical amino acid sequences.
14. The concatemer according to any of claims 1-13 wherein the at least two beta solenoid domains have a cross-section selected from the group consisting of a tesselatable cross-section, optionally a square, a rectangle, a parallelogram, a hexagon, or a triangle cross-section; or a circle or an oval cross-section.
15. The concatemer according to any of claims 1-14 wherein the beta solenoid domains are capable of forming a lyotropic liquid crystal mesophase when expressed as monomers.
16. The concatemer according to any of claims 1-14 wherein the concatemer is capable of forming a lyotropic liquid crystal mesophase.
17. The concatemer according to any of claims 1-16 wherein at least one of the beta solenoid domains comprises units of a consensus motif selected from the group consisting of: TABLE-US-00043 [SEQIDNO:147] I)AX.sub.1L/FX.sub.2X.sub.3 wherein X.sub.1 is any residue X.sub.2 is any residue X.sub.3 is any residue; optionally wherein X, is A, C, D, E, I, K, L, M, N, R, S, T or V X.sub.2 is A, C, D, E, F, G, H, I, K, N, Q, R, S, T, V, W, or Y X.sub.3 is A, C, D, E, G, H, I, K, N, Q, R, S, Y; [SEQ ID NO: 148] TABLE-US-00044 [SEQIDNO:149] ii)AX.sub.1LX.sub.2X.sub.3 wherein X.sub.1 is any residue X.sub.2 is any residue X.sub.3 is any residue; optionally wherein X.sub.1 is A, C, D, E, I, K, L, M, N, R, S, T or V X.sub.2 is A, C, D, E, F, G, H, I, K, N, Q, R, S, T, V, W, or Y X.sub.3 is A, C, D, E, G, H, I, K, N, Q, R, S, Y; [SEQ ID NO: 150] TABLE-US-00045 [SEQIDNO:151] iii)A(N/D)L*X where * is a polar residue (C, R, H, K, D, E, S, T, N, Q) and X is any residue; TABLE-US-00046 [SEQIDNO:152] iv)(S/G/T/A)X(A/V/G)X(G/A)XX where X is any residue; TABLE-US-00047 [SEQIDNO:153] v)(S/G/T/A)X(A/V/G)X(G/A)XX(S/G/T/A)*(A/V/G)* (G/A)XX where * is a hydrophobic residue and where X is any residue; TABLE-US-00048 [SEQIDNO:154] vi)SXAXGXXS*A*GXX where * is a hydrophobic residue and where X is any residue; TABLE-US-00049 [SEQIDNO:155] vii)NXAXGXXST(I/V)(G/A)GGXX where X is any residue; TABLE-US-00050 [SEQIDNO:156] viii)NXAXGXXSTIGGGXX where X is any residue; TABLE-US-00051 [SEQIDNO:157] ix)GXQX(V/I/L)XXGGXAXXTX(V/I/L)XXG where X is any residue; optionally wherein (i), (ii) and (iii) generate a beta solenoid domain with a square cross-section; (iv), (v), (vi), (vii) and (viii) generate an oval shaped cross-section; and (ix) forms a triangular cross-section.
18. The concatemer according to any of claims 1-17 wherein at least one of the at least two beta solenoid domains comprises or consists of the sequence of SEQ ID NO: 1, 8-28 and 169, optionally wherein at least two of the beta solenoid domains comprise or consist of the sequence of any one of SEQ ID NO: 1, 8-28 and 169, optionally wherein all of the beta solenoid domains comprise or consist of the sequence of any one of SEQ ID NO: 1, 8-28 and 169, optionally wherein the at least one, the at least two or all of the beta solenoid domains have a) at least 75% homology to any one of the sequences of SEQ ID NO: 1, 8-28 and 169, optionally at least 80% homology, optionally at least 85% homology, optionally at least 90% homology, optionally at least 95% homology, optionally at least 96% homology, optionally at least 97% homology, optionally at least 98% homology, optionally at least 99% homology, optionally 100% homology or b) at least 75% sequence identity to any one of the sequences of SEQ ID NO: 1, 8-28 and 169, optionally at least 80% sequence identity, optionally at least 85% sequence identity, optionally at least 90% sequence identity, optionally at least 95% sequence identity, optionally at least 96% sequence identity, optionally at least 97% sequence identity, optionally at least 98% sequence identity, optionally at least 99% sequence identity, optionally 100% sequence identity.
19. The concatemer according to any of claims 1-18 wherein at least one of the beta solenoid domains comprises a capping sequence at a first end or a second end, or both the first end and the second end of the beta solenoid domain, optionally wherein at least two of the beta solenoid domains comprise a capping sequence at a first end or a second end, or both the first end and the second end of each beta solenoid domains, optionally wherein all of the beta solenoid domains comprise a capping sequence at a first end or a second end, or both the first end and the second end of each beta solenoid domain, optionally wherein the capping sequence prevents the beta solenoid domains from joining end-to-end and aggregating, optionally wherein the capping sequence is an alpha helix.
20. The concatemer according to claim 19 wherein at least one of or both of or all of the capping sequence comprises: a) a sequence that conforms to the consensus sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83, optionally wherein the first cap conforms to SEQ ID NO: 82 and the second cap conforms to SEQ ID NO: 83; and/or b) any one or more of SEQ ID NO: 2, 3, 84-104 and 105-125 optionally wherein at least one of or both of or all of the capping sequence comprises a sequence with a) at least 80% homology, optionally at least 85% homology, optionally at least 90% homology, optionally at least 95% homology, optionally at least 96% homology, optionally at least 97% homology, optionally at least 98% homology, optionally at least 99% homology, optionally 100% homology to SEQ ID NO: 2, 3, 84-104 or 105-125; orb) at least 80% sequence identity, optionally at least 85% sequence identity, optionally at least 90% sequence identity, optionally at least 95% sequence identity, optionally at least 96% sequence identity, optionally at least 97% sequence identity, optionally at least 98% sequence identity, optionally at least 99% sequence identity, optionally 100% sequence identity to SEQ ID NO: 2, 3, 84-104 or 105-125.
21. The concatemer according to any of claims 1-20 wherein the linker is a flexible linker optionally wherein the linker or the flexible linker is formed from amino acid residues.
22. The concatemer according to any of claims 1-21 wherein the beta solenoid domains and linker are transcribed and translated as a single unit.
23. The concatemer according to any of claims 1-22 wherein the linker sequence is selected from the group consisting of a poly glycine linker, a poly alanine linker, a glycine rich linker, an alanine rich linker, a glycine and alanine rich linker, a glycine/alanine/serine rich linker, SEQ ID NO: 34 (GGGS), or SEQ ID NO: 168 (GGGSAAAAAAGGGS).
24. The concatemer according to any of claims 1-23 wherein at least one of the at least two beta solenoid domains comprises hidden length, optionally wherein two of the at least two beta solenoid domains comprises hidden length, optionally wherein all of the beta solenoid domains comprises hidden length.
25. The concatemer according to any of claims 1-24 wherein at least one of the at least two beta solenoid domains comprises a first and a second conformation, wherein the second conformation is extended relative to the first conformation, optionally wherein the length of the second conformation is at least 1.5 times longer than the length of the first conformation, optionally at least 2 times longer, optionally at least 3 times longer, optionally at least 4 times longer, optionally at least 5 times longer, optionally at least 10 times longer, optionally at least 20 times longer, optionally at least 30 times longer, optionally at least 40 times longer, optionally at least 50 times longer, optionally at least 100 times longer than the length of the first conformation.
26. The concatemer according to claim 25 wherein when in the first conformation the concatemer comprises sacrificial hydrogen bonds and wherein in moving between the first and second conformation at least one of the sacrificial hydrogen bonds is broken.
27. The concatemer according to any of claims 1-24 wherein when in the first conformation the concatemer comprises a high degree of coiling.
28. The concatemer according to any of claims 1-27 wherein the concatemer moves between the first conformation and the second conformation when a defined force is applied parallel to the longitudinal axis of the concatemer.
29. The concatemer according to any of claims 1-28 wherein the concatemer has a molecular weight of at least 30 kDa, optionally at least 40 kDa, optionally at least 50 kDa, optionally at least 60 kDa, optionally at least 70 kDa, optionally at least 80 kDa, optionally at least 90 kDa, optionally at least 100 kDa, optionally at least 120 kDa, optionally at least 150 kDa, optionally at least 200 kDa, optionally at least 250 kDa, optionally at least 300 kDa, optionally at least 350 kDa, optionally at least 400 kDa, optionally at least 450 kDa, optionally at least 500 kDa, optionally at least 600 kDa, optionally at least 650 kDa, optionally at least 700 kDa, optionally at least 800 kDa, optionally at least 900 kDa, optionally at least 1000 kDa.; optionally between 30 kDa and 1000 kDa.
30. The concatemer according to any of claims 1-29 wherein the concatemer is soluble at high concentrations, optionally soluble at a concentration of at least 20 mg/ml, optionally at least 30 mg/ml, optionally at least 40 mg/ml, optionally at least 50 mg/ml, optionally at least 60 mg/ml, optionally at least 70 mg/ml, optionally at least 80 mg/ml, optionally at least 90 mg/ml, optionally at least 100 mg/ml, optionally at least 120 mg/ml, optionally at least 130 mg/ml, optionally at least 150 mg/ml, optionally at least 170 mg/ml, optionally at least 190 mg/ml, optionally at least 200 mg/ml, optionally at least 250 mg/ml, optionally at least 300 mg/ml, optionally at least 350 mg/ml, optionally at least 400 mg/ml, optionally soluble in a solvent selected from the group consisting of: a) a buffer selected from the group consisting of any one or more of: MES, Bis-tris methane, ADA, ACES, Bis-tris propane, PIPES, MOPSO, Cholamine chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, Acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, Glycinamide, Glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO CAPSO, CAPS, CABS, Bicine, sodium citrate buffer, sodium bicarbonate buffer, phosphate buffer, borate buffer, optionally wherein the buffer comprises one or more salts, optionally sodium chloride and/or potassium chloride, optionally wherein the buffer is at a pH range of 3 to 10, optionally pH 7; b) an organic solvent, optionally HFIP.
31. The concatemer according to any of claims 1-30 wherein at least one of the at least two beta solenoid domains has a high net charge, optionally has a net charge of at least +3 charge units, optionally at least +5 charge units, optionally at least +7 charge units, optionally at least +10 charge units, optionally at least +15 charge units, optionally at least +20 charge units, optionally at least +25 charge units, optionally at least +30 charge units, optionally at least +35 charge units, optionally at least +40 charge units, optionally at least +45 charge units, optionally at least +50 charge units, optionally at least +55 charge units, optionally at least +60 charge units, optionally at least +65 charge units, optionally at least +70 charge units, optionally at least +75 charge units, optionally at least +80 charge units, optionally at least +85 charge units, optionally at least +90 charge units, optionally at least +95 charge units, optionally at least +100 charge units, optionally ata pH range of 3 to 10, optionally pH 7, optionally wherein two of the at least two beta solenoid domains have a high net charge, optionally have a net charge of at least +3 charge units, optionally at least +5 charge units, optionally at least +7 charge units, optionally at least +10 charge units, optionally at least +15 charge units, optionally at least +20 charge units, optionally at least +25 charge units, optionally at least +30 charge units, optionally at least +35 charge units, optionally at least +40 charge units, optionally at least +45 charge units, optionally at least +50 charge units, optionally at least +55 charge units, optionally at least +60 charge units, optionally at least +65 charge units, optionally at least +70 charge units, optionally at least +75 charge units, optionally at least +80 charge units, optionally at least +85 charge units, optionally at least +90 charge units, optionally at least +95 charge units, optionally at least +100 charge units, optionally at a pH range of 3 to 10, optionally pH 7, optionally wherein all of the beta solenoid domains have a high net charge, optionally have a net charge of at least +3 charge units, optionally at least +5 charge units, optionally at least +7 charge units, optionally at least +10 charge units, optionally at least +15 charge units, optionally at least +20 charge units, optionally at least +25 charge units, optionally at least +30 charge units, optionally at least +35 charge units, optionally at least +40 charge units, optionally at least +45 charge units, optionally at least +50 charge units, optionally at least +55 charge units, optionally at least +60 charge units, optionally at least +65 charge units, optionally at least +70 charge units, optionally at least +75 charge units, optionally at least +80 charge units, optionally at least +85 charge units, optionally at least +90 charge units, optionally at least +95 charge units, optionally at least +100 charge units, optionally at a pH range of 3 to 10, optionally pH 7.
32. The concatemer according to any of claims 1-30 wherein at least one of the at least two beta solenoid domains has a high negative net charge, optionally has a net charge of less than 3 charge units, optionally at less than 5 charge units, optionally less than 7 charge units, optionally less than 10 charge units, optionally less than 15 charge units, optionally less than 20 charge units, optionally less than 25 charge units, optionally less than 30 charge units, optionally less than 35 charge units, optionally less than 40 charge units, optionally less than 45 charge units, optionally less than 50 charge units, optionally less than 55 charge units, optionally less than 60 charge units, optionally less than 65 charge units, optionally less than 70 charge units, optionally less than 75 charge units, optionally less than 80 charge units, optionally less than 85 charge units, optionally less than 90 charge units, optionally less than 95 charge units, optionally less than 100 charge units, optionally at a pH range of 3 to 10, optionally pH 7, optionally wherein two of the at least two beta solenoid domains have a high negative net charge, optionally has a net charge of less than 3 charge units, optionally at less than 5 charge units, optionally less than 7 charge units, optionally less than 10 charge units, optionally less than 15 charge units, optionally less than 20 charge units, optionally less than 25 charge units, optionally less than 30 charge units, optionally less than 35 charge units, optionally less than 40 charge units, optionally less than 45 charge units, optionally less than 50 charge units, optionally less than 55 charge units, optionally less than 60 charge units, optionally less than 65 charge units, optionally less than 70 charge units, optionally less than 75 charge units, optionally less than 80 charge units, optionally less than 85 charge units, optionally less than 90 charge units, optionally less than 95 charge units, optionally less than 100 charge units, optionally at a pH range of 3 to 10, optionally pH 7, optionally wherein all of the two beta solenoid domains have a high negative net charge, optionally has a net charge of less than 3 charge units, optionally at less than 5 charge units, optionally less than 7 charge units, optionally less than 10 charge units, optionally less than 15 charge units, optionally less than 20 charge units, optionally less than 25 charge units, optionally less than 30 charge units, optionally less than 35 charge units, optionally less than 40 charge units, optionally less than 45 charge units, optionally less than 50 charge units, optionally less than 55 charge units, optionally less than 60 charge units, optionally less than 65 charge units, optionally less than 70 charge units, optionally less than 75 charge units, optionally less than 80 charge units, optionally less than 85 charge units, optionally less than 90 charge units, optionally less than 95 charge units, optionally less than 100 charge units optionally at a pH range of 3 to 10, optionally pH 7.
33. The concatemer according to any of claims 1-31 wherein the concatemer has a high net charge, optionally has a net charge of at least +3 charge units, optionally at least +5 charge units, optionally at least +7 charge units, optionally at least +10 charge units, optionally at least +15 charge units, optionally at least +20 charge units, optionally at least +25 charge units, optionally at least +30 charge units, optionally at least +35 charge units, optionally at least +40 charge units, optionally at least +45 charge units, optionally at least +50 charge units, optionally at least +55 charge units, optionally at least +60 charge units, optionally at least +65 charge units, optionally at least +70 charge units, optionally at least +75 charge units, optionally at least +80 charge units, optionally at least +85 charge units, optionally at least +90 charge units, optionally at least +95 charge units, optionally at least +100 charge units, optionally at least +200 charge units, optionally at least +300 charge units, optionally at least +400 charge units, optionally at least +500 charge units, optionally at least +600 charge units, optionally at least +700 charge units, optionally at least +800 charge units, optionally at least +900 charge units, optionally at least +1000 charge units, optionally at least +2000 charge units, optionally at least +3000 charge units, optionally at least +4000 charge units, optionally at least +5000 charge units, optionally at least +6000 charge units, optionally at least +7000 charge units, optionally at least +8000 charge units, optionally at least +9000 charge units, optionally at least +10000 charge units.
34. The concatemer according to any of claims 1-30 and 32 wherein the concatemer has a high negative net charge, optionally has a net charge of less than 3 charge units, optionally at less than 5 charge units, optionally less than 7 charge units, optionally less than 10 charge units, optionally less than 15 charge units, optionally less than 20 charge units, optionally less than 25 charge units, optionally less than 30 charge units, optionally less than 35 charge units, optionally less than 40 charge units, optionally less than 45 charge units, optionally less than 50 charge units, optionally less than 55 charge units, optionally less than 60 charge units, optionally less than 65 charge units, optionally less than 70 charge units, optionally less than 75 charge units, optionally less than 80 charge units, optionally less than 85 charge units, optionally less than 90 charge units, optionally less than 95 charge units, optionally less than 100 charge units, optionally less than 200 charge units, optionally less than 300 charge units, optionally less than 400 charge units, optionally less than 500 charge units, optionally less than 600 charge units, optionally less than 700 charge units, optionally less than 800 charge units, optionally less than 900 charge units, optionally less than 1000 charge units, optionally less than 2000 charge units, optionally less than 3000 charge units, optionally less than 4000 charge units, optionally less than 5000 charge units, optionally less than 6000 charge units, optionally less than 7000 charge units, optionally less than 8000 charge units, optionally less than 9000 charge units, optionally less than 10000 charge units.
35. The concatemer according to any of claims 1-34 wherein the concatemer comprises at least three beta solenoid domains, optionally at least four beta solenoid domains, optionally at least 5 beta solenoid domains, optionally at least 6 beta solenoid domains, optionally at least 7 beta solenoid domains, optionally at least 8 beta solenoid domains, optionally at least 9 beta solenoid domains, optionally at least 10 beta solenoid domains, optionally at least 11 beta solenoid domains, optionally at least 12 beta solenoid domains, optionally at least 13 beta solenoid domains, optionally at least 14 beta solenoid domains, optionally at least 15 beta solenoid domains, optionally at least 16 beta solenoid domains, optionally at least 17 beta solenoid domains, optionally at least 18 beta solenoid domains, optionally at least 19 beta solenoid domains, optionally at least 20 beta solenoid domains, optionally at least 21 beta solenoid domains, optionally at least 22 beta solenoid domains, optionally at least 23 beta solenoid domains, optionally at least 24 beta solenoid domains, optionally at least 25 beta solenoid domains, optionally at least 26 beta solenoid domains, optionally at least 27 beta solenoid domains, optionally at least 28 beta solenoid domains, optionally at least 29 beta solenoid domains, optionally at least 30 beta solenoid domains, optionally at least 35 beta solenoid domains, optionally at least 40 beta solenoid domains, optionally at least 45 beta solenoid domains, optionally at least 50 beta solenoid domains, optionally at least 55 beta solenoid domains, optionally at least 60 beta solenoid domains, optionally at least 65 beta solenoid domains, optionally at least 70 beta solenoid domains, optionally at least 75 beta solenoid domains, optionally at least 80 beta solenoid domains, optionally at least 85 beta solenoid domains, optionally at least 90 beta solenoid domains, optionally at least 95 beta solenoid domains, optionally at least 100 beta solenoid domains.
36. The concatemer according to any of claims 1-35 wherein the concatemer comprises or consists of one or more of the sequences according to SEQ ID NO: 29-32 and 158-162.
37. The concatemer according to any of claims 1-36 wherein the concatemer is capable of being expressed by a cell or a cell-free transcription and translation system.
38. The concatemer according to claim 37 wherein the cell is a prokaryotic cell, optionally a bacterial cell, optionally an E. coli cell, a Bacillus subtilis cell, a Bacillus megaterium cell, a Vibrio natriegens cell, or a Pseudomonas fluorescens cell.
39. The concatemer according to claim 37 wherein the cell is a eukaryotic cell, optionally a yeast cell, optionally Pichia pastoris or Saccharomyces cerevisiae; an insect cell, optionally a baculovirus infected insect cell; or a mammalian cell, optionally a baculovirus infected mammalian cell, a HEK293 cell, a HeLa cell, or CHO cells.
40. A beta solenoid domain that comprises an amino acid sequence that comprises at least 4 units of a consensus motif.
41. The beta solenoid domain according to claim 40 wherein the beta solenoid domain is rod-shaped, optionally wherein the beta solenoid domain has a longitudinal axis and a transverse axis and the ratio of longitudinal length to transverse length is at least 2:1, optionally at least 3:1, optionally at least 4:1, optionally at least 5:1, optionally at least 6:1, optionally at least 7:1, optionally at least 8:1, optionally at least 9:1, optionally 10:1.
42. The beta solenoid domain according to any of claims 40 and 41 wherein the beta solenoid domain has an aspect ratio of 10:1 or less.
43. The beta solenoid domain according to any of claims 40 to 42 wherein the beta solenoid domain comprises between 4 and 3000 units of the consensus motif, optionally between 5 and 2800, optionally between 6 and 2600, optionally between 7 and 2400, optionally between 8 and 2200, optionally between 9 and 2000, optionally between 10 and 1800, optionally between 11 and 1600, optionally between 12 and 1400, optionally between 13 and 1200, optionally between 14 and 1000, optionally between 15 and 800, optionally between 16 and 600, optionally between 17 and 500, optionally between 18 and 400, optionally between 19 and 300, optionally between 21 and 200, optionally between 22 and 150, optionally between 23 and 100, optionally between 24 and 90, optionally between 25 and 85, optionally between 26 and 80, optionally between 27 and 75, optionally between 28 and 70, optionally between 29 and 65, optionally between 30 and 60, optionally between 35 and 55, optionally between 40 and 50, optionally 45 units of the consensus motif; optionally wherein the units of the consensus motif are arranged consecutively.
44. The beta solenoid domain according to any of claims 40-43 wherein the beta solenoid domain comprises at least 5 units of the consensus motif, optionally at least 6 units, optionally at least 7 units, optionally at least 8 units, optionally at least 9 units, optionally at least 10 units, optionally at least 11 units, optionally at least 12 units, optionally at least 13 units, optionally at least 14 units, optionally at least 15 units, optionally at least 16 units, optionally at least 17 units, optionally at least 18 units, optionally at least 19 units, optionally at least 20 units, optionally at least 21 units, optionally at least 24 units, optionally at least 25 units, optionally at least 26 units, optionally at least 27 units, optionally at least 28 units, optionally at least 29 units, optionally at least 30 units, optionally at least 40 units, optionally at least 50 units, optionally at least 60 units, optionally at least 70 units, optionally at least 80 units, optionally at least 90 units, optionally at least 100 units, optionally at least 200 units of the consensus motif; optionally wherein the units of the consensus motif are arranged consecutively.
45. The beta solenoid domain according to any of claims 40-44 wherein the beta solenoid domain comprises less than 3000 units of the consensus motif, optionally less than 2000 units, optionally less than 1000 units, optionally less than 500 units, optionally less than 400 units, optionally less than 300 units, optionally less than 200 units, optionally less than 100 units, optionally less than 90 units, optionally less than 80 units, optionally less than 70 units, optionally less than 60 units, optionally less than 50 units, optionally less than 40 units, optionally less than 30 units, optionally less than 29 units, optionally less than 28 units, optionally less than 27 units, optionally less than 26 units, optionally less than 25 units, optionally less than 24 units, optionally less than 23 units, optionally less than 22 units, optionally less than 21 units, optionally less than 20 units, optionally less than 19 units, optionally less than 18 units, optionally less than 17 units, optionally less than 16 units, optionally less than 15 units, optionally less than 14 units, optionally less than 13 units, optionally less than 12 units, optionally less than 11 units, optionally less than 10 units, optionally less than 9 units, optionally less than 8 units, optionally less than 7 units, optionally 6 units of the consensus motif; optionally wherein the units of the consensus motif are arranged consecutively.
46. The beta solenoid domain according to any of claims 40-45 wherein the beta solenoid domain comprises at least two identical units of the consensus motif, optionally wherein all of the units of the consensus motif are identical.
47. The beta solenoid domain according to any of claims 40-46 wherein the beta solenoid domain comprises non-identical units of the consensus motif, optionally wherein all of the units of the consensus motif are different.
48. The beta solenoid domain according to any of claims 40-47 wherein the beta solenoid domains have a cross-section selected from the group consisting of a tesselatable cross-section, optionally a square, a rectangle, a parallelogram, a hexagon, or a triangle cross-section; or a circle or an oval cross-section.
49. The beta solenoid domain according to any of claims 40-48 wherein the beta solenoid domain is capable of forming a lyotropic liquid crystal mesophase.
50. The beta solenoid domain according to any of claims 40-49 wherein the beta solenoid domain comprises units of the consensus motif selected from the group consisting of: TABLE-US-00052 [SEQIDNO:147] i)AX.sub.1L/FX.sub.2X.sub.3 wherein X.sub.1 is any residue X.sub.2 is any residue X.sub.3 is any residue; optionally wherein X.sub.1 is A, C, D, E, I, K, L, M, N, R, S, T or V X.sub.2 is A, C, D, E, F, G, H, I, K, N, Q, R, S, T, V, W, or Y X.sub.3 is A, C, D, E, G, H, I, K, N, Q, R, S, Y; [SEQ ID NO: 148] Or TABLE-US-00053 [SEQIDNO:149] ii)AX.sub.1LX.sub.2X.sub.3 wherein X.sub.1 is any residue X.sub.2 is any residue X.sub.3 is any residue; optionally wherein X.sub.1 is A, C, D, E, I, K, L, M, N, R, S, T or V X.sub.2 is A, C, D, E, F, G, H, I, K, N, Q, R, S, T, V, W, or Y X.sub.3 is A, C, D, E, G, H, I, K, N, Q, R, S, Y; [SEQ ID NO: 150] Or TABLE-US-00054 [SEQIDNO:151] iii)A(N/D)L*X where * is a polar residue (C, R, H, K, D, E, S, T, N, Q) and X is any residue TABLE-US-00055 [SEQIDNO:152] iv)(S/G/T/A)X(A/V/G)X(G/A)XX where X is any residue TABLE-US-00056 [SEQIDNO:153] v)(S/G/T/A)X(A/V/G)X(G/A)XX(S/G/T/A)*(A/V/G)* (G/A)XX where * is a hydrophobic residue and where X is any residue TABLE-US-00057 [SEQIDNO:154] vi)SXAXGXXS*A*GXX where * is a hydrophobic residue and where X is any residue TABLE-US-00058 [SEQIDNO:155] vii)NXAXGXXST(I/V)(G/A)GGXX where X is any residue TABLE-US-00059 [SEQIDNO:156] viii)NXAXGXXSTIGGGXX where X is any residue TABLE-US-00060 [SEQIDNO:157] ix)GXQX(V/I/L)XXGGXAXXTX(V/I/L)XXG where X is any residue optionally wherein (i), (ii) and (iii) generate a beta solenoid domain with a square cross-section; (iv), (v), (vi), (vii) and (viii) generate an oval shaped cross-section; and (ix) forms a triangular cross-section.
51. The beta solenoid domain according to any of claims 40-50 wherein the beta solenoid domain comprises or consists of any of the sequences of SEQ ID NO: 8-28 and 169:
52. The beta solenoid domain according to any of claims 40-51 wherein the beta solenoid domain does not consist of any of the sequences of SEQ ID NO: 1, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62; and/or SEQ ID NO: 66-80.
53. The beta solenoid domain according to any of claims 40-52 wherein the beta solenoid domains comprises a capping sequence at a first end or a second end, or both the first end and the second end of the beta solenoid domain, optionally wherein the capping sequence prevents the beta solenoid domain from joining end-to-end with another beta solenoid domain and aggregating, optionally wherein the capping sequence contains an alpha helix.
54. The beta solenoid domain according to claim 53 wherein the capping sequence comprises: a) a sequence that conforms to the consensus sequence of SEQ ID NO: 82 and/or SEQ ID NO: 83, optionally wherein the first cap conforms to SEQ ID NO: 82 and the second cap conforms to SEQ ID NO: 83; and/or b) any one or more of SEQ ID NO: 2, 3, 84-104 and 105-125, optionally wherein any one or more of the capping sequence comprises a sequence with a) at least 80% homology, optionally at least 85% homology, optionally at least 90% homology, optionally at least 95% homology, optionally at least 96% homology, optionally at least 97% homology, optionally at least 98% homology, optionally at least 99% homology, optionally 100% homology to SEQ ID NO: 2, 3, 84-104 or 105-125; or b) at least 80% sequence identity, optionally at least 85% sequence identity, optionally at least 90% sequence identity, optionally at least 95% sequence identity, optionally at least 96% sequence identity, optionally at least 97% sequence identity, optionally at least 98% sequence identity, optionally at least 99% sequence identity, optionally 100% sequence identity to SEQ ID NO: 2, 3, 84-104 or 105-125.
55. The beta solenoid domain according to any of claims 40-54 wherein the beta solenoid domain comprises hidden length.
56. The beta solenoid domain according to any of claims 40-55 wherein the beta solenoid domain comprises a first and a second conformation, wherein the second conformation is extended relative to the first conformation, optionally wherein the length of the second conformation is at least 1.5 times longer than the length of the first conformation, optionally at least 2 times longer, optionally at least 3 times longer, optionally at least 4 times longer, optionally at least 5 times longer, optionally at least 10 times longer, optionally at least 20 times longer, optionally at least 30 times longer, optionally at least 40 times longer, optionally at least 50 times longer, optionally at least 100 times longer than the length of the first conformation.
57. The beta solenoid domain according to claim 56 wherein when in the first conformation the beta solenoid domain comprises sacrificial hydrogen bonds and wherein in moving between the first and second conformation at least one of the sacrificial hydrogen bonds is broken.
58. The beta solenoid domain according to any of claim 56 or 57 wherein when in the first conformation the beta solenoid domain comprises a high degree of coiling.
59. The beta solenoid domain according to any of claims 56-58 wherein the beta solenoid domain moves between the first conformation and the second conformation when a defined force is applied parallel to the longitudinal axis of the beta solenoid domain.
60. The beta solenoid domain according to any of claims 40-59 wherein the beta solenoid domain has a molecular weight of at least 30 kDa, optionally at least 40 kDa, optionally at least 50 kDa, optionally at least 60 kDa, optionally at least 70 kDa, optionally at least 80 kDa, optionally at least 90 kDa, optionally at least 100 kDa, optionally at least 120 kDa, optionally at least 150 kDa, optionally at least 200 kDa, optionally at least 250 kDa, optionally at least 300 kDa, optionally at least 350 kDa, optionally at least 400 kDa, optionally at least 450 kDa, optionally at least 500 kDa, optionally at least 600 kDa, optionally at least 650 kDa, optionally at least 700 kDa, optionally at least 800 kDa, optionally at least 900 kDa, optionally at least 1000 kDa.; optionally between 30 kDa and 1000 kDa.
61. The beta solenoid domain according to any of claims 40-60 wherein the beta solenoid domain is soluble at high concentrations, optionally soluble at a concentration of at least 20 mg/ml, optionally at least 30 mg/ml, optionally at least 40 mg/ml, optionally at least 50 mg/ml, optionally at least 60 mg/ml, optionally at least 70 mg/ml, optionally at least 80 mg/ml, optionally at least 90 mg/ml, optionally at least 100 mg/ml, optionally at least 120 mg/ml, optionally at least 130 mg/ml, optionally at least 150 mg/ml, optionally at least 170 mg/ml, optionally at least 190 mg/ml, optionally at least 200 mg/ml, optionally at least 250 mg/ml, optionally at least 300 mg/ml, optionally at least 350 mg/ml, optionally at least 400 mg/ml, optionally soluble in: a) a buffer selected from the group consisting of any one or more of: MES, Bis-tris methane, ADA, ACES, Bis-tris propane, PIPES, MOPSO, Cholamine chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, Acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, Glycinamide, Glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO CAPSO, CAPS, CABS, Bicine, sodium citrate buffer, sodium bicarbonate buffer, phosphate buffer, borate buffer; or b) an organic solvent, optionally HFIP; optionally wherein the buffer comprises one or more salts, optionally sodium chloride and/or potassium chloride; optionally wherein the buffer is at a pH range of 3 to 10, optionally pH 7.
62. The beta solenoid domain according to any of claims 40-61 wherein the beta solenoid domain has a high net charge, optionally has a net charge of at least +3 charge units, optionally at least +5 charge units, optionally at least +7 charge units, optionally at least +10 charge units, optionally at least +15 charge units, optionally at least +20 charge units, optionally at least +25 charge units, optionally at least +30 charge units, optionally at least +35 charge units, optionally at least +40 charge units, optionally at least +45 charge units, optionally at least +50 charge units, optionally at least +55 charge units, optionally at least +60 charge units, optionally at least +65 charge units, optionally at least +70 charge units, optionally at least +75 charge units, optionally at least +80 charge units, optionally at least +85 charge units, optionally at least +90 charge units, optionally at least +95 charge units, optionally at least +100 charge units, optionally at a pH range of 3 to 10, optionally pH 7.
63. The beta solenoid domain according to any of claims 40-61 wherein the beta solenoid domain has a high negative net charge, optionally has a net charge of less than 3 charge units, optionally at less than 5 charge units, optionally less than 7 charge units, optionally less than 10 charge units, optionally less than 15 charge units, optionally less than 20 charge units, optionally less than 25 charge units, optionally less than 30 charge units, optionally less than 35 charge units, optionally less than 40 charge units, optionally less than 45 charge units, optionally less than 50 charge units, optionally less than 55 charge units, optionally less than 60 charge units, optionally less than 65 charge units, optionally less than 70 charge units, optionally less than 75 charge units, optionally less than 80 charge units, optionally less than 85 charge units, optionally less than 90 charge units, optionally less than 95 charge units, optionally less than 100 charge units, optionally at a pH range of 3 to 10, optionally pH 7.
64. The beta solenoid domain according to any of claims 40-63 wherein the beta solenoid domain is capable of being expressed by a cell or a cell-free transcription and translation system.
65. The beta solenoid domain according to claim 64 wherein the cell is a prokaryotic cell, optionally a bacterial cell, optionally an E. coli cell, a Bacillus subtilis cell, a Bacillus megaterium cell, a Vibrio natriegens cell, or a Pseudomonas fluorescens cell.
66. The beta solenoid domain according to claim 64 wherein the cell is a eukaryotic cell, optionally a yeast cell, optionally Pichia pastoris or Saccharomyces cerevisiae; an insect cell, optionally a baculovirus infected insect cell; or a mammalian cell, optionally a baculovirus infected mammalian cell, a HEK293 cell, a HeLa cell, or CHO cells.
67. A composition comprising a concatemer according to any of claims 1-39 or a beta solenoid domain according to any of claims 40-66.
68. The composition according to claim 67 wherein the concatemer or beta solenoid domain is at a concentration of at least 20 mg/ml, optionally at least 30 mg/ml, optionally at least 40 mg/ml, optionally at least 50 mg/ml, optionally at least 60 mg/ml, optionally at least 70 mg/ml, optionally at least 80 mg/ml, optionally at least 90 mg/ml, optionally at least 100 mg/ml, optionally at least 120 mg/ml, optionally at least 130 mg/ml, optionally at least 150 mg/ml, optionally at least 170 mg/ml, optionally at least 190 mg/ml, optionally at least 200 mg/ml, optionally at least 250 mg/ml, optionally at least 300 mg/ml, optionally at least 350 mg/ml, optionally at least 400 mg/ml.
69. The composition according to any of claim 67 or 68 wherein the concatemer or the beta solenoid domain is not denatured.
70. The composition according to any of claims 67-69 wherein the composition comprises a solvent, optionally wherein the solvent is a) a buffer selected from the group consisting of any one or more of: MES, Bis-tris methane, ADA, ACES, Bis-tris propane, PIPES, MOPSO, Cholamine chloride, MOPS, BES, TES, HEPES, DIPSO, MOBS, Acetamidoglycine, TAPSO, TEA, POPSO, HEPPSO, EPS, HEPPS, Tricine, Tris, Glycinamide, Glycylglycine, HEPBS, Bicine, TAPS, AMPB, CHES, AMP, AMPSO CAPSO, CAPS, CABS, Bicine, sodium citrate buffer, sodium bicarbonate buffer, phosphate buffer, borate buffer; or b) an organic solvent, optionally HFIP; optionally wherein the buffer comprises one or more salts, optionally sodium chloride and/or potassium chloride; optionally wherein the buffer is at a pH range of 3 to 10, optionally pH 7.
71. A nucleic acid encoding the concatemer according to any of claims 1-39 or the beta solenoid domain according to any of claims 40-66.
72. A vector comprising the nucleic acid according to claim 71.
73. A host cell comprising the nucleic acid according to claim 71 and/or the vector according to claim 72, optionally wherein the host cell is a) a prokaryotic cell, optionally a bacterial cell, optionally an E. coli cell, a Bacillus subtilis cell, a Bacillus megaterium cell, a Vibrio natriegens cell, or a Pseudomonas fluorescens cell; or b) is a eukaryotic cell, optionally a yeast cell, optionally Pichia pastoris or Saccharomyces cerevisiae; an insect cell, optionally a baculovirus infected insect cell; or a mammalian cell, optionally a baculovirus infected mammalian cell, a HEK293 cell, a HeLa cell, or CHO cells.
74. A fibre comprising the concatemer according to any of claims 1-39 or the beta solenoid domain according to any of claims 40-66.
75. The fibre according to claim 74 wherein the fibre has been produced via a wet-spinning method.
76. The fibre according to any of claims 74 and 75 wherein the fibre has an average diameter of between 100 nm and 100 um, optionally between 500 nm and 50 um, optionally between 1000 nm and 40 um, optionally between 2.5 um and 35 um, optionally between 5 um and 30 um, optionally between 7.5 um and 25 um, optionally between 10 um and 20 um.
77. A fibre according to any of claims 74-76 wherein the fibre has a Young's modulus of between 0.5 GPa and 200 GPa, optionally between 1.0 GPa and 175 GPa, optionally between 2 GPa and 150 GPa, optionally between 5.0 GPa and 125 GPa, optionally between 10.0 GPa and 100.0 GPa, optionally between 15.0 GPa and 90.0 GPa, optionally between 20.0 GPa and 80.0 GPa, optionally between 25.0 GPa and 75.0 GPa, optionally between 30.0 GPa and 70.0 GPa, optionally between 35.0 GPa and 60.0 GPa, optionally between 40.0 GPa and 50.0 GPa; and/or a Young's modulus of at least 0.5 GPa, 1.0 GPa, 2 GPa, 5.0 GPa, 10.0 GPa, 15.0 GPa, 20.0 GPa, 25.0 GPa, 30.0 GPa, 35.0 GPa, 40.0 GPa, 50.0 GPa, 60.0 GPa, 70.0 GPa, 75.0 GPa, 80.0 GPa, 90.0 GPa, 100.0 GPa, 125 GPa, 150 GPa, 175 GPa or at least 200 GPa.
78. A fibre according to any of claims 74-77 wherein the fibre has an engineered strength of: between 0.1 GPa and 3 GPa, optionally between 0.2 GPa and 2.8 GPa, optionally between 0.3 GPa and 2.6 GPa, optionally between 0.4 GPa and 2.4 GPa, optionally between 0.5 GPa and 2.2 GPa, optionally between 0.6 GPa and 2.0 GPa, optionally between 0.7 GPa and 1.8 GPa, optionally between 0.8 GPa and 1.6 GPa, optionally between 0.9 GPa and 1.4 GPa, optionally between 1.0 GPa and 1.2 GPa; and/or at least 0.1 GPa, optionally at least 0.2 GPa, optionally at least 0.3 GPa, optionally at least 0.4 GPa, optionally at least 0.5 GPa, optionally at least 0.6 GPa, optionally at least 0.7 GPa, optionally at least 0.8 GPa, optionally at least 0.9 GPa, optionally at least 1.0 GPa, optionally at least 1.1 GPa, optionally at least 1.2 GPa, optionally at least 1.3 GPa, optionally at least 1.4 GPa, optionally at least 1.5 GPa, optionally at least 1.6 GPa, optionally at least 1.7 GPa, optionally at least 1.8 GPa, optionally at least 1.9 GPa, optionally at least 2.0 GPa, optionally at least 2.1 GPa, optionally at least 2.2 GPa, optionally at least 2.3 GPa, optionally at least 2.4 GPa, optionally at least 2.5 GPa, optionally at least 2.6 GPa, optionally at least 2.7 GPa, optionally at least 2.8 GPa, optionally at least 2.9 GPa, optionally at least 3.0 GPa.
79. A fibre according to any of claims 74-78 wherein the fibre has a strain to failure of between 2% and 300%, optionally between 5% and 275%, optionally between 10% and 250%, optionally between 15% and 225%, optionally between 20% and 200%, optionally between 25% and 175%, optionally between 30% and 150%, optionally between 35% and 125%, optionally between 40% and 100%, optionally between 45% and 80%, optionally between 50% and 75%, optionally between 55% and 70%; and/or a strain to failure of at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, 225%, 250%, 275% or at least 300%.
80. A fibre according to any of claims 74-79 wherein the fibre has a toughness of at least 10 J/g, optionally at least 20 J/g, optionally at least 40 J/g, optionally at least 60 J/g, optionally at least 80 J/g, optionally at least 100 J/g, optionally at least 120 J/g, optionally at least 140 J/g, optionally at least 160 J/g, optionally at least 180 J/g, optionally at least 200 J/g, optionally at least 250 J/g, optionally at least 300 J/g, optionally at least 350 J/g, optionally at least 400 J/g, optionally at least 450 J/g, optionally at least 500 J/g, optionally at least 550 J/g, optionally at least 600 J/g, optionally at least 650 J/g, optionally at least 700 J/g, optionally at least 750 J/g, optionally at least 800 J/g, optionally at least 850 J/g, optionally at least 900 J/g, optionally at least 950 J/g, optionally at least 1000 J/g.
81. A fibre according to any of claims 74-80 wherein the fibre comprises more than one different concatemer according to any of claims 1-39 and/or more than one beta solenoid domain according to any of claims 40-66.
82. A yarn comprising a fibre according to any of claims 74-81.
83. A yarn comprising at least two different fibres according to any of claims 74-81.
84. A textile comprising the concatemer according to any of claims 1-39, the beta solenoid domain according to any of claims 40-66, or the fibre according to any of claims 74-81.
85. Use of a concatemer according to any of claims 1-39, a beta solenoid domain according to any of claims 40-66, a fibre according to any of claims 74-81 or a textile according to claim 84 in the production of any one or more of: sportswear (e.g. for protective uses such as rash guards); Protective wear for motorcyclists and/or other motorsports; Personal protective equipment (e.g. body armour, protective underwear for soldiers); Blast containment; Blade containment (e.g. for aircraft engines); Textiles for parachutes; Ropes; Textiles for clothing; Textiles for shoes; general medical textiles; Wound dressings; Resorbable and non-resorbable surgical sutures; Resorbable and non-resorbable stents; Guide-wires for surgery (including non-metallic guide wires suitable for MRI); Biocompatible coatings for implanted medical devices; Surgical meshes; Scaffolds for 3D tissue engineering.
86. Sportswear (e.g. for protective uses such as rash guards); Protective wear for motorcyclists and/or other motorsports; Personal protective equipment (e.g. body armour, protective underwear for soldiers); Blast containment; Blade containment (e.g. for aircraft engines); Textiles for parachutes; Ropes; Textiles for clothing; Textiles for shoes; general medical textiles; Wound dressings; Resorbable and non-resorbable surgical sutures; Resorbable and non-resorbable stents; Guide-wires for surgery (including non-metallic guide wires suitable for MRI); Biocompatible coatings for implanted medical devices; Surgical meshes; or Scaffolds for 3D tissue engineering comprising a concatemer according to any of claims 1-39, a beta solenoid domain according to any of claims 40-66, a fibre according to any of claims 74-81 or a textile according to claim 84.
87. A beta solenoid capping structure comprising an amino acid sequence that conforms to any of the following the consensus sequences: TABLE-US-00061 a) [SEQIDNO:82] xMxxxxILxxYxxGxxxFxxIxLxx(I/A)xLxx where x is any residue or TABLE-US-00062 b) [SEQIDNO:83] Ax(T/L/F)xxAx(L/F)xxAx(L/F)xxAxVxPVLWxxAxLxGAx (M/V)xxxxxxx(Y/F)xxAxx where x is any residue optionally wherein capping structures that conform to the sequence of SEQ ID NO: 82 are suitable for capping the N-terminus of the beta solenoid domain, and capping structures that conform to the sequence of SEQ ID NO: 83 are suitable for capping the C-terminus of the beta solenoid domain; optionally wherein the capping structure comprises or consists of any of SEQ ID NO: 2, 3, 84-104 or 105-125, optionally wherein SEQ ID NO: 2 and 84-104 are suitable for capping the N-terminus of the beta solenoid domain and SEQ ID NO: 3 and 105-125 are suitable for capping the C-terminus of the beta solenoid domain.
Description
FIGURE LEGENDS
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[0340]
[0341]
[0342] Codon optimised genes were expressed in the KRX E. coli strain and induced with 0.1% filter sterilised rhamnose and grown overnight at 25 degrees C. Non-induced cultures were also grown in the same way for comparison. Cell were harvested and lysed by sonication. SDS-PAGE gels of the soluble and insoluble fractions showed significant protein over-expression bands for all proteins in the soluble fraction. L indicates the PageRuler plus ladder, S indicates the soluble supernatant, I insoluble fraction, and the + indicates that the culture had been induced.
[0343]
[0344] SynRFR24.nnum35.x4 was purified to high purity and concentration. L is the PageRuler plus ladder and the other lane is the concentrated soluble protein. The SynRFR24.nnum35.x4 sample was found to be highly soluble and could be concentrated to >200 mg/ml.
[0345]
EXAMPLES
Example 1
[0346] The original SynRFR series of artificial beta solenoid proteins were designed to form homodimers with the dimer interface at the C-terminal capping domain (MacDonald et al 2016 PNAS 37: 10346-10351).
[0347] The rigid rod-like SynRFR domain has an aspect ratio of around 3:1, potentially permitting spinning from a well-ordered nematic liquid crystalline phase. In order to achieve this, new monomeric versions of the SynRFR domain repeat unit were required that can then be concatenated into higher molecular weight polymers.
[0348] Using the high-resolution crystal structure of the SynRFR24.1 beta solenoid protein (PDB code: 4YC5), monomeric versions were created by completely redesigning the sequence of the terminal cap residues using computational protein design methods (using the RosettaDesign algorithm, https://www.rosettacommons.orq/).
[0349] Extra restraint terms were added to the Rosetta potential energy function to create designs biased towards a range of different net surface charges [SEQ ID NO: 3-23]. In addition to these designs, a more conservative design was created where only the C- and N-terminal caps were redesigned as we previously found that these caps are vital for the soluble folding of the protein by shielding the hydrophobic core of the solenoid. This design, called SynRFR24.new_caps [SEQ ID NO: 6], was used for initial expression tests. SynRFR24.new_caps (25.52 kDa) has a slightly higher molecular weight than SynRFR24.1 (24.95 KDa) [SEQ ID NO: 7]. (
[0350] Cloning, Expression and Purification of Repeat Units
[0351] The SynRFR24.new_caps design was ordered as a gBlock gene fragment from IDT and cloned into a standard pET11a expression plasmid. The existing SynRFR24.1 design previously cloned in to the same plasmid. Both plasmids were sequence verified and then transformed in the KRX E. coli strain.
[0352] 1 L of 2YT medium was inoculated with 5 ml LB overnight cultures for both SynRFR24.1 and SynRFR24.new_caps (all media with appropriate antibiotics). The 1 L cultures were grown at 37 degrees Celsius until reaching an OD600 reading of 1. Expression was then induced with 0.1% filter sterilised rhamnose and the cultures grown overnight at 21 degrees Celsius. Cells were harvested and resuspended in lysis buffer (100 mM bicine and 150 mM NaCl buffer titrated to pH 9.0 with NaOH). The cells were sonicated and clarified by centrifugation at 40,000g for 40 min. The proteins were purified with a nickel-nitrilotriacetic acid (Ni-NTA) column; washed with 100 mM bicine, 150 mM NaCl, and 50 mM imidazole at pH 9.0; and eluted in 100 mM bicine, 150 mM NaCl, and 500 mM imidazole at pH 9.0. The new protein design was found to be well expressed and soluble (
[0353] Crystallisation Trials
[0354] The concentrated SynRFR24,new_caps samples were used to set up vapour diffusion sparse-matrix crystallization trials with a Mosquito robot (TTP Labtech) in 864 different conditions. After two days crystals were observed in four conditions (
[0355] Polarised Optical MicroscopyLiquid Crystal Formation
[0356] Polarised optical microscopy was utilised in order to observe any liquid crystalline behaviour of the protein monomers. 1 l of each protein (namely SynRFR24.1 and SynRFR24.new_caps) were dropped on a clean glass slide with a cover slip placed on top. The concentration of the SynRFR24.1 and SynRFR24.new_caps was ca. 60 mg ml- and 40 mg ml-1, respectively, in bicine buffer. The samples were stored in the fridge at ca. 4 degrees. Once drop cast, the samples were viewed immediately using polarised optical microscopy (POM) and what potentially is the beginning of a Schlieren textured nematic liquid crystal was evident in small areas for the SynRFR24.new_caps (
[0357] 1D Liquid SAXS
[0358] Small angle X-ray scattering (SAXS) measurements were performed using a PANalytical Empyrean diffractometer equipped with ScatterX78 module, from 0-4 20, and the data analysis was performed using PANalytic EasySAXS software. For SAXS data collection, a step size of 0.003 2, scan step time of 350 s, and scan speed of 0.002/s were used. Samples were prepared and stored in the fridge 20 h before running. Approximately 50 l of each protein was used for filling the capillary. The SynRFR24.new_caps was diluted to ca. 10 mg ml-1, which is a lower concentration than the protein liquid crystal observed in
[0359] Potential Precipitants
[0360] Ten different coagulant solvents were investigated to observe how and if either of the proteins would precipitate. Several of the solvents were successful at coagulating the proteins, as summarised in Table 1. Both proteins produced white fibrous-like precipitate in several of the solvents, especially ethanol and propanol. 1.5 l of each protein was injected into 5 ml of the coagulation solution. It is very plausible that the amount of precipitation of the SynRFR24.1 compared to the SynRFR24.new_caps is concentration dependent (60 mg ml-1 to 40 mg ml-1). From initial results, it is likely that higher concentrations of the protein monomers and/or concatenating several protein monomers would lead to a higher degree of coagulation and fibre formation. The degree of precipitation using ethanol was significant for both proteins (the difference in degree of precipitation is most likely concentration dependent between the 2 proteins) and it is possible to attempt spinning a fibre with slightly larger volumes (ca. 0.5-1 ml).
TABLE-US-00039 TABLE 1 Summary of potential coagulation solvents for fibre formation (WP-white precipitate). Solvent SynRFR24.1 SynRFR24.new_caps Ethanol plenty of WP present WP present Acetone WP present WP present Propanol WP present some WP present Diethyl ether Yes after 1 h None Methanol WP present small amount WP present CaCl 20 mM WP HC of salt? None KCl 20 mM clear then dissolved None MgSO.sub.4 20 mM None transparent swirl DMSO V small amount None WP present DMSO:H.sub.2O (50:50) None None
Example 2Design of High Molecular Weight Concatenated Versions of SynRFR24.New_Caps
[0361] The SynRFR24.new_caps monomer described above was concatenated with flexible poly-glycine linker sequences to produce a new high molecular weight version, labelled SynRFR24.nc.x4.1 (880 amino acid residues and 95.2 kDa molecular weight;
[0362] Cloning, Expression and Purification of SynRFR24.Nc.x4.1
[0363] The SynRFR24.nc.x4.1 design was ordered as a codon-optimised synthetic gene from GeneArt/Thermo Fisher with flanking T7 promoter and terminator sequences. The plasmid was transformed into the KRX E. coli strain. 1 L of Terrific Broth medium was inoculated with 5 ml LB overnight cultures (all media with appropriate antibiotics). The 1 L cultures were grown at 37 degrees Celsius until reaching an OD600 reading of 1. Expression was then induced with 0.1% filter sterilised rhamnose and the cultures grown overnight at 25 degrees Celsius. Cells were harvested and resuspended in lysis buffer (100 mM bicine and 150 mM NaCl buffer titrated to pH 9.0 with NaOH). The cells were sonicated and clarified by centrifugation at 40,000g for 40 min. The proteins were purified with a nickel-nitrilotriacetic acid (Ni-NTA) column; washed with 100 mM bicine, 150 mM NaCl, and 0 to 5 mM imidazole at pH 9.0; and eluted in 100 mM bicine, 150 mM NaCl, and 500 mM imidazole at pH 9.0. The concatemer design was found to be well expressed and highly soluble (
[0364] Thermofluor Assay for Protein Stability
[0365] To measure the thermal stability of the SynRFR24.nc.x4.1 protein, melting experiments were performed in the presence of 10 final concentration of SYPRO Orange dye (Life Technologies) and a final protein concentration of approximately 1 mg/ml in 100 mM bicine and 150 mM NaCl buffer titrated to pH 9.0 with NaOH. The samples were heated from degrees Celsius to 99 degrees Celsius at rate of 1 degrees Celsius min-1 in a StepOnePlus Real-Time PCR system (Applied Biosystems) with fluorescence measured at standard excitation/emission wavelengths. The protein melting temperature, T.sub.m, was estimated using the R statistical package Rstats by fitting a smoothing cubic spline and finding the maximum of the first derivative. The protein was found to have a melting temperature of around 67 degrees Celsius (
[0366] Size Exclusion Chromatography-Multi-Angle Light Scattering (SEC-MALS)
[0367] An Agilent 1260 system (Agilent Technologies) with a miniDAWN TREOS (Wyatt Technologies) light scattering detector and an Optilab T-rEX (Wyatt Technologies) refractive index detector were used together with a Superdex 200 Increase 10/300 GL column (GE Healthcare). The column was pre-equilibrated with bicine buffer (100 mM bicine and 150 mM NaCl buffer titrated to pH 9.0 with NaOH). 100 l of approximately 5 mg/ml SynRFR24.nc.x4.1 protein was injected. Data was analysed using the ASTRA software (Wyatt Technologies) in order to estimate the molar mass of the protein. The MALS estimated molar mass of the protein was consistent with being monomeric (
[0368] Cloning, Expression and Purification of SynRFR24.Nc.x8.1
[0369] The gene encoding the double length SynRFR24.nc.x8.1 design was created from the synthetic SynRFR24.nc.x4.1 gene using a combination of PCR amplification and Golden Gate assembly. This new plasmid construct was validated using restriction digests and partial sequencing. The new protein was expressed and purified using the same protocol as described for SynRFR24.nc.x4.1 in the previous section (
[0370] Polarised Optical MicroscopyLiquid Crystal Formation
[0371] Following the initial polarised optical microscopy of the monomer SynRFr24.new_caps (discussed in the first report) concatenated SynRFR24.new_caps, labelled SynRFR24.nc.x4.1 herein, were also observed for any liquid crystalline behaviour. 1 l of SynRFR24.nc.x4.1 at several different concentrations were dropped on clean glass slides with a cover slip placed on top. Concentrations 160 mg ml-1, 115 mg ml-1, 70 mg ml-1 and 140 mg ml-1 obtained through an ion exchange purification method (described above) were observed over a few days to identify LC behaviour. The samples were left at room temperature (colder temperatures caused precipitation of the polypeptide). As previously observed with the monomer, the nucleation of a Schlieren textured chiral nematic liquid crystal was evident in several areas (
[0372] Initial Fibre Formation
[0373] Following the initial coagulation solvent results, ethanol was a positive candidate to coagulate the monomer. With the first concatenated sample, ethanol and acetone were used as coagulating agents. Ethanol yielded extremely promising results, and was therefore chosen as the coagulation agent for the spinning optimisation process. Initially 2 l of SynRFR24.nc.x4.1 were drawn into a glass petri dish filled with ethanol, using a micropipette. It was possible to manipulate the SynRFR24.nc.x4.1 and stretch the material (when wet) up to 300%. The ethanol was left to dry and short (ca. 3-5 cm) fibres remained. It was possible to handle these fibres and perform tensile measurements to ascertain their mechanical properties. Under POM, the fibres are birefringent (
[0374] Tensile properties of single composite fibres were determined following the British standard (BS ISO 11566:1996). Fibres were loaded on cardboard holders (
[0375] On completion of the tensile test, the data was analysed to obtain mechanical performance (
[0376] Wet-Spinning Fibres
[0377] Optimisation of continuous fibre spinning has also been conducted. The prepared SynRFR24.nc.x4.1 of varying concentrations (average volume of each batch 0.5 ml) were drawn into a 1 ml glass syringe and wet-spun into a stream of ethanol through a 24 gauge (0.31 mm inner diameter) needle using a syringe pump (KDS 100, KDS Scientific), which resulted in coagulation and fibre formation (solid). The injection rate of spinning dopes was between 5 and 7 ml h.sup.1 and the rotating speed of the ethanol bath was ca. 4 rpm (spinning parameters have been summarised in Table 2). Each batch of SynRFR24.nc.x4.1 had a different concentration. The higher concentration dope (160 mg ml.sup.1), was diluted to 100 mg ml.sup.1 after a few days as the dope was too viscous to draw into the needle and a lot of air bubbles were trapped which prevented continuous spinning. The ultimate strengths summarised in Table 2 and
[0378] SynRFR24.nc.x4.1, following an ion exchange purification method (discussed above), was also wet-spun at 4 rpm with an injection speed of 6 ml h.sup.1 into ethanol, at a concentration of 140 mg ml. It is possible to wind the fibre (
TABLE-US-00040 TABLE 2 Spinning parameters for wet-spun SynRFR24.nc.x4.1 Valid Tests Dope Spinning Youngs Ultimate Strength Strain Sample # concentration parameters Modulus (MPa) (%) 3 115 Hand inject, 3 rpm 1.7 40.6 48.6 5 160 day 1 6 ml h.sup.1, 5 rpm 6.2 96.3 37.0 8 100 day 5 7 ml h.sup.1, 3.5 rpm 2.4 52.3 9.5 12 100 day 5 6 ml h.sup.1, 4 rpm 5.9 51.5 9.4 14 100 day 5 6 ml h.sup.1, 4 rpm 2.1 48.1 6.7 15 100 day 5 6 ml h.sup.1, 4 rpm 7.1 49.2 8.7 17 100 day 5 6.5 ml h.sup.1, 4 rpm 4.9 43.8 25.3 19 100 day 5 6.5 ml h.sup.1, 4 rpm 8.0 82.86 10.3 20 100 day 5 6.5 ml h.sup.1, 4 rpm 6.1 50.10 9.6
Example 3
[0379] Optimising the Expression and Purification of SynRFR24.x4.1
[0380] In order to obtain extremely pure samples of SynRFR24.x4.1, a three-step purification protocol of the clarified E. coli lysate was developed. Previously, it was found that SynRFR24.x4.1 does not bind very tightly to Ni-NTA columns, eluting at around 50 mM imidazole concentration. However, very little protein was found in the flow-through, indicating that binding is occurring and this method still has value as an initial purification step. In order to purify the protein further, anion exchange and gel filtration steps were added. KRX cells (Promega) expressing SynRFR24.x4.1 under an inducible T7 promoter were grown, harvested, lysed and clarified as described previously, but the resuspension buffer was slightly altered to decrease the salt concentration (50 mM bicine, 150 mM NaCl, pH 9). The lysate was loaded on to a Ni-NTA column as before, then immediately eluted (50 mM bicine, 150 mM NaCl, 500 mM imidazole, pH 9) without any intermediate wash steps. The eluate was diluted two-fold (in 50 mM bicine, 0 mM NaCl, pH 9 buffer) to reduce the salt concentration. A HiTrap Q HP column (25 ml) was equilibrated with buffer A (50 mM bicine, pH 9). The eluate from the Ni-NTA purification was applied to the column at 5 ml/min. The column was then washed with 5% buffer B (50 mM bicine, 1 M NaCl, pH 9). Protein was found to elute at around 300 mM NaCl concentration. Fractions containing the protein were pooled and concentrated to around 5 ml. A S200 gel filtration column was equilibrated with two column volumes of 20 mM bicine, 150 mM NaCl, pH 9 buffer. The pooled fractions from the anion exchange step were loaded on to the column and the appropriate fractions collected. The fractions were pooled and concentrated to around 160 mg/ml.
[0381] Polarised Optical MicroscopyPhase Diagram
[0382] A phase diagram of the concatenated monomers with concentration has been developed to identify the nematic region, this has been conducted as the goal is to wet-spin the LC SynRFR24. Several concentrations have been investigated and the purification methods improved (as discussed above).
[0383] 1 l of the concatenated SynRFR24 at several different concentrations were dropped on clean glass slides with a cover slip placed on top. Three edges of the cover slips were sealed with grease with only one edge exposed to control the evaporation rate. The samples were left at room temperature (colder temperatures caused precipitation of the polypeptide). As previously observed with the monomer, the nucleation of a Schlieren textured chiral nematic liquid crystal was evident in several areas (
[0384] Wet-Spinning Fibres
[0385] Optimisation of continuous fibre spinning has also been conducted. The prepared SynRFR24.nc.x4.1 of varying concentrations (average volume of each batch 0.5 ml) were drawn into a 1 ml glass syringe and wet-spun into a stream of ethanol through a 24 gauge (0.31 mm inner diameter) needle using a syringe pump (KDS 100, KDS Scientific), which resulted in coagulation and fibre formation (solid). The injection rate of spinning dope was 7 ml h.sup.1 and the rotating speed of the ethanol bath was ca. 4 rpm. The ultimate strengths summarised in Table 3 and
TABLE-US-00041 TABLE 3 Mechanical strength of wet-spun SynRFR24.nc.x4.1 at concentration of 100 mg ml-1and spinning parameters; 7 ml h-1, 4.5 rpm with a 24 g needle. Valid Tests Youngs Ultimate Strength Strain Sample # Modulus(GPa) (MPa) (%) 28 7.2 132.4 24.8 29 8.7 120.7 2.5 30 8.8 132.1 2.0 32 10.1 116.0 2.6 34 7.6 119.3 2.0 37 11.6 185.7 2.3 39 11.4 267.4 3.1 40 9.1 167.8 11.1 41 11.9 186.9 2.2 42 9.3 180.0 6.2
Example 4
[0386] Sec-Mals
[0387] An Agilent 1260 system (Agilent Technologies) with a miniDAWN TREOS (Wyatt Technologies) light scattering detector and an Optilab T-rEX (Wyatt Technologies) refractive index detector were used together with a Superdex 200 Increase 10/300 GL column (GE Healthcare) or a Superose 6 10/300 column (GE Healthcare). The column was pre-equilibrated with bicine buffer (20 mM bicine and 150 mM NaCl buffer titrated to pH 9.0 with NaOH). 100 l of approximately 5-15 mg/ml protein was injected for each of the different designs. Data was analysed using the ASTRA software (Wyatt Technologies) in order to estimate the molar mass of the protein and all measured designs (SynRFR24.nc.x6, SynRFR24.nc.x4, SynRFR24.nc.x2, SynRFR24_nnum35, SynRFR24_nnum36, SynRFR24_nnum40) were confirmed to be monomeric (
[0388] 1D Proton NMR
[0389] Protein samples in bicine buffer were concentrated to >300 M and D.sub.2O was added to the final concentration of around 10% v/v (for instrumental lock). 500 L samples were loaded in glass NMR tubes (Norell, part no. 507-HP-7). The NMR measurements were performed at 298 K on an Avance III 600 MHz spectrometer (Bruker) and processed using TopSpin (Bruker;
[0390] Circular Dichroism
[0391] 0.1-0.2 mg/ml of purified protein was exchanged into 20 mM borate, 150 mM NaF, pH 9.0 buffer using 0.5 mL 7K MWCO Zeba Spin Desalting Columns (Thermo Scientific). Spectra were collected in triplicate using a Chirascan (Applied Photophysics) from 190 to 260 nm wavelengths with 0.5 nm intervals, 1 nm band width, and a 1 second time per point. Measurements were taken using a quartz cuvette (QS-284) with a 0.1 cm path length. Spectra were collected for the SynRFR24.1, SynRFR24.new_caps, SynRFR24.new_caps.x4.1, SynRFR24_neg_num_36_0005, SynRFR24_neg_num_38_0005, SynRFR24_neg_num_40_0004 (
[0392] Secondary structure content was estimated using the K2D3 server http://cbdm-01.zdv.uni-mainz.de/andrade/k2d3/(https://onlinelibrary.wiley.com/doi/full/10.1002/prot.23188) with the following values: [0393] SynRFR24.1: 4.6% alpha helix, 34.7% beta strand [0394] SynRFR24.new_caps: 4.5% alpha helix, 35.9% beta strand [0395] SynRFR24.new_caps.x4: 5.8% alpha helix, 20.6% beta strand [0396] SynRFR24_nnum36: 4.6% alpha helix, 34.7% beta strand [0397] SynRFR24_nnum38: 4.9% alpha helix, 34.0% beta strand [0398] SynRFR24_nnum40: 4.9% alpha helix, 34.0% beta strand
[0399] The distance to the closest spectrum in the K2D3 database was large and so the secondary structure decompositions were flagged as potentially having high error. It is unclear why the SynRFR24.new_caps and SynRFR24.new_caps.x4 estimates were so different given the spectra overlay almost exactly. All the variants show differences from SynRFR24.1 in the 190-210 nm region, possible due to less structure in the C-terminal in the capping regions which form a dimeric interface in SynRFR24.1 but not in any of the variants (which are all monomeric by design).
[0400] Thermofluor Assay
[0401] To measure the thermal stability of each of the SynRFR proteins, melting experiments were performed in the presence of 10 final concentration of SYPRO Orange dye (Life Technologies) and a final protein concentration of approximately 0.75 mg/ml in 100 mM bicine and 150 mM NaCl buffer titrated to pH 9.0 with NaOH. The samples were heated from 25 deg C. to 99 deg C. at rate of 1 deg C/minute in a StepOnePlus Real-Time PCR system (Applied Biosystems) with fluorescence measured at standard excitation/emission wavelengths. The protein melting temperature, T.sub.m, was estimated using the R statistical package (Rstats) by fitting a smoothing cubic spline and finding the maximum of the first derivative (
Example 5
[0402] Liquid Crystal (Polarised Optical Microscopy, SAXS/WAXS)
[0403] The morphology of the fibers was investigated by polarized optical microscope (DM2500P, Leica Microsystems Ltd., GB) fitted with a DFC295 camera (Leica Application Suite v4.0.0, cc/1.1 HI PLAN 40/0.50). Concentrated protein samples were set up on glass slides or in 0.2 mm internal depth/2 mm wide flat glass capillaries (Part 3520, VitroCom;
TABLE-US-00042 TABLE 4 Number of solenoid domains versus protein concentration and sublocation in FIG. 25. Number of solenoid Protein concentration FIG. 25 domains (mg ml.sup.1) subsection (a.-r.) 1 (SynRFR24.nc.x1) 40 c. 2 (SynRFR24.nc.x2) 92 h. 117 l. 205 q. 213 r. 4 (SynRFR24.nc.x4) 30 a. 35 b. 63 e. 83 f. 89 g. 101 i. 106 j. 113 k. 130 m. 159 n. 160 o. 176 p. 8 (SynRFR24.nc.x8) 70 d.
Example 6
[0404] Wet Spinning of Fibres
[0405] Fibres were spun through injection into a rotating bath of ethanol, used as coagulant (at room temperature). In a typical experiment, injection was performed at 7 ml h.sup.1 (SynRFR24.nc.x4 concentration at 100 mg ml.sup.1) through a 24-gauge cannula (0.5588 mm inner diameter, stainless-steel, Central Surgical Co. Ltd., GB) using a syringe pump (KDS100, KD scientific, US), while the bath rotated at 4.5 rpm at the point of injection to provide extensional flow during fibre coagulation. The fibres were removed from the bath within 1 minute of coagulation and drawn before evaporation to improve alignment and strength. The fibres were stored at room temperature at ambient temperature and pressure.
[0406] Mechanical Testing
[0407] Tensile properties of single fibres were determined following the British standard (BS ISO 11566:1996). Fibres were loaded onto cardboard struts, with gauge lengths 15 mm+/0.5 mm, using an epoxy adhesive (50/50 hardener to resin, Araldite Rapid Adhesive, Bostik Fingley Ltd., UK). The tensile properties of single fibres were determined using a Linkam TST 350 tester equipped with a 20 N load cell operating at 1 mm min.sup.1 crosshead speed.
[0408] Fibre WAXS
[0409] X-ray diffraction experiments were carried out using an in-house x-ray source (Rigaku Micromax 007HF-M high-flux generator with Osmic VariMax optics) and images were collected on a Rigaku Saturn 944+ CCD detector. Image processing was carried out using the Python libraries FabIO (http://journals.iucrorg/j/issues/2013/02/00/kk5124/) and PyFAI (https://journals.iucr.org/j/issues/2015/02/00/fv5028/). Single fibres were supported on cardboard struts (see Mechanical testing section) and mounted on the goniometer and aligned in the X-ray beam.
[0410] Ten images were collected with the fibre in the X-ray beam and ten background images with no sample present were also collected and averaged to reduce noise. All images were taken with a 120 second exposure time with a 5 degrees oscillation angle and with a 110 cm distance between the sample to the detector. The mean of the ten background images was subtracted from the mean of the ten fibre diffraction images to remove background artefacts (
[0411] FTIR
[0412] Infra-red spectra of both the liquid crystalline spinning dope and the spun fibre were recorded on a Perkin Elmer Frontier FTIR instrument with a diamond ATR head with a resolution of 4 cm.sup.1 and 10 acquisitions. The spectra were taken to compare the secondary structure of the solenoid before (
[0413] Polarized Raman Spectroscopy
[0414] Polarised Raman spectroscopy was performed to identify the different amide regions of the fibre (which support the FTIR secondary structure findings) and polarising the monochromatic light can elucidate structural motifs of the fibre and potentially the orientation of the solenoids in the fibre. Raman data obtained with three different polarization configurations, IVV, IVH, and IHH, where V and H refer to orientation parallel and perpendicular, respectively (
Example 7
[0415] Test Expressions of New Concatemer Variants
[0416] Codon optimised genes encoding the proteins SynRFR24.nnum35.x4, SynRFR24.nnum36.x4, SynRFR24.nnum40.x4 were synthesised by GeneArt with flanking T7 promoter and T7 terminator sequences in the vector backbone pMK-RQ (kanR). These plasmids were transformed into the KRX E. coli strain. 100 uL of 5 ml LB overnight cultures (all media were supplemented with appropriate antibiotics) were used to inoculate 5 mL of fresh LB medium. This fresh culture was grown for 3 hours at 37 degrees C. before being induced with 0.1% filter sterilised rhamnose and grown overnight at 25 degrees C. Non-induced cultures were also grown in the same way for comparison. Cells were harvested in the morning and resuspended in 100 uL of lysis buffer (100 mM bicine and 150 mM NaCl buffer titrated to pH 9.0 with NaOH) then sonicated and clarified by centrifugation at 20,000g for 10 min in a benchtop mini-centrifuge. SDS-PAGE gels of the soluble and insoluble fractions showed significant protein over-expression bands for all proteins in the soluble fraction. See
[0417] Purification of SynRFR24.Nnum35.x4 Variant
[0418] Larger scale (3 litres of TB medium) expression and purification of SynRFR24.nnum35.x4 was carried out in an identical fashion as described for SynRFR24.x4.1 (three-step purification: Ni-NTA, anion exchange, gel filtration as described in the section: Optimising the expression and purification of SynRFR24.x4.1 in Example 3.
[0419] SynRFR24.nnum35.x4 was purified to high purity and quantity (200 mg); see
[0420] Summary of Data to Date
[0421] Data shows that it is possible to design a wide range of different SynRFR protein domains that show similar CD spectra (
[0422] The SynRFR24.nc.x.4.1 concatemer has been consistently spun into strong fibres, and the concentration range for liquid crystalline dopes has been investigated thoroughly. The fibres are being spun from dope solutions at concentrations at which liquid crystalline mesophases are formed, and the polymers are strongly aligned in the fibre (shown using fibre WAXS (