Prokaryotic expression system and methods of using the same

Abstract

To provide improved compositions and methods for producing synthetic spider silk with mechanical properties similar to natural spider silk.SOLUTION: The present invention provides a recombinant bacterium having the ability to produce a MaSp based protein organized in b-sheet crystalline structures, methods of producing MaSp-based polymers using the same, and compositions comprising the same. According to one aspect, the present invention provides a composition comprising a synthetic major ampullate spidroin protein (MaSp)-based polymer in the form of particles having a size in a range of 0.5 μm to 1.5 μm. In some embodiments, the MaSp-based polymer is a water insoluble polymer.SELECTED DRAWING: Figure 21A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 870,750, entitled "PROKARYOTIC EXPRESSION SYSTEM AND METHODS OF USING THE SAME," filed July 4, 2019, the contents of which are incorporated herein by reference in their entirety.

[0002] The present invention relates to synthetic dragline spider silk polymers. [Background technology]

[0003] Dragline spider silk is known in the art as the silk used by orb-web weaving spiders to construct web frames and radii and lifelines when they fall or escape danger. To enable them to perform these tasks, dragline fibers exhibit exceptionally high toughness, a combination of high elasticity and strength that ranks them among the toughest fibers, natural or man-made. For example, dragline diameters are six times stronger than high-tensile steel and three times stronger than Kevlar, one of the strongest synthetic fibers ever produced.

[0004] Dragline silk consists of two major polypeptides, often referred to as major ampullate spidroins (MaSp) 1 and 2, and also referred to as ADF-3 and ADF-4 in Araneus diadematus. These proteins have apparent molecular weights ranging from 200 to 720 kDa, depending on the age of the sample and analytical conditions. Known dragline silk spidroins consist of alternating alanine-rich segments that form crystalline β-sheets in the fibers, and glycine-rich segments that are more flexible and largely lack ordered structure. The C-terminal region is non-repetitive, highly conserved across species, and adopts an α-helical conformation. The N-terminal region of dragline silk proteins has been found to be highly conserved among different spidroins and even among different spider species.

[0005] Many attempts have been made to synthetically produce spider silk, including by genetic engineering using bacteria, yeast, plant, and mammalian cells in tissue culture, and even transgenic goats.

[0006] U.S. Patent No. 8,461,301 relates to isolated amino acid sequences, particularly those containing multiple repeats of semisynthetic spider silk protein domains, or any functional homologs, variants, derivatives, fragments, or mutants thereof. Additional publications related to dragline spider silk include, but are not limited to, Ittah, S., et al., Biopolymers, 93(5), 458-468, 2010; Ittah, S., et al., Biomacromolecules, 8(9), 2768-2773, 2007; Ittah, S., et al., Biomacromolecules, 7(6), 1790-1795, 2006; and Huemmerich, D., Ittah, S., et al., Current Biology, 14, 2070-2074, 2004.

[0007] There is an unmet need for improved compositions and methods for producing synthetic spider silk with mechanical properties similar to natural spider silk. Summary of the Invention

[0008] The present invention relates to recombinant bacteria capable of producing MaSp-based proteins organized into β-sheet crystalline structures, methods of using same to produce MaSp-based polymers, and compositions comprising MaSp-based polymers.

[0009] According to one aspect, the present invention provides a composition comprising a synthetic major ampullate spidroin protein (MaSp)-based polymer in the form of particles having a size ranging from 0.5 μm to 1.5 μm.

[0010] In some embodiments, the MaSp-based polymer is a water-insoluble polymer.

[0011] In some embodiments, the composition has a DSC pattern that exhibits at least one endothermic peak in the range of 200°C to 280°C.

[0012] In some embodiments, the particles are porous particles and are characterized by a BET surface area of ​​at least 10 m2 / g.

[0013] In some embodiments, the particles comprise a plurality of nanofibrils.

[0014] In some embodiments, the composition further comprises an additional compound in contact with the MaSp-based polymer.

[0015] In some embodiments, the compound is selected from a biologically active agent and a functional food.

[0016] In some embodiments, the weight / weight (w / w) ratio of the MaSp-based polymer to the additional compound is 10:1 to 1:10.

[0017] In some embodiments, the MaSp-based polymer comprises the amino acid sequence set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS).

[0018] In some embodiments, the MaSp-based polymer comprises the amino acid sequence set forth in SEQ ID NO: 3 (AAAAAAAASGPGGYGPGSQGPSGPGGYGPGGPGSS).

[0019] In some embodiments, the MaSp-based polymer comprises 10-20 repeats of SEQ ID NO:3.

[0020] In some embodiments, the MaSp-based polymer comprises a single N-terminal region selected from the group consisting of SEQ ID NO: 4 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLV).

[0021] In some embodiments, the MaSp-based polymer comprises a single C-terminal region selected from the group consisting of SEQ ID NO: 7 (VAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS).

[0022] In some embodiments, the composition is obtained by expression of MaSp in bacteria.

[0023] In some embodiments, the bacterium is Escherichia coli.

[0024] In another embodiment, there is a composition comprising a MaSp-based polymer of the present invention conjugated to an additional polymer, wherein the w / w ratio of said MaSp-based polymer to said additional polymer is from 1:1 to 1:100.

[0025] In some embodiments, the w / w concentration of the additional polymer is 50% to 95% (w / w) of the total composition.

[0026] In some embodiments, the additional polymer is selected from synthetic polymers, thermoplastic polymers, thermosets, film formers, epoxies, polyesters, polyamides, polyols, polyurethanes, polyethylene, silicones, liquid crystal polymers, maleic anhydride grafted polypropylene, polyacrylates, polycarbonates, polyamides, nylon 4,6, nylon 6, nylon 6,6, nylon 11, nylon 12, poly(arylamides), polyethylene, polybutylene terephthalate, polyethylene terephthalate, polyphenylene sulfide, polyphthalamide, polypropylene, poly(vinylidene fluoride), poly(2-hydroxyethyl methacrylate) (pHEMA), polyurethanes, polyvinyl butyral, ethylene vinyl alcohol copolymers, polylactide acid (PLA) or copolymers thereof, polycaprolactone (PCL), xanthan, cellulose, collagen, elastin, keratin, cotton, rubber, cellulose, wool, and any combination thereof.

[0027] In some embodiments, the film-forming agent is a solid film-forming agent.

[0028] In some embodiments, the w / w ratio between the film-forming agent and the MaSp-based fibers is between 5:1 and 50:1.

[0029] In some embodiments, the composition is characterized by at least one improved mechanical property compared to the properties of the additional polymer without the MaSp-based polymer, the property being selected from the group consisting of Young's modulus, tensile strength, strain at break, yield point, toughness, work to failure, impact strength, tear strength, flexural modulus, flexural strain and stress at a specific elongation, and wear.

[0030] According to another aspect, the present invention provides an article comprising the composition described herein, in the form of a film, suture, surgical mesh, medical adhesive strip, electrospun mesh, skin graft, fat graft, cosmetic, dermal filler, drug eluting / delivery device, replacement ligament, clothing fabric, bulletproof vest lining, cable, tubing, film, rope, fishing line, tire, sporting goods, and reinforced plastic.

[0031] According to another aspect, the present invention provides a recombinant bacterium capable of expressing a MaSP-based polymer comprising the amino acid sequence set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS).

[0032] According to another aspect, the present invention provides a process comprising: (i) providing a recombinant bacterium as described herein; (ii) providing conditions for expression of MaSP by the bacterium; and (iii) isolating the expressed protein, thereby producing synthetic dragline spider silk.

[0033] In some embodiments, step (ii) comprises providing a solution having a pH in the range of 5 to 6.5.

[0034] In some embodiments, step (ii) comprises providing an inducer.

[0035] In some embodiments, step (ii) comprises waiting a period of time to obtain an insoluble polymer.

[0036] In some embodiments, step (iii) further comprises drying the synthetic dragline spider silk.

[0037] In some embodiments, the process further comprises a toughening step with an additional polymer.

[0038] In some embodiments, the strengthening step comprises mixing a solution of synthetic dragline spider silk with a solution of a second polymer.

[0039] Further embodiments and the full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0040] [Figures 1A-1C] UV spectrum of the obtained protein (Figure 1A), a graph representing measured vs. predicted amino acid content (Figure 1B), and Fourier transform infrared spectroscopy (FTIR) spectrum of the obtained protein (SVX-E) (Figure 1C). [Figure 2] Graph showing particle size distribution of proteins obtained using the described expression system. [Figure 3] FTIR analysis of the obtained particles. [Figure 4] FTIR spectra of spider silk protein (SVX-E) expressed in bacteria versus spider silk SVX expressed in Sf9 cells. [Figure 5] FTIR spectrum of spider silk protein expressed in bacteria (SVX-E) compared to spider silk (SVX) and silk fibers expressed in Sf9 cells. [Figures 6A-6C] Differential scanning calorimetry (DSC) curves of the spider silk protein SVX-E expressed in bacteria (FIGS. 6A and 6B) and the DSC curve of the SVX protein (FIG. 6C). [Figures 7A-7B] Transmission electron microscopy image of SVXE (Fig. 7B) compared with Sf9-derived SVX (Fig. 7A). [Figure 8A-8B]Graph of the dose-dependent effect of SVXE on the mechanical properties of polymer-P490RSJT composites. Figure 8A shows a bar graph illustrating the increase in stress (10-20%) at 500% modulus for P490RSJT reinforced with 10, 20, and 30% SVXE compared to the control (pristine P490RSJT polymer). Figure 8B shows the stress-strain curves for P490RSJT reinforced with 5, 10, 20, and 30% SVXE compared to the control (pristine P490RSJT polymer). [Figures 9A-9E] Graphs of the dose-dependent effect of SVXE on the mechanical properties of polymer-E394POTA composites. Figure 9A is a bar graph showing the increase in Young's modulus (approximately 50% to approximately 400% increase) for E394POTA reinforced with 10, 20, and 30% SVXE compared to the control (pristine E394POTA polymer). Figures 9B-D are bar graphs showing the decrease in UTS (Figure 9B), elongation at break % (Figure 9C), and toughness (Figure 9D) for E394POTA reinforced with 10, 20, and 30% SVXE compared to the control (pristine E394POTA polymer). Figure 9E shows the stress-strain curves for E394POTA reinforced with 10, 20, and 30% SVXE compared to the control (pristine E394POTA polymer). [Figures 10A-10E] Graphs comparing the mechanical properties of polymer P490RSJT PU composites reinforced with 20% SVX, crushed SVX, and SVX-E. Figure 10A is a bar graph showing the increase in Young's modulus (approximately 150% to approximately 300%) of the reinforced P490RSJT PU compared to the control (pristine P490RSJT PU polymer). Figures 10B-D are bar graphs showing the decrease in UTS (Figure 10B), elongation at break % (Figure 10C), and toughness (Figure 10D) of P490RSJT PU reinforced with 10, 20, and 30% SVXE compared to the control (pristine P490RSJT PU polymer). Figure 10E shows the stress-strain curves of P490RSJT PU reinforced with 20% SVX, crushed SVX, and SVX-E compared to the control (pristine E394POTA polymer). [Figures 11A-11E]Graph showing a comparison of the mechanical properties of polymer E394POTA PU and composites reinforced with 20% SVX, crushed SVX, and SVX-E, respectively. [Figure 12] Bar graph showing the size of SVX-E particles in aqueous suspension as measured by laser diffraction. *Refers to the average size of isolated SVX-E particles. **Refers to the average size of porous or non-aggregated SVX-E particles after drying and resuspension. ***Refers to the average size of "non-porous" or aggregated SVX-E particles after drying and resuspension. [Figure 13] Graph showing comparative stress-strain curves of PU samples reinforced with 15% w / w SVX-E. The graph shows the stress-strain behavior of PU samples reinforced with non-porous or agglomerated particles (SVX agglomerated), porous or non-agglomerated particles (SVX fully dispersed) compared to the control PU sample (solid). [Figure 14] Table summarizing the mechanical properties of film formers reinforced with 10% w / w SVX-E. [Figure 15] Graph of SVX-E levels in E. coli after 30 hours of incubation. SVX-E was determined using a specific beta-sheet crystal-binding fluorescent dye. [Figure 16] Photographs of SVX-E staining at different incubation times at pH 5.8 and pH 7.5. The green spots are due to specific beta-crystal-bound fluorescent dyes entering the cells. [Figure 17] Graph of growth rate and beta-sheet crystal staining difference over time at pH 5.8. [Figure 18] Graph of absolute growth curve over time at pH 5.8. [Figure 19] Graph of growth rate and beta-sheet crystal staining difference over time at pH 7.5. [Figure 20] Graph showing the release of hyaluronic acid (HA) from SVX-E fibers compared to cellulose and silk. The graph shows the calculated ratio between the HA-specific peak and the SVX-E / silk / cellulose-specific peak (Y-axis) and the number of washes (X-axis). [Figures 21A-21E]Scanning electron micrographs (SEM) of MaSp-based (SVX) fibers. Figures 21A and 21B show images of porous fibers. Figures 21C and 21D show images of non-porous particles. Figure 21E shows fibers resulting from expression of a different sequence (SEQ ID NO: 10). DETAILED DESCRIPTION OF THE INVENTION

[0041] In some embodiments, provided herein are bacteria expressing compositions comprising major ampullate spidroin protein (MaSp)-based proteins useful for preparing synthetic dragline spider silk. The invention further provides articles and composites comprising these compositions.

[0042] The present invention is based in part on the unexpected discovery that microorganisms such as E. coli are unexpectedly efficient hosts for the production of MaSp proteins. MaSp proteins are not only expressed in E. coli, but also self-aggregate to form functional spider silk within the bacteria. Surprisingly, this self-aggregation occurs only at an external pH <7, but not at a neutral pH of 7-7.5.

[0043] The present invention is further based in part on the unexpected discovery that the microbially produced MaSp protein (also used herein as SVX-E) unexpectedly possesses exceptional mechanical properties compared to natural dragline spider silk or compared to production of dragline spider silk proteins in other expression systems.

[0044] The present invention is further based, in part, on the unexpected discovery that SVX-E-based fibers are in the form of particles having sizes ranging from 0.5 μm to 1.5 μm. As illustrated herein (e.g., in FIG. 12), these particles remain stable (e.g., substantially free of agglomerates) in aqueous dispersions, in contrast to control MaSp-based polymers. Furthermore, SVX-E-based fibers exhibit increased porosity and very large BET surface areas (e.g., approximately 180 m). 2 / g).

[0045] composition According to some embodiments, a composition is provided comprising a synthetic major ampullate spidroin protein (MaSp)-based polymer in the form of particles having a size ranging from 0.5 μm to 1.5 μm. In some embodiments, the MaSp-based polymer is an insoluble polymer. In some embodiments, the composition has a DSC pattern exhibiting at least one endothermic peak in the range of 200° C. to 280° C. In some embodiments, the composition has a DSC pattern exhibiting at least one endothermic peak at 1615 cm as measured by FTIR analysis. -1 ~1635cm -1 The amide peak ranges from 0.1 to 1.0.

[0046] According to some embodiments, a composition is provided comprising a synthetic MaSp-based polymer, wherein the MaSp-based polymer has at least one property selected from the following: a) being an insoluble polymer; b) in the form of particles having a size in the range of 0.5 μm to 1.5 μm; c) At least 10m 2 / g BET surface area, d) having a DSC pattern showing at least one endothermic peak in the range of 200°C to 280°C; and e) 1615 cm as measured by FTIR analysis -1 ~1638cm -1 The amide peak should be within the range of

[0047] According to some embodiments, a composition comprising a synthetic MaSp-based polymer in the form of particles is provided. In some embodiments, the particles have a size in the range of 0.5 μm to 1.5 μm, 0.7 μm to 1.5 μm, 0.8 μm to 1.5 μm, 0.9 μm to 1.5 μm, 0.5 μm to 1 μm, 0.7 μm to 1 μm, 0.8 μm to 1 μm, 0.9 μm to 1 μm, 0.5 μm to 1.3 μm, 0.5 μm to 1.2 μm, 0.7 μm to 1.3 μm, 0.7 μm to 1.2 μm, or 0.9 μm to 1.2 μm (including any range therebetween). In some embodiments, particle size refers to the average particle size in an aqueous medium (e.g., an aqueous dispersion) as measured by laser diffraction (see the Examples section). In some embodiments, particle size refers to the dry particle size (e.g., the size of particles substantially lacking an outer shell containing water molecules).

[0048] In some embodiments, a composition comprising an insoluble MaSp-based polymer is provided. In some embodiments, the insoluble MaSp-based polymer is in the form of particles. In some embodiments, the insoluble MaSp-based polymer is insoluble in organic solvents. In some embodiments, the insoluble MaSp-based polymer is insoluble in aqueous solutions.

[0049] As used herein, the term "insoluble" refers to a material that does not dissolve when exposed to an excess of solvent, but may disperse to varying degrees. In some embodiments, the term "insoluble" refers to a material that is less than 10%, less than 5%, less than 2%, or less than 1% soluble in the solvent. In some embodiments, "insoluble" refers to a material that can only be partially dissolved in the solvent at a concentration of less than 0.01% by weight. Solvents according to the present invention include organic solvents and aqueous solutions. In some embodiments, the solvent comprises an aqueous surfactant solution. Surfactants (e.g., ionic surfactants) are well known in the art. In some embodiments, the solvent comprises an aqueous urea solution.

[0050] In some embodiments, compositions of the present disclosure are characterized by a defined differential scanning calorimetry (DSC) pattern. In some embodiments, "DSC pattern" refers to the location of a peak. In some embodiments, "peak" refers to an exothermic peak. Throughout this specification, "peak location" or "peak position" refers to a peak along the temperature axis of a thermogram pattern, and in some embodiments, may refer to the peak position at any peak intensity. Those skilled in the art will understand that the data obtained in a DSC measurement will depend, in part, on the instrument used and the environmental conditions (e.g., humidity) when the measurement is performed.

[0051] In some embodiments, compositions of the present disclosure are characterized by a DSC pattern exhibiting at least one endothermic peak in the range of 200° C. to 280° C. In some embodiments, compositions of the present disclosure are characterized by a DSC pattern exhibiting at least one endothermic peak in the range of 200° C. to 270° C., 200° C. to 260° C., 200° C. to 250° C., 210° C. to 280° C., 212° C. to 280° C., 215° C. to 280° C., 216° C. to 280° C., 220° C. to 280° C., 210° C. to 250° C., 212° C. to 250° C., 215° C. to 250° C., 216° C. to 250° C., 220° C. to 250° C., 210° C. to 245° C., 210° C. to 242° C., or 215° C. to 245° C. (including any range therebetween).

[0052] In some embodiments, compositions of the present disclosure are characterized by a DSC pattern exhibiting at least one endothermic peak that is at least 5°C to 100°C, at least 10°C to 100°C, at least 15°C to 100°C, at least 12°C to 100°C, at least 25°C to 100°C, at least 5°C to 80°C, at least 10°C to 80°C, at least 15°C to 80°C, at least 12°C to 80°C, at least 25°C to 80°C, at least 5°C to 50°C, at least 10°C to 50°C, at least 15°C to 50°C, at least 12°C to 50°C, or at least 25°C to 50°C lower than a DSC pattern of a corresponding composition comprising a (MaSp)-based fiber.

[0053] In some embodiments, the compositions of the present disclosure lack a DSC peak in the range of about -100°C to about 190°C. In some embodiments, the compounds of the present disclosure lack a DSC peak in the range of about -100°C to about 25°C. In some embodiments, the compositions of the present disclosure are characterized by a DSC pattern lacking an exothermic peak in the range of at least 40°C to 70°C.

[0054] In some embodiments, the compositions of the present disclosure lack a DSC peak in the range of about -100°C to about -50°C. In some embodiments, the compounds of the present disclosure lack a DSC peak in the range of about -50°C to about 0°C. In some embodiments, the compounds of the present disclosure lack a DSC peak in the range of about -0°C to about -25°C.

[0055] In some embodiments, the compositions of the present disclosure have a spectral reflectance of 1615 cm as measured by FTIR analysis. -1 ~1635cm -1 In some embodiments, the compositions of the present disclosure are characterized by having an amide peak in the range of 1620 cm as measured by FTIR analysis. -1 ~1635cm -1 , 1620cm -1 ~1630cm -1 , 1621cm -1 ~1630cm -1 , or 1620cm -1 ~1625cm -1 (including any range therebetween).

[0056] In some embodiments, the compositions of the present disclosure have a spectral reflectance of 1700 cm as measured by FTIR analysis. -1 ~1800cm -1 There is no peak in the range.

[0057] According to one embodiment, the MaSp-based polymers of the present invention assemble by self-aggregation. By "self-aggregation" is meant that the monomers, i.e., the synthetic spider silk proteins of the present invention, spontaneously combine with each other in an energetically favorable manner under normal physiological conditions or at room temperature to create a macromolecular structure having the properties described herein. Furthermore, the MaSp-based polymers of the present invention are highly resilient and, once assembled, can withstand extreme chemical attack, such as solubilization in a 10% w / w detergent solution and boiling for at least 1 hour.

[0058] "Tenacity" or "tensile strength" refers to the weight a filament can withstand before breaking. The maximum specific stress generated is typically determined by a tensile test on a filament, yarn, or fabric to break the material. According to certain embodiments, the MaSp-based polymers of the present invention have a tensile strength of about 100-3000 MPa (MPa = N / mm2), about 300-3000 MPa, about 500-2700 MPa, about 700-2500 MPa, about 900-2300 MPa, about 1100-2000 MPa, about 1200-1800 MPa, about 1300-1700 MPa, or about 1400-1600 MPa. More specifically, about 1500 MPa.

[0059] "Toughness" refers to the energy required to break a MaSp-based polymer. This is the area under the stress-strain curve and is sometimes called the "energy to break" or work to rupture. According to certain embodiments, the MaSp-based polymers of the present invention have a toughness of about 20-1000 MJ / m, about 50-950 MJ / m, about 100-900 MJ / m, about 120-850 MJ / m, about 150-800 MJ / m, about 180-700 MJ / m, about 180-750 MJ / m, about 250-700 MJ / m, about 280-600 MJ / m, about 300-580 MJ / m, about 310-560 MJ / m, about 320-540 MJ / m, or about 350-520 MJ / m, most particularly about 350-520 MJ / m.

[0060] "Elasticity" refers to the property of an object that tends to return to its original size and shape after deformation. Plasticity, or the inability to return to its original shape after deformation, is the opposite of elasticity. The molecular configuration of MaSp-based polymers allows for reversible or elastic deformation by stretching (reorienting) the structural bonds between atoms and molecules. Conversely, irreversible or plastic deformation occurs by breaking the intermolecular bonds and reforming them in new, stable positions.

[0061] "Elongation" refers to an increase in length expressed as a percentage or fraction of the original length.

[0062] "Fineness" refers to the average diameter of a MaSp-based polymer or filament (eg, biofilament), which is usually expressed in microns (micrometers).

[0063] According to some aspects, MaSp-based proteins or MaSp-based polymers, used interchangeably herein, are in the form of fibers. As used herein, "fiber" refers to a thin cord of fibrous material composed of two or more filaments twisted together. "Filament" refers to a thin, elongated, thread-like object or structure, ranging in length from microscopic to over a mile. Specifically, synthetic spider silk filaments are microscopic and proteinaceous. "Biofilaments" refer to filaments made from proteins, including recombinantly produced spider silk proteins. In some embodiments, the term "fiber" does not include unstructured aggregates or precipitates.

[0064] In some embodiments, the MaSp-based fiber comprises a plurality of MaSp-based polymers. In some embodiments, the plurality of MaSp-based polymers comprises polymers having different chemical compositions and / or different molecular weights (MW). In some embodiments, the plurality of MaSp-based polymers comprises polymers having different numbers of repeat regions.

[0065] In some embodiments, the MaSp-based polymer or MaSp-based fiber is substantially devoid of additional non-MaSp-based proteins, hi one embodiment, the MaSp-based polymer or MaSp-based fiber is substantially devoid of additional polymers (e.g., synthetic polymers, non-MaSp-based peptides, non-MaSp-based proteins).

[0066] In some embodiments, the protein fibers are characterized by the size of at least one dimension (e.g., diameter, length). For example, but not limited to, the fiber diameter is 10 nm to 1 μm, 20 to 100 nm, or 10 to 50 nm.

[0067] In one embodiment, the MaSp-based fiber is composed of a monomer. In one embodiment, a plurality of MaSp-based polymers are arranged in nanofibrils. In one embodiment, a plurality of nanofibrils are arranged in or comprise a fiber. In one embodiment, the monomers or nanofibrils in the MaSp-based fiber have a diameter of 4-16 nm. In one embodiment, the monomers or nanofibrils in the MaSp-based fiber have a diameter of 6-14 nm. In one embodiment, the monomers or nanofibrils in the MaSp-based fiber have a diameter of 8-12 nm. In some embodiments, the MaSp-based fiber comprises a plurality of fibrils (e.g., nanofibrils), as illustrated below (Figures 21A-B). In some embodiments, fibers having a mutant amino acid sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 10 (MSYYHHHHHHDYDIPTTENLYFQGAMPRKSPFPRPEL)) are substantially devoid of nanofibrils, as illustrated in FIG. 21E. In some embodiments, fibers having a mutant amino acid sequence (e.g., the amino acid sequence set forth in SEQ ID NO: 10 (MSYYHHHHHHDYDIPTTENLYFQGAMPRKSPFPRPEL)) are in the form of non-porous particles, as illustrated in FIG. 21E.

[0068] In some embodiments, the nanofibrils have a diameter of, for example, 1 nm, about 2 nm, about 3 nm, about 4 nm, about 5 nm, about 6 nm, about 7 nm, about 8 nm, about 9 nm, about 10 nm, about 11 nm, about 12 nm, about 13 nm, about 14 nm, about 15 nm, about 16 nm, about 17 nm, about 18 nm, about 19 nm, about 20 nm, about 21 nm, about 22 nm, about 23 nm, about 24 nm, about 25 nm, about 26 nm, about 27 nm, about 28 nm, about 29 nm, about 30 nm, about 31 nm, about 32 nm, about 33 nm, about 34 nm, about 35 nm, about 36 nm, about 37 nm, about 38 nm, about 40 nm, about 42 nm, about 44 nm, about 46 nm, about 48 nm, or about 50 nm (including any value or range therebetween). In one embodiment, the nanofibrils have a diameter of 3 to 7 nm, hi one embodiment, the nanofibrils have a diameter of 4 to 6 nm.

[0069] In one embodiment, the MaSp-based fiber has a diameter of 70 to 450 nm. In one embodiment, the MaSp-based fiber has a diameter of 80 to 350 nm. In one embodiment, the MaSp-based fiber has a diameter of 80 to 300 nm. In one embodiment, the MaSp-based fiber has a diameter of 150 to 250 nm. In one embodiment, the MaSp-based fiber or MaSp-based polymer is arranged as a coil. In one embodiment, a single fiber or a single MaSp-based polymer is arranged as a coil. In one embodiment, the coil has a diameter of 5 to 800 micrometers. In one embodiment, the coil has a diameter of 5 to 500 micrometers. In one embodiment, the coil has a diameter of 5 to 30 micrometers. In one embodiment, the coil has a diameter of 5 to 20 micrometers. In one embodiment, the MaSp-based fiber or MaSp-based polymer has a length of 5 to 800 micrometers. In one embodiment, the MaSp-based fiber or MaSp-based polymer has a length of 30 to 300 micrometers. In some embodiments, the length of the MaSp-based fibers is 1-200 μm, 10-100 μm, 100-500 μm, or 200-500 μm (including any range therebetween).

[0070] In some embodiments, the MaSp-based fiber comprises a plurality of pores. In some embodiments, the MaSp-based fiber is in the form of a particle as described herein. In some embodiments, the composition comprises a plurality of MaSp-based fibers. In some embodiments, the plurality of MaSp-based fibers comprises fibers having different chemical compositions. In some embodiments, the plurality of MaSp-based fibers is in the form of particles having different sizes and / or different structures. In some embodiments, the plurality of MaSp-based fibers is in the form of particles having different porosities (represented by BET surface areas).

[0071] In some embodiments, the MaSp-based fibers are characterized by a porous structure. In some embodiments, the MaSp-based fibers are porous fibers. In some embodiments, the porous structure or porous MaSp-based fibers are characterized by a porosity of at least 30% (e.g., 30-99%). In some embodiments, the porous structure is characterized by a porosity of at least 50% (e.g., 50-99%). In some embodiments, the porous structure is characterized by a porosity of at least 60% (e.g., 60-99%). In some embodiments, the porous structure is characterized by a porosity of at least 70% (e.g., 70-99%). In some embodiments, the porous structure is characterized by a porosity of at least 80% (e.g., 80-99%). In some embodiments, the porous structure is characterized by a porosity of at least 90% (e.g., 90-99%). In some embodiments, the porous structure is characterized by a porosity of about 90%.

[0072] As used herein, the term "porosity" refers to the percentage of a volume of a material that consists of voids (e.g., a "sponge-like" material). In another embodiment, porosity is measured by dividing the voids within the surface area by the total surface area (porous and non-porous).

[0073] In some embodiments, the porous structure of the disclosed fibers allows for efficient water absorption at the fiber surface. Without being bound by any particular theory, this surprising finding can be explained in view of the disclosed fiber structure and its porosity, which is distinct from native spider silk found in nature.

[0074] In some embodiments, the porous MaSp-based fibers are in the form of particles as described herein. In some embodiments, the particles are porous particles. In some embodiments, the particles are substantially non-agglomerated particles. In some embodiments, the particles comprise a plurality of pores (i.e., spaces or cavities) formed by entangled polymer chains of the MaSp-based polymer. In some embodiments, the entangled or entangled MaSp-based polymer forms a matrix. In some embodiments, the cosmetic active fills at least a portion of the pores within the matrix or particle. In some embodiments, the cosmetic active is encapsulated by the plurality of pores.

[0075] In some embodiments, the MaSp-based fiber has a length of at least 10 m. 2 / g, at least 20m 2 / g, at least 30m 2 / g, at least 40m 2 / g, at least 50m 2 / g, at least 60m 2 / g, at least 70m 2 / g, at least 80m 2 / g, at least 100m 2 / g, at least 130m 2 / g, at least 150m 2 / g, at least 160m 2 / g, at least 170m 2 / g, at least 180m 2 / g (including any range or value therebetween).

[0076] In some embodiments, the porous MaSp-based fibers have a thickness of 10 to 200 mm. 2 / g, 10-50m 2 / g, 10-20m 2 / g, 20-50m 2 / g, 50-70m 2 / g, 70-100m 2 / g, 100-120m 2 / g, 120-150m 2 / g, 150-170m 2 / g, 170-190m 2 / g, 150-190m 2 / g, 160-190m 2 / g, 170-190m 2 / g, 100-190m 2 / g, 180-190m 2 / g, 170-180m 2 / g, 180-200m 2 / g, 190-200m 2 / g (including any range or value therebetween). In some embodiments, the porous MaSp-based fibers are characterized by a BET surface area of ​​100-200 m 2 / g, 150-200m 2 / g (including any range or value therebetween).

[0077] In some embodiments, the MaSp-based fibers are in the form of non-porous MaSp-based fibers or non-porous particles. In some embodiments, the MaSp-based fibers are in the form of agglomerated particles. In some embodiments, the non-porous MaSp-based fibers are up to 10 m 2 / g, up to 8m 2 / g, up to 6m 2 / g, up to 5m 2 / g, up to 3m 2 / g, up to 2m 2 / g, up to 1m 2 / g, max. 0.5m 2 / g, max. 0.3m 2 / g, max. 0.1m 2 / g (including any range or value therebetween). In some embodiments, the non-porous MaSp-based fibers or particles have a BET surface area of ​​0.01 to 1 m 2 It is characterized by a BET surface area in g / g.

[0078] Particles characterized by a large surface area (e.g., porous particles) are more easily absorbed than non-porous particles (e.g., 10 m 2 It should be appreciated that the porous or non-agglomerated particles have an increased encapsulation capacity compared to particles with a BET surface area less than 1 / g. Comparative SEM images of porous or non-agglomerated particles and non-porous or agglomerated particles are shown in Figures 21A-D.

[0079] In some embodiments, the terms "porous particles" and "non-agglomerated particles" are used interchangeably herein. In some embodiments, the terms "non-porous particles" and "agglomerated particles" are used interchangeably herein.

[0080] In some embodiments, the porous particles are stable in the dispersion. In some embodiments, the porous particles are significantly more stable in the dispersion compared to non-porous particles. In some embodiments, the dispersion is an aqueous dispersion. In some embodiments, the dried porous particles substantially maintain their size upon redispersion in an aqueous solution. In some embodiments, the porous particles lack cohesion in the aqueous dispersion. In some embodiments, the porous particles lack agglomerates in the aqueous dispersion.

[0081] In some embodiments, the non-porous particles are characterized by a particle size of greater than 10 μm, greater than 20 μm, greater than 30 μm, greater than 40 μm, greater than 50 μm, greater than 60 μm, greater than 70 μm, greater than 80 μm or greater (including any range therebetween). In some embodiments, the particle size is as described herein. In some embodiments, the non-porous particles are in the form of aggregates or agglomerates in aqueous solution. In some embodiments, the non-porous particles (e.g., dried non-porous particles) form aggregates upon redispersion in aqueous solution.

[0082] As illustrated below (FIG. 12), dry non-porous (or agglomerated) particles tend to form aggregates upon redispersion in aqueous solution. Such aggregates can have particle sizes of tens of micrometers (up to 100 μm). Without being bound to a particular theory or mechanism of action, it is hypothesized that the exceptional stability of porous particles in dispersion may be related to increased interaction with the aqueous medium due to the increased surface area of ​​the porous particles. Those skilled in the art will understand that the stability of porous particles in dispersion may be advantageous in terms of the shelf life of the composition, the maximum concentration of particles in the composition (e.g., aqueous dispersion), and / or the loading capacity of the composition (e.g., a composition including an active agent incorporated within or encapsulated by multiple particles). Furthermore, it is hypothesized that the porous particles may be formed exclusively by any one of the bacterial expression systems described herein.

[0083] In some embodiments, the porous particles have a size in the range of 0.5 μm to 1.5 μm, 0.7 μm to 1.5 μm, 0.8 μm to 1.5 μm, 0.9 μm to 1.5 μm, 0.5 μm to 1 μm, 0.7 μm to 1 μm, 0.8 μm to 1 μm, 0.9 μm to 1 μm, 0.5 μm to 1.3 μm, 0.5 μm to 1.2 μm, 0.7 μm to 1.3 μm, 0.7 μm to 1.2 μm, or 0.9 μm to 1.2 μm (including any range therebetween). In some embodiments, the size or particle size is as described above.

[0084] In some embodiments, a material (e.g., a polymeric material) rich in porous particles is characterized by at least one improved mechanical property compared to a material rich in non-porous (or agglomerated) particles (see FIG. 13). In some embodiments, the mechanical property is as described herein. Without being bound by a particular theory or mechanism of action, it is hypothesized that porous particles are more advantageous than non-porous particles for material reinforcement with MaSp-based fibers (e.g., polymers reinforced with MaSp-based fibers, as described below). It is hypothesized that materials reinforced with porous (or non-agglomerated) particles have a higher enrichment rate compared to non-porous (or agglomerated) particles, thereby enhancing the reinforcement performance of MaSp-based fibers.

[0085] Enclosed In some embodiments, the composition further comprises an additional compound in contact with the MaSp-based polymer or MaSp-based fiber. In some embodiments, the additional compound is bound to the MaSp-based polymer or MaSp-based fiber. In some embodiments, the composition comprises an additional compound substantially bound to the porous MaSp-based fiber by non-covalent bonding, physical interactions, or both. In some embodiments, the cosmetic active fills at least a portion of the pores within the matrix or particles, the matrix being formed by the intertwined polymer chains of the MaSp-based polymer. In some embodiments, the additional compound is encapsulated by the pores.

[0086] In some embodiments, porous particles are characterized by an increased encapsulation capacity compared to non-porous particles. In some embodiments, encapsulation capacity refers to the ability of the particles to incorporate additional compounds. In some embodiments, the increased encapsulation capacity is related to the large surface area of ​​the particles, as described above.

[0087] In some embodiments, the additional compound is stably encapsulated within the plurality of pores of the particle. In some embodiments, the encapsulated additional compound is characterized by a sustained release profile (e.g., at the site of application in solution or dispersion). In some embodiments, the particle encapsulating the additional compound substantially prevents rapid release of the additional compound from the particle.

[0088] As used herein, the term "stably encapsulated" refers to the ability of a composition to substantially prevent the release of an active ingredient (e.g., an additional compound) from the composition. As used herein, the term "substantially prevent" refers to the total amount of active ingredient released from the composition, such as upon subsequent washing (as described in the Examples section).

[0089] In some embodiments, substantially comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% by weight of the additional compound. In some embodiments, substantially comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% by weight of the additional compound, which is bonded to the MaSp-based fiber by non-covalent bonds, physical interactions, or both. Non-covalent bonds are well known in the art and include hydrogen bonding, pp stacking, van der Waals interactions, and the like, among others.

[0090] In some embodiments, physical interaction refers to the encapsulation (i.e., entrapment) of the additional compound within the matrix formed by the MaSp-based polymer. In some embodiments, the matrix is ​​bound to or in contact with the additional compound.

[0091] In some embodiments, the additional compound is selected from a biologic, a pharmaceutical, a nutrient, and a dietary supplement.

[0092] As used herein, the term "biological agent (also referred to as biological material)" relates to any substance or material having a biological origin. For example, the term "biological agent" encompasses cells (including stem cells), proteins, peptides, or nucleic acids (including nucleic acid analogs). As used herein, the term "pharmaceutical agent (also referred to as pharmaceutical compound)" refers to any biological or chemical substance that can be used to treat, cure, prevent, prevent, or diagnose a pathological condition, e.g., a disease or disorder, or that can otherwise be used to enhance physical, psychological, or spiritual well-being. Thus, the term "pharmaceutical agent" as contemplated in the context of the present invention includes any agent that has a therapeutic, diagnostic, or prophylactic effect, i.e., any therapeutic, diagnostic, or prophylactic agent.

[0093] A pharmaceutical agent may be an agent that affects or is involved in tissue growth, cell proliferation, cell differentiation, an agent that can cause a biological effect such as an immune response, or an agent that can play any other role in one or more biological processes.

[0094] Non-limiting examples of pharmaceutical agents include, but are not limited to, antimicrobial agents, e.g., antibacterial agents (e.g., antibiotics), antiviral agents, or antifungal agents, immunosuppressants, anti-inflammatory agents, antiallergic agents, anticoagulants, antirheumatic agents, antipsoriatic agents, sedatives, muscle relaxants, antimigraine agents, antidepressants, insect repellents, growth factors, hormones, hormone antagonists, antibodies, adjuvants, combinations with immunologically active compounds such as antibodies, antioxidants, proteins, e.g., glycoproteins, lipoproteins, or enzymes (e.g., hyaluronidase), polysaccharides, free radical scavengers, radiotherapeutic agents, photodynamic therapy agents, dyes (e.g., fluorescent dyes), contrast agents, disinfectants, preservatives, or any combination thereof.

[0095] Pharmaceuticals can also be small molecule compounds. The term "small molecule compound" refers to a molecule that can act to affect biological processes. Small molecules can include any number of currently known and used therapeutic agents, or can be small molecules synthesized in libraries of such molecules for the purpose of screening biological functions. Small molecule compounds usually have a molecular weight of less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1,000 Da, and most preferably less than about 500 Da.

[0096] As used herein, a "nutrient" is a chemical that an organism needs to live and grow, or a substance used in the organism's metabolism that it must obtain from its environment. Organic nutrients include carbohydrates, fats, proteins (amino acids), and vitamins. Inorganic nutrients are dietary minerals, water, and oxygen. Preferred nutrients are macronutrients such as carbohydrates, amino acids, or proteins, and micronutrients such as vitamins.

[0097] Non-limiting examples of carbohydrates include, but are not limited to, monosaccharides such as glyceraldehyde, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, dihydroxyacetone, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, or stereoisomers thereof, amino sugars such as galactosamine, glucosamine, sialic acid, N-acetylglucosamine, sulfosugars such as sulfoquinovose, disaccharides such as sucrose, lactulose, lactose, maltose, trehalose, or maltobiose, and oligosaccharides such as fructooligosaccharides (FOS), galactooligosaccharides (GOS), or mannanoligosaccharides (MOS).

[0098] As used herein, the term "dietary supplement" (also referred to as a food supplement or nutritional supplement) refers to a preparation intended to provide nutrients, such as vitamins, minerals, fiber, fatty acids, or amino acids, that are deficient or not consumed in sufficient amounts in a person's diet.

[0099] Non-limiting examples of dietary supplements include steroids, such as dehydroepiandrosterone (DHEA), pregnenolone, or derivatives thereof, hormones, such as melatonin, and other substances, such as hydrazine sulfate, caffeine, catechin, soy isoflavones, glucosamine, coenzyme-Q. 10 , or ephedrine-type alkaloids, such as ephedrine, synephrine, norephedrine, or pseudoephedrine.

[0100] The active agent can be positively or negatively charged. The active agent can also be electrically neutral. Preferably, the active agent is positively or negatively charged. The terms "positive charge" and "cation" and "negative charge" and "anion" can be used interchangeably.

[0101] In some embodiments, the weight / weight (w / w) ratio of the MaSp-based polymer to the additional compound is 10:1 to 1:10, 10:1 to 8:1, 8:1 to 6:1, 6:1 to 4:1, 4:1 to 3:1, 3:1 to 2:1, 2:1 to 1:1, 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:5, 1:5 to 1:10 (including any range therebetween).

[0102] Polymer Reinforced In another aspect, there is a composition comprising a MaSp-based polymer and an additional polymer. In some embodiments, the additional polymer is in contact with the MaSp-based fiber or the MaSp-based polymer. In some embodiments, the additional polymer is bound to the MaSp-based fiber or the MaSp-based polymer.

[0103] In some embodiments, the composition comprises an additional polymer bonded to the MaSp-based fiber to form a composite material, in some embodiments, the contacting comprises bonding or adhering, wherein the bonding is as described herein.

[0104] In some embodiments, the additional polymer fills 50% to 100% of the pore volume. In some embodiments, the additional polymer substantially fills 50% to 100% of the pore volume, where substantially is defined above. In some embodiments, the additional polymer fills 55% to 100%, 60% to 100%, 55% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 50% to 99%, 50% to 98%, 50% to 97%, 50% to 95%, 50% to 90%, 70% to 90%, or 70% to 95% of the pore volume (including any range therebetween).

[0105] In some embodiments, a composition comprises a MaSp-based polymer of the present invention conjugated to an additional polymer, wherein the w / w ratio of MaSp-based polymer to additional polymer is 1:1 to 1:100, 1:1 to 1:5, 1:1 to 1:3, 1:3 to 1:5, 1:5 to 1:10, 1:5 to 1:7, 1:7 to 1:10, 1:10 to 1:2, 1:12 to 1:15, 1:15 to 1:20, 1:20 to 1:30, 1:30 to 1:40, 1:40 to 1:50, 1:50 to 1:70, 1:70 to 1:100 (including any range or value therebetween).

[0106] In some embodiments, the additional polymer is a synthetic polymer. In some embodiments, the synthetic polymer is selected from thermoplastic polymers and thermosetting polymers. Non-limiting examples of synthetic polymers include, but are not limited to, epoxy, polyester, polyamide, polyol, polyurethane, polyethylene, silicone, liquid crystal polymer, maleic anhydride-grafted polypropylene, polyacrylate, polycarbonate, polyamide, nylon 4,6, nylon 6, nylon 6,6, polymer 11, nylon 12, poly(arylamide), polyethylene, polybutylene terephthalate, polyethylene terephthalate, polyphenylene sulfide, polyphthalamide, polypropylene, poly(vinylidene fluoride), poly(2-hydroxyethyl methacrylate) (pHEMA), polyurethane, polyvinyl butyral, ethylene-vinyl alcohol copolymer, polylactide acid (PLA) or its copolymer, polycaprolactone (PCL), xanthan, cellulose, collagen, elastin, keratin, cotton, rubber, cellulose, wool, and film-forming agents, or any combination thereof.

[0107] In some embodiments, the additional polymer is a synthetic polymer. In some embodiments, the additional polymer is a film-forming agent. In some embodiments, the w / w content of MaSp-based fibers in the composition is 0.1 and 20%, 0.1-1%, 1-2%, 2-5%, 5-7%, 4-6%, 6-8%, 8-10%, 10-12%, 12-15%, 15-20%, or any range or value therebetween. In some embodiments, compositions containing greater than 20% w / w MaSp base are heterogeneous compositions. In some embodiments, compositions containing greater than 20% w / w MaSp base are characterized by the formation of aggregates.

[0108] In some embodiments, the film-forming agent is selected from a liquid film-forming agent and / or a solid film-forming agent, hi some embodiments, the film-forming agent is a solid film-forming agent.

[0109] In some embodiments, the w / w concentration of the film-forming agent in a composition comprising the film-forming agent and MaSp-based fibers is 20-95%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-85%, 85-90%, 90-95% (including any range or value therebetween).

[0110] Non-limiting examples of film-forming agents include polysaccharides, such as pullulan, agave-based polysaccharides (Gosulin agave®), Intensyl®, Osilift®, Liftonin Xpress®, LiftLiss®, Trik®, and others. Examples of suitable resins include, but are not limited to, Fision®, polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyvinyl acetate, polyalkyl acrylates, dextrins, cellulose derivatives such as alkylcelluloses and nitrocelluloses, siliconized polysaccharides such as pullulan tri(trimethylsiloxy)silylpropylcarbamate, polyphenols, gums, acrylic-silicone graft copolymers such as alkylacrylate-dimethicone copolymers, silicone resins such as trimethylsiloxysilicate or fluorine-modified silicone resins, silicone-modified polynorbornenes, fluorocarbon resins, aromatic hydrocarbon resins, polymer emulsion resins, terpene resins, polybutene, polyisoprene, alkylated resins, polyvinylpyrrolidone-modified polymers, rosin-modified resins, and polyurethanes, or any combination thereof.

[0111] Other non-limiting examples of film-forming agents include, but are not limited to, pullulan tri(trimethylsiloxy)silylpropylcarbamate (e.g., TSPL-30-D5), alkyl acrylate-dimethicone copolymers (e.g., KP-543, 545, 549, 550, and 545L), trimethylsiloxysilicate (e.g., KF-7312J and X-21-5250), and silicon-modified polynorbornene, or any combination thereof. In some embodiments, the film-forming agent is at least one of pullulan, Osilift®, agave-based polysaccharide (Gosulin agave®), and Intensyl® (a manioca-based polysaccharide).

[0112] In some embodiments, the film-forming agent is in the form of a purified compound, a plant extract, an at least partially enriched plant extract, or any combination thereof. In some embodiments, an appropriate concentration of the film-forming agent in the composition provides it with flexibility. In some embodiments, an appropriate concentration of the film-forming agent allows the composition to be applied (e.g., by spreading) to the skin of a subject. In some embodiments, an appropriate concentration of the film-forming agent enhances the film-forming properties of the composition. In some embodiments, an appropriate concentration of the film-forming agent is as described herein. Exemplary compositions containing a film-forming agent are provided in the Examples section.

[0113] In some embodiments, the composition comprises a MaSp-based polymer bound to a film-forming agent. In some embodiments, the w / w ratio between the film-forming agent in the composition and the MaSp-based fibers is 5:1 to 50:1, 5:1 to 7:1, 7:1 to 8:1, 8:1 to 9:1, 9:1 to 10:1, 10:1 to 11:1, 11:1 to 12:1, 12:1 to 15:1, 15:1 to 20:1, 20:1 to 30:1, 30:1 to 40:1, or 40:1 to 50:1 (including any range therebetween). In some embodiments, the composition is in the form of a film. In some embodiments, the composition is flexible. In some embodiments, the composition can form a film (e.g., a layer) when applied to a substrate. In some embodiments, an appropriate concentration of the MaSp-based polymer in the composition enhances the flexibility of the composition. In some embodiments, the appropriate concentration of the MaSp-based polymer is as described herein. In some embodiments, the MaSp-based polymer improves at least one mechanical property of a composition that includes a film-former (as illustrated by Figure 14). Exemplary compositions that include a film-former and a MaSp-based polymer are provided in the Examples section.

[0114] In some embodiments, the content of the additional polymer is 10% to 99% (w / w), 10% to 90% (w / w), 10% to 80% (w / w), 30% to 99% (w / w), 30% to 98% (w / w), 30% to 95% (w / w), 30% to 90% (w / w), 30% to 85% (w / w), 30% to 80% (w / w), 30% to 75% (w / w), 30% to 70% (w / w), 30% to 65% (w / w), 30% to 50% (w / w), or the like, based on the total composition. (w / w), 30%-45% (w / w), 30%-40% (w / w), 40%-70% (w / w), 40%-65% (w / w), 40%-50% (w / w), 40%-45% (w / w), 50%-70% (w / w), 50%-80% (w / w), 50%-90% (w / w), 50%-95% (w / w), 70%-80% (w / w), 70%-90% (w / w), or 70%-95% (w / w) (including any ranges therebetween).

[0115] In some embodiments, the additional polymer according to the present invention is enriched with an insoluble MaSp-based polymer as described herein, ie, the additional polymer is enriched with more than 1% (w / w), more than 2% (w / w), more than 4% (w / w), more than 5% (w / w), more than 10% (w / w), more than 12% (w / w), more than 15% (w / w), more than 20% (w / w), more than 25% (w / w), more than 30% (w / w), more than 40% (w / w), more than 45% (w / w), or more than 50% (w / w) of the insoluble MaSp-based polymer.

[0116] In some embodiments, the additional polymer is attached to the MaSp-based fiber by non-covalent bonds, physical interactions, or both. Non-covalent bonds are well known in the art and include hydrogen bonding, pp stacking, van der Waals interactions, and the like, among others.

[0117] In some embodiments, physical interaction refers to additional polymers that are entrapped within, entangled or intertwined with the MaSp-based fibers within a mesh or matrix formed by the MaSp-based fibers, hi some embodiments, the matrix is ​​formed by a cosmetic active ingredient that fills at least a portion of the pores within the MaSp-based fibers.

[0118] In some embodiments, the composition is a composite material, the composite material comprising MaSp-based fibers bound to an additional polymer, the binding being by non-covalent bonds, covalent bonds, physical interactions, or any combination thereof.

[0119] In some embodiments, the additional polymer is in contact with or bound to the fibrils. In some embodiments, the MaSp-based fiber is embedded within the additional polymer. In some embodiments, the MaSp-based fiber is embedded within the additional polymer. In some embodiments, the additional polymer is doped by the MaSp-based fiber. In some embodiments, the MaSp-based fiber is encapsulated by the additional polymer.

[0120] In some embodiments, at least a portion of the additional polymer fills 20% to 100% of the volume (e.g., lumen) of the particle, hi some embodiments, at least a portion of the additional polymer fills 55% to 100%, 60% to 100%, 55% to 100%, 70% to 100%, 75% to 100%, 80% to 100%, 85% to 100%, 90% to 100%, 95% to 100%, 50% to 99%, 50% to 98%, 50% to 97%, 50% to 95%, 50% to 90%, 70% to 90%, or 70% to 95% (including any range therebetween) of the volume of the particle.

[0121] In some embodiments, the additional polymer is substantially devoid of any additional biologically active moieties. In some embodiments, the additional polymer is substantially devoid of proteins, such as MaSp-based proteins. In some embodiments, the additional polymer of the present invention consists essentially of the polymers listed above.

[0122] In some embodiments, the compositions of the present invention are substantially homogeneous.

[0123] In some embodiments, the composition or composite is a solid. In some embodiments, the composition or composite is a semi-solid. In some embodiments, the composition or composite is a gel. In some embodiments, the composition (e.g., a solid composition) is substantially devoid of any one of solvent, surfactant, carrier, or particles, where substantially is at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, or at least 99% by weight of the composition.

[0124] In some embodiments, the composition is characterized by at least one improved mechanical property compared to the property of the additional polymer without the MaSp-based polymer.

[0125] In some embodiments, the property is selected from the group consisting of Young's modulus, tensile strength, strain at break, yield point, toughness, work of failure, impact strength, tear strength, flexural modulus, flexural strain and stress at a specific elongation, and wear, hi some embodiments, the mechanical property is selected from storage modulus and loss modulus, or any combination thereof.

[0126] In some embodiments, the present invention provides abrasion-resistant compositions. In some embodiments, the present invention provides compositions with improved abrasion resistance. As used herein, the term "abrasion resistance" refers to the ability of a material to stop movement when exposed to the relative movement of hard particles or protrusions. Abrasion resistance can be measured by various tests known in the art, such as, for example, the Taber abrasion test, the Gardner scrubber test, and the falling sand test.

[0127] In some embodiments, one or more properties selected from Young's modulus, tensile strength, yield point, abrasion resistance, and stress at elongation are enhanced, for example, by at least 1%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0128] In some embodiments, one or more properties selected from Young's modulus, tensile strength, yield point, abrasion resistance, and stress at elongation are enhanced, for example, by at least 100%, at least 150%, at least 250%, at least 250%, at least 260%, at least 270%, at least 280%, at least 290%, at least 300%, at least 350%, at least 400%, at least 450%, at least 500%, at least 550%, at least 600%, at least 650%, at least 700%, at least 750%, at least 800%, at least 850%, at least 900%, at least 1000%, at least 1500%, at least 2000%, at least 2500%, or at least 3000%.

[0129] In some embodiments, the composition is characterized by a Young's modulus in the range of 50 MPa to 170 MPa, 52 MPa to 170 MPa, 60 MPa to 170 MPa, 68 MPa to 170 MPa, 90 MPa to 170 MPa, 100 MPa to 170 MPa, 101 MPa to 170 MPa, 105 MPa to 170 MPa, 101 MPa to 160 MPa, or 105 MPa to 160 MPa, including any range therebetween.

[0130] In some embodiments, at least two properties selected from Young's modulus, tensile strength, yield point, abrasion resistance, and stress at elongation are enhanced, for example, by at least 1%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0131] In some embodiments, at least three properties selected from Young's modulus, tensile strength, yield point, abrasion resistance, and stress at elongation are enhanced, for example, by at least 1%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0132] In some embodiments, the Young's modulus is enhanced by, for example, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0133] In some embodiments, the tensile strength is increased by, for example, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or at least 50%.

[0134] In some embodiments, the yield point is increased by, for example, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or at least 50%.

[0135] In some embodiments, the wear resistance is enhanced by, for example, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, or at least 50%.

[0136] In some embodiments, the compositions are characterized by structural strength, with greater than 20% of the structural strength attributable to the incorporated MaSp-based polymer. In some embodiments, the composite materials are characterized by structural strength, with greater than 30% of the structural strength attributable to the incorporated MaSp-based polymer.

[0137] In some embodiments, the compositions are characterized by structural strength, with more than 1% of the tensile strength coming from the incorporated MaSp-based polymer. In some embodiments, the composite materials are characterized by structural strength, with more than 5% of the tensile strength coming from the incorporated MaSp-based polymer. In some embodiments, the composite materials are characterized by structural strength, with more than 10% of the tensile strength coming from the incorporated MaSp-based polymer. In some embodiments, the composite materials are characterized by structural strength, with more than 20% of the tensile strength coming from the incorporated MaSp-based polymer. In some embodiments, the composite materials are characterized by tensile strength, with more than 30% of the structural strength coming from the incorporated MaSp-based polymer.

[0138] In some embodiments, the phrase "structural strength" as used herein refers to mechanical properties such as, but not limited to, elastic modulus, tensile stress, elongation (strain), and toughness (e.g., a combination of tensile stress and elongation (strain)).

[0139] In some embodiments, the MaSp-based polymer improves at least one mechanical property of a composition comprising a film former, the mechanical property being as described herein. Experimental results of the mechanical properties of exemplary compositions comprising a MaSp-based polymer and a film former are summarized in Figure 14.

[0140] Manufacturing method In some embodiments, methods are provided for producing spider silk (MaSp)-based polymers of the present invention. In some embodiments, the bacteria used in the compositions and / or methods of the present invention are recombinant bacteria. Recombinant bacterial proteins can be artificially produced by recombinant DNA technology known in the art.

[0141] As used herein, a "recombinant nucleic acid" is a nucleic acid molecule encoding a polypeptide of interest that has been modified in vitro so that its sequence does not occur in nature or corresponds to a naturally occurring sequence that is not positioned as it would be positioned in the unmodified genome.

[0142] In one embodiment, the bacteria described herein are genetically modified bacteria. In one embodiment, the artificial dragline spider silk is not endogenously produced in bacteria such as those described herein. The artificial dragline spider silk of the invention described herein produced in bacteria has unexpected and unique properties.

[0143] In some embodiments, the method of the present invention comprises: a. providing an expression vector comprising a nucleic acid sequence encoding an amino acid sequence, wherein the nucleic acid is under the expression control of an operably linked promoter, and optionally regulatory sequences; b. Transforming a microbial (e.g., bacterial) host with the expression vector of (a); c. Providing conditions for expression of the heterologous protein by the microorganism of (b); d. isolating the expressed protein; thereby obtaining the synthetic amino acid sequence of the present invention.

[0144] According to some embodiments, (i) providing a microorganism (e.g., a recombinant bacterium) described herein; (ii) providing conditions for the expression of MaSp by the microorganism; (iii) isolating the expressed protein; and producing synthetic dragline spider silk, thereby providing a process (e.g., a process for producing synthetic dragline spider silk).

[0145] In some embodiments, step (ii) of providing conditions for bacterial expression of MaSp comprises providing a solution with a pH in the range of 5 to 6.5, hi some embodiments, step (ii) comprises providing a solution with a pH below 6.5.

[0146] In some embodiments, step (ii) of providing conditions for expression of MaSp by the bacterium comprises providing an expression inducer. In some embodiments, the expression inducer comprises lactose. In some embodiments, the expression inducer comprises isopropyl β-D-1-thiogalactopyranoside (IPTG). As used herein, the term "inducer" refers to a compound that induces and / or increases protein expression. In some embodiments, the expression is constitutive expression.

[0147] In some embodiments, step (ii) of providing conditions for bacterial expression of MaSp includes waiting a period of time to obtain an insoluble MaSp-based polymer. In some embodiments, the period of time is in the range of 15 to 48 hours, 17 to 48 hours, 18 to 48 hours, 18 to 24 hours, 20 to 48 hours, 22 to 48 hours, 24 to 48 hours, 22 to 36 hours, 24 to 36 hours, or 24 to 32 hours, including any range therebetween. In some embodiments, waiting a period of time allows for the formation of synthetic dragline spider silk in two phases. In some embodiments, soluble spidroin protein is formed in the first phase. Upon accumulation of a critical intracellular concentration of soluble protein, self-aggregation of SVX-E occurs, forming the desired insoluble MaSp-based polymer.

[0148] In some embodiments, step (iii) of isolating the expressed protein comprises solubilizing the bacteria in a 0.1-10% detergent solution and centrifuging the mixture. In some embodiments, step (iii) of isolating the expressed protein comprises solubilizing the bacteria in a 0.1-5% detergent solution and centrifuging the mixture. In some embodiments, the resulting pellet is suspended in a 6 M urea solution. In some embodiments, after further centrifugation, the pellet is suspended in a 0.07% detergent solution. In some embodiments, after resuspension in the detergent solution and urea, the protein is separated from the cellular debris according to well-known procedures (e.g., by using a 10-90% w / w mono- or disaccharide solution).

[0149] In some embodiments, the process further comprises a strengthening step with an additional polymer and / or additional compound. In some embodiments, the additional polymer and additional compound are as described herein. In some embodiments, the strengthening step comprises mixing a solution of synthetic dragline spider silk with a solution of the additional polymer.

[0150] In some embodiments, the strengthening step comprises degassing a mixture of a solution comprising synthetic dragline spider silk and an additional polymer.

[0151] In some embodiments, degassing is performed by leaving the resulting suspension at a temperature between 20 and 50° C. without shaking for a period of time. In some embodiments, the period of time is in the range of 30 minutes to 24 hours, 1 hour to 24 hours, or 1 hour to 12 hours, including any range therebetween.

[0152] In some embodiments, the synthetic dragline spider silk (e.g., MaSp-based polymer or MaSp-based fiber) and the additional polymer are mixed at a ratio of 1:4 to 4:1, 1:3.9 to 4:1, 1:3.8 to 4:1, 1:3.5 to 4:1, 1:3 to 4:1, 1:2.8 to 4:1, 1:2.5 to 4:1, 1:2 to 4:1, 1.5:4 to 4:1, 1.5:3.9 to 4:1, 1.5:3.8 to 4:1, 1.5:3.5 to 4:1, 1.5:3 to 4:1, 1.5:2.8 to 4:1, 1.5:2.5 to 4:1, 1.5:2 to 4:1, 2:4 to 4:1 , 2:3.9-4:1, 2:3.8-4:1, 2:3.5-4:1, 2:3-4:1, 2:2.8-4:1, 2:2.5-4:1, 2:2-4:1, 1:4-3:1, 1:3.9-3:1, 1:3.8-3:1, 1:3.5-3:1, 1:3-3:1, 1:2.8-3:1, 1:2.5-3:1, 1:2-3:1, 1:4-4:3, 1:3.9-4:3, 1:3.8-4:3, 1:3.5-4:3, 1:3-4:3, 1:2.8-4:3, 1:2.5-4:3, or 1:2-4:3 (including any ranges between these).

[0153] In some embodiments, synthetic dragline spider silk (e.g., MaSp-based polymer or MaSp-based fiber) and additional polymer are used in ratios of 1:1 to 1:100, 1:1 to 1:5, 1:1 to 1:3, 1:3 to 1:5, 1:5 to 1:10, 1:5 to 1:7, 1:7 to 1:10, 1:10 to 1:2, 1:12 to 1:15, 1:15 to 1:20, 1:20 to 1:30, 1:30 to 1:40, 1:40 to 1:50, 1:50 to 1:70, 1:70 to 1:100 (including any range therebetween).

[0154] In some embodiments, synthetic dragline spider silk (e.g., MaSp-based polymer or MaSp-based fiber) and additional compounds are used in ratios of 10:1 to 1:10, 10:1 to 8:1, 8:1 to 6:1, 6:1 to 4:1, 4:1 to 3:1, 3:1 to 2:1, 2:1 to 1:1, 1:1 to 1:2, 1:2 to 1:3, 1:3 to 1:5, 1:5 to 1:10 (including any range therebetween).

[0155] In some embodiments, the polymer is selected from synthetic polymers, thermoplastic polymers, thermosets, film formers, epoxies, polyesters, polyamides, polyols, polyurethanes, polyethylene, nylons, polyacrylates, polycarbonates, polylactide acid (PLA) or copolymers thereof, silicones, liquid crystal polymers, maleic anhydride grafted polypropylene, polycaprolactone (PCL), rubber, cellulose, or any combination thereof.

[0156] In some embodiments, step (iii) further comprises drying the synthetic dragline spider silk (e.g., SVX-E-based fibers). In some embodiments, drying comprises partially drying the fibers. In some embodiments, drying is by evaporation of at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 97% by weight of the solvent (e.g., aqueous solvent). In some embodiments, drying is by exposing the fibers to temperatures between -180 and 200°C. In some embodiments, drying is by exposing the fibers to electromagnetic radiation in the visible and / or infrared light spectrum.

[0157] In some embodiments, drying is by exposing the fibers to a temperature of 40-200°C, 40-50°C, 40-60°C, 60-80°C, 40-80°C, 60-100°C, 100-150°C, 150-200°C (including any range or value therebetween).

[0158] In some embodiments, drying is by exposing the fibers to microwave radiation. In some embodiments, drying is by convection drying, e.g., by applying a hot gas stream to the fibers. In some embodiments, drying is by low temperature drying, e.g., by applying a dehumidified gas stream to the fibers. In some embodiments, drying is by freeze-drying. Generally, the drying method and exact drying conditions selected will depend, among other things, on the chemical and / or physical stability (e.g., thermal stability) of the fibers.

[0159] MaSp-based fibers The terms "major ampullate gland spidroin protein" and "spidroin protein" are used interchangeably throughout the description and encompass all known major ampullate gland spidroin proteins, typically abbreviated as "MaSp" or, in the case of Araneus diadematus, "ADF." These major ampullate gland spidroin proteins generally come in two types: type 1 and type 2. These terms also encompass non-naturally occurring proteins disclosed herein that share a high degree of identity and / or similarity to at least the repeat region of known major ampullate gland spidroin proteins. Additional suitable spider silk proteins include MaSp2, MiSp, MiSp2, AcSp, FLYS, FLAS, and flagelliform.

[0160] As used herein, the terms "repeat region," "repeat sequence," or "repeat" refer to a recombinant protein sequence derived from a repeat unit that occurs multiple times in nature in spider silk amino acid sequences (e.g., MaSp-1 protein). Those skilled in the art will understand that the primary structure of spider silk proteins is believed to consist mostly of a series of small variations of unit repeats. Unit repeats in naturally occurring proteins often differ from one another; that is, there is little or no exact overlap of unit repeats along the length of the protein. In some embodiments, the synthetic spider silks of the present invention are produced such that the primary structure of the protein contains many exact repeats of a single unit repeat. In additional embodiments, the synthetic spider silks of the present invention contain many repeats of one unit repeat along with many repeats of a second unit repeat. Such structures resemble typical block copolymers. Unit repeats of several different sequences can be combined to provide synthetic spider silk proteins with properties suitable for particular applications. As used herein, the term "direct repeat" refers to a tandem repeat (head-to-tail arrangement) with similar repeats. In another embodiment, the repeats used to form the synthetic spider silks of the present invention are direct repeats. In some embodiments, the repeats are not found in nature (ie, are not naturally occurring amino acid sequences).

[0161] An exemplary sequence comprising a repeat sequence is ADF-4: (SEQ ID NO: 1). In some embodiments, the synthetic repeat sequence of the present invention is based on (e.g., has a high percentage of identity as defined below) one or more repeat sequences derived from ADF-4 (SEQ ID NO: 1). As used herein, the term "based on" refers to a sequence that has a high percentage of homology to the repeat sequence.

[0162] In some embodiments, each repeat sequence contains up to 60 amino acids, up to 55 amino acids, up to 50 amino acids, up to 49 amino acids, up to 48 amino acids, up to 47 amino acids, up to 46 amino acids, up to 45 amino acids, up to 44 amino acids, up to 43 amino acids, up to 42 amino acids, up to 41 amino acids, up to 40 amino acids, up to 39 amino acids, up to 38 amino acids, up to 37 amino acids, up to 36 amino acids, or up to 35 amino acids, with each alternative representing a separate embodiment of the present invention. In some embodiments, each repeat sequence contains 5 to 60 amino acids, 10 to 55 amino acids, 15 to 50 amino acids, 20 to 45 amino acids, 25 to 40 amino acids, 25 to 39 amino acids, or 28 to 36 amino acids, with each alternative representing a separate embodiment of the present invention. In some embodiments, each repeat sequence contains 30 to 40 amino acids, 31 to 39 amino acids, 32 to 38 amino acids, 33 to 37 amino acids, or 34 to 36 amino acids, with each alternative representing a separate embodiment of the present invention. In additional embodiments, each repeat sequence contains 35 amino acids.

[0163] In some embodiments, the repeat regions independently comprise an amino acid sequence set forth in Formula 1: (X1) Z X2GPGGYGPX3X4X5GPX6GX7GGX8GPGGPGX9X 10 (wherein X1 is independently at each occurrence in A or G).

[0164] In some embodiments, (X1) Z at least 50% of the groups are A, Z is an integer from 5 to 30, X2 is S or G, X3 is G or E, X4 is G, S, or N, X5 is Q or Y, X6 is G or S, X7 is P or R, X8 is Y or Q, X9 is G or S, and X 10 is S or G.

[0165] In another embodiment, the repeat region of the MaSP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS). In another embodiment, the repeat region of the MaSP1 protein comprises the amino acid sequence set forth in SEQ ID NO: 3 (AAAAAAAASGPGGYGPGSQGPSGPGGYGPGGPGSS).

[0166] In another embodiment, a homologue of the repeat region of MaSP1 protein is provided that shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO:1.

[0167] In some embodiments, a homologue shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with SEQ ID NO:2.

[0168] In another embodiment, the repeat region of the MaSP1 protein has the amino acid sequence set forth in SEQ ID NO:1.

[0169] In another embodiment, the MaSP1 protein comprises a single N-terminal region selected from the group consisting of SEQ ID NO: 4 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLV), SEQ ID NO: 5 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVRPLSNLDNAP), and SEQ ID NO: 6 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVDPPGCRNSARAGSS), or any functional homolog, variant, derivative, or fragment thereof. In another embodiment, a homolog of the C-terminal region shares at least 70% homology with any one of SEQ ID NOs: 4-6.

[0170] In another embodiment, the MaSP1 protein further comprises a single C-terminal region selected from the group consisting of SEQ ID NO: 7 (VAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS) and SEQ ID NO: 8 (GPSGPGAYGPSPSASASVAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS), or any functional homolog, variant, derivative, fragment, or mutant thereof. In another embodiment, the homolog of the N-terminal region shares at least 70% homology with SEQ ID NOs: 7-8.

[0171] In some embodiments, MaSp-based fibers comprising a mixture of proteins as disclosed under WO2017 / 025964, the entire contents of which are incorporated herein by reference.

[0172] In some embodiments, the MaSP1 protein further comprises at least one tag sequence. Non-limiting examples of tags that can be used in the present invention include His tags, HA tags, T7 tags, etc. Those skilled in the art will be familiar with alternative suitable tags or other fusion partners.

[0173] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analog" refers to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon bonded to a hydrogen atom, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have modified R groups or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. "Amino acid mimetics" refer to compounds that have a structure different from the general chemical structure of an amino acid but function similarly to a naturally occurring amino acid. Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.

[0174] An "amino acid sequence" or "peptide sequence" is the order in which amino acid residues connected by peptide bonds are arranged in a peptide or protein chain. The sequence is generally reported from the N-terminus, containing the free amino group, to the C-terminus, containing the free carboxyl group. Although amino acid sequences are often referred to as peptide or protein sequences when describing the primary structure of a protein, the terms "amino acid sequence" or "peptide sequence" must be distinguished from the term "protein," because proteins are defined as amino acid sequences that fold into specific three-dimensional configurations and typically undergo post-translational modifications such as phosphorylation, acetylation, glycosylation, sulfhydryl bond formation, and cleavage.

[0175] As used herein, "isolated" or "substantially purified" in the context of the synthetic spider silk amino acid sequences exemplified by the present invention or the nucleic acid molecules encoding them means that the amino acid sequences or polynucleotides have been removed from their natural environment or altered from their natural state. Thus, "isolated" does not necessarily reflect the extent to which the amino acid sequence or nucleic acid molecule has been purified. However, it will be understood that such molecules that have been purified to some degree are "isolated." If the molecule is not present in its natural environment, i.e., not present in nature, then the molecule is "isolated" regardless of where it is found. By way of example, an amino acid sequence or polynucleotide that does not naturally occur in humans is "isolated" even if it is present in humans.

[0176] The terms "isolated" or "substantially purified," when applied to an amino acid sequence or nucleic acid, mean that the amino acid sequence or nucleic acid is essentially free from other cellular components with which it is naturally associated. It may be in a homogeneous state, or in either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis and high performance liquid chromatography. An amino acid sequence or nucleic acid that is the predominant species present in a preparation is substantially purified.

[0177] In some embodiments, the repeats are of a homolog, variant, or derivative of the repeat region of the MaSp1 protein or a fragment thereof, hi some embodiments, the repeats are of a homolog, variant, or derivative of the repeat region of the ADF-4 protein or a fragment thereof.

[0178] As used herein, the term "functional" in "functional homolog, variant, derivative, or fragment" refers to an amino acid sequence that has a biological function or activity that is identified by a defined functional assay. More specifically, the defined functional assay is the formation of self-aggregating fibers in cells expressing the functional homolog, variant, derivative, or fragment.

[0179] An amino acid sequence or nucleic acid sequence is a homolog of the corresponding amino acid sequence or nucleic acid if the homology is determined to be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99%.

[0180] The terms "identical," "substantial identity," "substantial homology," or percent "identity," in the context of two or more amino acid or nucleic acid sequences, refer to two or more sequences or subsequences that have a specified percentage of amino acid residues or nucleotides that are the same or identical (i.e., about 60% identity, or at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity over a designated region (e.g., amino acid sequence SEQ ID NO: 2 or 3) when compared and aligned for maximum correspondence over a comparison window or designated region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters described below, or by manual alignment and visual inspection. Such sequences are then "substantially identical." This definition may also refer to or apply to the complement of a test sequence. The definition also includes sequences that have deletions and / or additions, as well as sequences that have substitutions. Preferred algorithms can account for gaps, etc.

[0181] In sequence comparison, typically, one sequence serves as reference sequence, and test sequence is compared to it.When using sequence comparison algorithm, test sequence and reference sequence are input into computer, and if necessary, subsequence coordinate is designated, and sequence algorithm program parameters are designated.Preferably, default program parameters can be used, or alternative parameters can be designated.Then, sequence comparison algorithm calculates the percent sequence identity of test sequence with reference sequence based on program parameters.

[0182] It should be understood that the present invention further encompasses amino acid sequences comprising n repeats of any one of SEQ ID NOs: 1, 2, or 3 variants. As used herein, the term "variant" or "substantially similar" includes amino acid or nucleotide sequences that differ from a specifically identified sequence by one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or 25) amino acid residues or nucleotides being deleted, substituted, or added. Variants may be naturally occurring allelic variants or non-naturally occurring variants. Variants or substantially similar sequences refer to amino acid sequences or nucleic acid fragments that can be characterized by the percentage of identity between their amino acid or nucleotide sequence and the amino acid or nucleotide sequences described herein, as determined by common algorithms used in the state of the art. Preferred amino acid or nucleic acid fragments are those having an amino acid or nucleotide sequence that has at least about 40 or 45% sequence identity, preferentially about 50% or 55% sequence identity, more preferentially about 60% or 65% sequence identity, more preferentially about 70% or 75% sequence identity, more preferentially about 80% or 85% sequence identity, and even more preferentially about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity when compared to a reference sequence.

[0183] The terms derivative and functional derivative as used herein refer to the amino acid sequences of the invention having any insertions, deletions, substitutions and modifications.

[0184] The term "insertion" as used herein should be understood to mean any addition of amino acid residues of 1 to 50 amino acid residues, particularly 20 to 1 amino acid residues, more particularly 1 to 10 amino acid residues, to a sequence of the invention. Most particularly, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 amino acid residues. Furthermore, the amino acid sequences of the invention can be extended at their N-terminus and / or C-terminus with various identical or different amino acid residues.

[0185] Amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., conservative amino acid substitutions. Amino acid substitutions can be made based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the involved residues. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0186] In another embodiment, the repeat sequence of the invention has no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than 12, no more than 11, no more than 10, no more than 9, no more than 8, or no more than 7 amino acid substitutions relative to any one of SEQ ID NOs: 2 or 3. In one embodiment, the repeat sequence of the invention has at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, or at least 13 amino acid substitutions relative to any one of SEQ ID NOs: 1, 2, or 3.

[0187] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to amino acid, nucleic acid, peptide, polypeptide, or protein sequences that modify, add, or delete a single amino acid or a small percentage of amino acids in the encoded sequence are "conservatively modified variants," in which the modification replaces an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, polymorphic variants, interspecies homologs, and alleles of the present invention.

[0188] For example, substitutions can be made by substituting an aliphatic amino acid (G, A, I, L, or V) for another member of the group, or by substituting one polar residue for another, e.g., arginine for lysine, glutamine for aspartic acid, or glutamine for asparagine. Each of the following eight groups includes other exemplary amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G), 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), 6) phenylalanine (F), tyrosine (Y), tryptophan (W), 7) serine (S), threonine (T), and 8) cysteine ​​(C), methionine (M).

[0189] Conservative nucleic acid substitutions are nucleic acid substitutions that result in conservative amino acid substitutions as defined above.

[0190] Variants of the amino acid sequences of the invention may have at least 80% sequence similarity, at least 85% sequence similarity, 90% sequence similarity, or at least 95%, 96%, 97%, 98%, or 99% sequence similarity at the amino acid level to the repeating unit set forth in any one of SEQ ID NOs: 1, 2 or 3.

[0191] The amino acid sequences of the present invention may include fragments of SEQ ID NO: 1. A "fragment" constitutes a portion of a particular region of an amino acid or DNA sequence. An amino acid fragment may contain at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 24, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, or at least 34 amino acids of SEQ ID NO: 1, 2, or 3.

[0192] Variants of the amino acid sequences of the present invention are characterized by one (point mutations) or multiple, up to about 10, exchanges of that amino acid for one or more other amino acids, which are the result of corresponding mutations at the DNA level resulting in different codons.

[0193] Furthermore, the present invention relates to derivatives of the amino acid sequences of the present invention. Derivatives of the amino acid sequences of the present invention are those in which functional groups such as amino, hydroxyl, mercapto, or carboxyl groups are derivatized, for example, glycosylated, acylated, amidated, or esterified, respectively. In glycosylated derivatives, oligosaccharides are usually linked to asparagine, serine, threonine, and / or lysine. Acylated derivatives are particularly acylated with naturally occurring organic or inorganic acids, such as acetic acid, phosphoric acid, or sulfuric acid, usually at the N-terminal amino or hydroxy group, particularly tyrosine or serine, respectively. Esters are esters of naturally occurring alcohols, such as methanol or ethanol. Further derivatives are salts, particularly pharmaceutically acceptable salts, such as metal salts, such as alkali metal and alkaline earth metal salts, for example, sodium, potassium, magnesium, calcium, or zinc salts, or ammonium salts formed with ammonia or suitable organic amines, such as lower alkylamines, for example, triethylamine, hydroxy-lower alkylamines, for example, 2-hydroxyethylamine, etc.

[0194] Although the present invention generally relates to synthetic spider silk proteins, or fragments or portions thereof, derived from Araneus diadematus dragline silk, it should be understood that synthetic spider silk can be obtained in a similar manner using many other spider species. More preferably, the dragline protein is derived from one or more of the following spiders: Arachnura higginsi, Araneus circulissparsus, Araneus diadematus, Argiope picta, Banded Garden Spider (Argiope trifasciata), Batik Golden Web Spider (Nephila antipodiana), Beccari's Tent Spider (Cyrtophora beccarii), Bird-dropping Spider (Celaenia excavata), Black-and-White Spiny Spider (Gasteracantha kuhlii), Black-and-yellow Garden Spider (Argiope aurantia), Bolas Spider (Ordgarius furcatus), Bolas Spiders Magnificent Spider (Ordgarius magnificus), Brown Sailor Spider (Neoscona nautica), Brown-Legged Spider (Neoscona rufofemorata), Capped Black-Headed Spider (Zygiella calyptrata), Common Garden Spider(Parawixia dehaani), Common Orb Weaver(Neoscona oxancensis), Crab-like Spiny Orb Weaver(Gasteracantha cancriformis(elipsoides)), Curved Spiny Spider(Gasteracantha arcuata), Cyrtophora moluccensis, Cyrtophora parnasia, Dolophones conifera, Dolophonesturrigera、Doria’s Spiny Spider(Gasteracantha doriae)、Double-Spotted Spiny Spider(Gasteracantha mammosa)、Double-Tailed Tent Spider(Cyrtophora exanthematica)、Aculeperia ceropegia、Eriophora pustulosa、Flat Anepsion(Anepsion depressium)、Four-spined Jewel Spider(Gasteracantha quadrispinosa)、Garden Orb Web Spider(Eriophora transmarina)、Giant Lichen Orbweaver(Araneus bicentenarius)、Golden Web Spider(Nephila maculata)、Hasselt’s Spiny Spider(Gasteracantha hasseltii)、Tegenaria atrica、Heurodes turrita、Island Cyclosa Spider(Cyclosa insulana)、Jewel or Spiny Spider(Astracantha minax)、Kidney Garden Spider(Araneus mitificus)、Laglaise’s Garden Spider(Eriovixia laglaisei)、Long-Bellied Cyclosa Spider(Cyclosa bifida)、Malabar Spider(Nephilengys malabarensis)、Multi-Coloured St Andrew’s Cross Spider(Argiope versicolor)、Ornamental Tree-Trunk Spider(Herennia ornatissima)、Oval St.Andrew’s Cross Spider(Argiope aemula)、Red Tent Spider(Cyrtophora unicolor)、Russian Tent Spider(Cyrtophora hirta)、Saint Andrew’s CrossSpider(Argiope keyserlingi), Scarlet Acusilas(Acusilas coccineus), Silver Argiope(Argiope argentata), Spinybacked Orbweaver(Gasteracantha cancriformis), Spotted Orbweaver(Neoscona domiciliorum), St.Andrews Cross(Argiope aetheria), St.Andrew's Cross Spider(Argiope Nephila spp., such as Nephila clavipes, Nephila senegalensis, Nephila madagascariensis, and many others.

[0195] Furthermore, synthetic spider silk can be enhanced not only by selecting different spider species from which it is derived, but also by using various compounds other than proteins. Pyrrolidine has hygroscopic properties and helps keep the silk moist. This occurs especially at high concentrations in adhesive silk. Potassium hydrogen phosphate releases protons in aqueous solution, which results in a pH of approximately 4, making the silk acidic and thus protecting it from fungi and bacteria that digest proteins. Potassium nitrate is thought to prevent protein denaturation in an acidic environment.

[0196] In some embodiments, the bacterial system of the present invention may utilize a number of expression vectors, advantageously selected depending on the intended use for the expressed protein. In one embodiment, large quantities of protein are desired. In one embodiment, vectors that direct high-level expression of protein products are desirable, perhaps as fusions with hydrophobic signal sequences that direct the expressed product to the periplasm of the bacteria or to the medium from which the protein product can be easily purified. In one embodiment, certain fusion proteins have been engineered with specific cleavage sites to aid in recovery of the polypeptide. In one embodiment, vectors amenable to such engineering include, but are not limited to, the pET series expression vectors.

[0197] "Nucleic acid" refers to molecules that can be single- or double-stranded, composed of monomers (nucleotides) containing sugars, phosphates, and either purines or pyrimidines. In bacteria, "deoxyribonucleic acid" (DNA) refers to the genetic material, while "ribonucleic acid" (RNA) is involved in translating information from DNA into proteins.

[0198] It is clear that due to the degeneracy of the genetic code, multiple different nucleic acid sequences can be used to encode the amino acid sequences of the present invention. It should be understood that the codons contained in the nucleic acid sequences of the present invention can be optimized for expression in a bacterial host cell.

[0199] The term "codon optimization," referring to genes or coding regions of nucleic acid molecules for transformation into various hosts, refers to the modification of codons in the genes or coding regions of nucleic acid molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA. In the context of the present invention, genes and DNA coding regions are codon-optimized for optimal expression in host bacterial cells.

[0200] As used herein, the term "expression" is intended to mean the transcription and translation of a gene encoding a sequence of a gene product into a gene product. In expression, a DNA strand encoding a sequence of a gene product is first transcribed into a complementary RNA, often a messenger RNA, and then the messenger RNA thus transcribed is translated into the gene product if the gene product is a protein.

[0201] In some embodiments, the present invention relates to one or more expression vectors comprising a nucleic acid sequence encoding a protein of the present invention.

[0202] As used herein, a "vector," "expression vector," or "plasmid" refers to an extrachromosomal element that often carries an exogenous gene that is not part of the central metabolism of a bacterial cell and is usually in the form of a circular double-stranded DNA molecule. It can be any of a number of nucleic acids into which a desired sequence can be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is capable of replicating in a host cell and is further characterized by one or more endonuclease restriction sites at which a desired DNA sequence is ligated so that the vector can be cleaved in a determinable manner and the new recombinant vector retains the ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence can occur multiple times as the plasmid increases in copy number within the host bacterium, or it can occur only once per host before the host replicates by mitosis. In the case of phages, replication can occur actively during the lytic phase or passively during the lysogenic phase. Expression vectors are those into which a desired DNA sequence can be inserted by restriction and ligation so that it is operably linked to regulatory sequences and can be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in identifying and selecting cells transformed or transfected with the vector. As used herein, "transformation" or "transfection" refers to the acquisition of new genes in a cell by the incorporation of nucleic acid. Markers include, for example, genes encoding proteins that increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes encoding enzymes whose activity can be detected by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes that clearly affect the phenotype of transformed or transfected cells, hosts, colonies, or plaques.Preferred vectors are those capable of autonomous replication and expression of structural gene products present in the DNA segments to which they are operably linked, i.e., expression of synthetic spider silk proteins.

[0203] As described above, the expression vectors of the present invention are operably linked to a promoter. The terms "promoter" and "promoter region" refer to a sequence of DNA, usually upstream (5') of the protein-coding sequence of a structural gene, that controls expression of the coding region by providing recognition for RNA polymerase and / or other factors necessary for transcription to begin at the correct site. A promoter sequence is necessary, but not necessarily sufficient, to drive gene expression. The term "suitable promoter" refers to any prokaryotic promoter capable of driving expression of the synthetic spider silk variant gene.

[0204] Promoters useful for driving expression of heterologous DNA fragments in bacteria are numerous and well known to those skilled in the art. Virtually any bacterial promoter capable of driving a gene encoding a silk variant protein is suitable for the present invention.

[0205] A coding sequence and a regulatory sequence are "operably linked" or "operably associated" when they are covalently linked in such a way that the expression or transcription of the coding sequence is under the influence or control of the regulatory sequence. A regulatory sequence is operably linked to a gene when it is positioned relative to the gene so that it can have a measurable effect on the amount of gene product produced. When it is desired to translate the coding sequence into a functional protein, induction of a promoter in the 5' regulatory sequence results in transcription of the coding sequence, and two DNA sequences are operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frameshift mutation, (2) interfere with the ability of the promoter region to direct transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into protein. Thus, a promoter region would be operably linked to a coding sequence if it is capable of effecting transcription of that DNA sequence such that the resulting transcript can be translated into the desired protein or polypeptide.

[0206] The exact nature of the regulatory sequences required for gene expression may vary depending on the species or cell type, but generally include 5' non-transcribed and 5' non-translated sequences involved in initiation of transcription and translation, respectively, such as the TATA box, capping sequence, CAAT sequence, etc. In particular, such 5' non-transcribed regulatory sequences will include a promoter region containing a promoter sequence for transcriptional control of an operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences.

[0207] "Regulation" and "regulate" refer to the modulation of gene expression controlled by DNA sequence elements located primarily but not exclusively upstream (5') of the transcription start of the gene. Regulation can result in an all-or-none response to a stimulus or can result in fluctuations in gene expression levels.

[0208] In a further aspect, the present invention provides a host cell transformed with an expression vector according to the invention.

[0209] "Cell," "host cell," or "recombinant host cell" are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in successive generations due to either mutation or environmental influences, such progeny may not actually be identical to the parent cell, but still be within the scope of the terms as used herein.

[0210] As used herein, "host cell" refers to a cell that can be recombinantly transformed with naked DNA or an expression vector constructed using recombinant DNA technology. Drug resistance or other selectable markers are intended, in part, to facilitate the selection of transformants. Additionally, the presence of a selectable marker, such as a drug resistance marker, can help prevent contaminating microorganisms from growing in the culture medium. Such a pure culture of transformed host cells may be obtained by culturing the cells under conditions that require the induced phenotype for survival.

[0211] Host cells of the present invention are transformed or transfected with the expression vectors described herein to express the synthetic spider silk proteins of the present invention. As used herein, "transformation" refers to the process by which a cell's genotype is changed as a result of cellular uptake of exogenous DNA or RNA; for example, the transformed cell expresses a recombinant form of the desired synthetic spider silk protein. The term "transfection" refers to the introduction of a nucleic acid, e.g., naked DNA or an expression vector, into a recipient cell by nucleic acid-mediated gene transfer.

[0212] In some embodiments, the spider silk proteins of the present invention lack post-translational modifications.

[0213] According to some aspects, the present invention provides an expression vector comprising a nucleic acid sequence of the present invention, wherein the nucleic acid sequence is under the expression control of an operably linked promoter and optionally regulatory sequences.

[0214] common terms As used herein, the term "about" refers to ±10%.

[0215] The term "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment described as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments and / or does not necessarily exclude the incorporation of features from other embodiments. The word "optionally" is used herein to mean "provided in some embodiments and not provided in other embodiments." Any particular embodiment of the present invention may include multiple "optional" features unless such features are inconsistent.

[0216] It should be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "MaSp" includes a plurality of such genes and variants, reference to "peptide" includes a reference to one or more peptides known to those skilled in the art, and so forth.

[0217] Also, unless otherwise stated, the use of "or" means "and / or." Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. It is further understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some specific instances, the embodiments could alternatively be described using language such as "consisting essentially of" or "consisting of."

[0218] Throughout this application, various embodiments of the present invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to have specifically disclosed subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the width of the range.

[0219] When a convention similar to "at least one of A, B, and C, etc." is used, generally such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). When a convention similar to "at least one of A, B, and C, etc." is used, generally such a configuration is intended in the sense that one of ordinary skill in the art would understand the convention (e.g., "a system having at least one of A, B, and C" includes, but is not limited to, systems having A alone, B alone, C alone, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). It will be further understood by those skilled in the art that virtually any disjunctive word and / or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibility of including one of the terms, either of the terms, or both terms. For example, the phrase "A or B" will be understood to include the possibilities of "A" or "B" or "A and B."

[0220] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice of the disclosed methods and compositions, exemplary methods, devices, and materials are described herein. [Example]

[0221] Experimental procedure Synthesis of a sequence encoding a single repeating unit of dragline spider silk protein: A 35 amino acid sequence was designed representing the average consensus sequence of the 15 repeats that make up the repeat region of the (MaSp)-based polymer (Genbank entry U47856). The average consensus peptide sequence is SGPGGYGPGSQGPSGPGGYGPGGPGSSAAAAAAAA (SEQ ID NO: 11), which is encoded by the 105 DNA base pair sequence: 5'-TCTGGTCCTGGAGGTTATGGCCCAGGAAGCCAAGGACCATCTGGTCCAGGAGGATATGGTCCAGGCGGACCTGGCTCTAGTGCAGCAGCTGCCGCAGCAGCTGCA-3' (SEQ ID NO: 9). The above synthetic DNA was obtained in the pPCR-ScriptAmpSK(+) plasmid.

[0222] Bacterial growth The pET24R-expressing bacteria were inoculated into 3 mL of LB medium starter containing chloramphenicol and kanamycin, grown to an OD of approximately 0.6 (measured in 100 μL in a 96-well plate), and plated onto growth medium.

[0223] Bacteria were grown at 37°C with shaking, and OD600 and beta-sheet specific staining were performed after approximately 20 hours. Significant beta-sheet staining was apparent after 24 hours.

[0224] Lysis and purification The bacteria were centrifuged and resuspended in deionized water. Detergent solution was then added, and the resulting suspension was shaken overnight at 37°C. After centrifugation, the pellet was resuspended in 6 M urea. After centrifugation, the pellet was resuspended and washed several times with detergent solution.

[0225] For quantification, 1 mL of the polymer suspension was washed with deionized water and dried on a microscope glass. The glass was weighed before and after application of the (MaSp)-based polymer, and the residue was calculated.

[0226] UV spectroscopy Spider silk (MaSp)-based polymers expressed in bacterial SVXE were dissolved by heating in 8 M LiBr and diluted 1:10 with 6 M urea. UV spectra were measured at 240-350 nm on an Ultrospec 2100 spectrophotometer in a disposable UV cuvette. 8 M LiBr diluted 1:10 with 6 M urea was used as a control.

[0227] staining The beta sheet-specific stain was dissolved in dimethyl sulfoxide (DMSO) at 0.8 mg / mL and stored at room temperature protected from light. To a suspension of E. coli in medium, 1–30% Triton X-100 was added, and the bacteria were centrifuged. The bacterial pellet was resuspended in PBS, and 1–50 μL / mL of the beta sheet-specific stain was added. The suspension was incubated at room temperature for 30 minutes, then centrifuged, and the bacterial pellet was resuspended in an equal volume of PBS. If necessary, the stained pellet was stored at 4°C. Fluorescence was read in a 96-well plate using a Cytation fluorometer. OD600 was measured in parallel, and fluorescence was normalized to the OD600 value. To stain the final preparation of SVX-E, the MaSp-based polymer was resuspended in PBS. The remaining steps for bacterial staining were the same.

[0228] Differential scanning calorimetry (DSC) Samples of SVX-E or Sf9-derived SVX were washed with the remaining detergent water, and the suspension was dried. Approximately 5 mg was weighed into capsules, and scanning calorimetry was performed in a Star calorimeter (Mettler Toledo) at a rate of 10° / min over the range of 25–300°C. In parallel, a sample of SVX-E was dissolved in 6 M guanidine thiocyanate and dialyzed against 6 M urea and then deionized water (partial precipitation occurred in water). The denatured protein was dried, and scanning calorimetry was performed under the same conditions.

[0229] TEM Spider silk fibers (SVX) or spider silk polymers expressed in bacterial SVX-E suspensions were deposited on carbon-coated copper grids and left for 5 minutes. The grids were washed with distilled water and stained with 2% (w / v) uranyl acetate for an additional 2 minutes. Analysis was performed using a TEM microscope at 120 keV.

[0230] SEM Scanning electron microscopy (SEM) of various samples was performed according to standard protocols.

[0231] Dynamic Light Scattering (DLS) SVX or SVX-E suspensions were diluted 1:100 with 0.07% surfactant solution and filtered through a 0.22 μm filter. DLS analysis was performed using a Malvern Zetasizer Nano device.

[0232] mass spectrometry SVX or SVX-E suspensions were precipitated by centrifugation, dissolved in 6 M guanidine thiocyanate, and dialyzed into two changes of 6 M urea. For analysis, samples were diluted 1:10 (final urea concentration 0.6 M), possible disulfide bonds were reduced with DTT, blocked with iodoacetamide, and proteins in two separate tubes were cleaved with either trypsin or chemotrypsin.

[0233] The sequences of the resulting peptides were analyzed on a Q Exactive mass spectrometer, and the sequences were analyzed against the predicted sequence of SVX and against databases of baculovirus proteins and Spodoptera frugiperda (sf9 source) for SVX, and a database of E coli proteins for SVX.

[0234] Amino acid analysis For amino acid analysis, SVX or SVXE samples were hydrolyzed with 6 M HCl at 110°C for 18–24 h.

[0235] FTIR The SVX or SVX-E samples were dried on microscope slides at 100 °C, and the IR spectra of the dried materials were measured using a Nicolet iS5 FTIR spectrometer (Thermo Fisher Scientific). The amide I and amide II peaks were used as markers for the presence of protein, and the precise position of the amide I peak was used to assess the secondary structure. The peaks were 1610–1630 cm. -1 The peak position is characteristic of amyloid-like β-sheets.

[0236] General Protocol for Reinforcement of Polyurethane with Spider Silk Polymers 1. Dispersion in organic solvents: Different amounts of spider silk polymer in aqueous suspension (Table 1) were centrifuged and resuspended in double distilled water (DDW). The suspension was allowed to settle for 3 minutes. It was then centrifuged and resuspended in ethanol. It was then centrifuged and resuspended twice in a sufficient amount of dry ethereal solvent (to stabilize the suspension). The suspension was then filtered through a 40 μm filter. It was then centrifuged and resuspended in dry ethereal solvent. The suspension was placed on a rotary shaker for 10 to 200 minutes (at room temperature).

[0237] In the case of cellulose, powdered cellulose was directly dispersed in a sufficient amount of ethereal solvent without prior steps of centrifugation and resuspension in water and ethanol.

[0238] 2. Polymer solubilization: Different amounts of polyurethane (Table 1) were dissolved in a sufficient amount of ethereal solvent in a clear glass vial equipped with a corkscrew (shaker: 37°C, 200 rpm, 1-24 hours).

[0239] 3. Mixing of spider silk suspension with polymer solution: The spider silk polymer suspension was centrifuged and resuspended in a sufficient amount of ethereal solvent. It was then sonicated using an ultrasonic generator. The size, disruption, and aggregation state of the spider silk polymer suspension were examined using an optical microscope. The suspension was poured into the polymer solution. The polymer and spider silk polymer suspension were thoroughly mixed until homogeneous and placed on a rotary shaker (room temperature, 50 rpm) for 60 minutes.

[0240] 4. Degassing: The suspension was left without shaking at 20-60°C for 0.5-5 hours (degassing). If bubbles were observed, longer degassing was continued overnight in a closed container.

[0241] 5. Casting: The size, breakage, and aggregation state of the spider silk polymer suspension were examined using an optical microscope. The contents of the flask were completely poured (18.2 g) into a 9 cm diameter glass Petri dish over a period of 14 hours. The Petri dish was covered with cardboard and placed under a plastic box in a hood. The reinforced polyurethane sheet was then removed from the Petri dish and placed under vacuum at 80 °C for 90 minutes. The reinforced polyurethane sheet was kept at room temperature for 2 days and then cut into 60 × 7 mm strips.

[0242] 6. Mechanical property measurements: The composites were analyzed on a Lloyd LS5 universal testing machine. The strips were pulled at a pulling rate (mm / min) of 50 mm / min. A 50 N load cell was used for accurate evaluation of Young's modulus, and a 5 kN load cell was used for elongation and (ultimate tensile strength) UTS. [Table 1]

[0243] Protocol for dissolution profiles of SVX and SVX-E in various denaturants Stock solutions of 8 M guanidine thiocyanate, 7.5 M guanidine hydrochloride, and 6 M urea were prepared in DDW, and different concentrations of the appropriate compounds were dispensed in 80 μL volumes into a 96-well plate. 20 μL of SVX (6.3 mg / mL) or SVXE (10 mg / mL) was added and mixed well, and the OD600 of each well was measured. Repeated measurements of the same well after 2 hours or overnight did not yield any significant differences.

[0244] Example 1 Bacterial protein expression The bacteria express lysozyme, which breaks down the cell wall. Bacteria expressing only lysozyme were used as a control. The neutral pH is 5.8.

[0245] β-sheet staining was used to follow the formation of insoluble polymers. After purification, the resulting product is protein. The average yield (before optimization) is 105 mg / L medium.

[0246] After solubilization in 6 M guanidine SCN (GuaSCN) and dialysis into urea, the UV spectrum is characteristic of tyrosine-containing proteins, with a maximum at 276 nm (Figure 1A).

[0247] The amino acid content corresponds to the predicted sequence (Fig. 1B).

[0248] The FTIR spectrum shows the characteristic peaks of amide bonds, amide I and amide II (Figure 1C).

[0249] The resulting product is a particle containing β-sheets. The particle size was determined to be 1.04 ± 0.2 μm using dynamic light scattering (DLS) (Figure 2), and FTIR analysis of the particles confirmed the β-sheet structure (Figure 3).

[0250] The amide I peak of the spider silk protein (SVXE) expressed in E. coli is at 1622 cm , similar to the spider silk protein (SVX) derived from Sf9. -1The SVX has a maximum at 1000 Hz, which is a wavelength characteristic of β-sheets. HFIP-solubilized SVX (resulting in an α-helical conformation) or native α-helical protein (BSA) exhibit a shifted amide I peak.

[0251] The FTIR spectrum of the spider silk protein (SVX) expressed in sf9 cells was similar to that of the spider silk protein (SVXE) expressed in E. coli (Figure 4).

[0252] When compared with worm silk, the spectrum of silkworm silk shows unique peaks that are not present in the spectra of SVX and SVX-E (FIG. 5).

[0253] Differential scanning calorimetry (DSC) of the particles confirms the crystalline structure.

[0254] The particles have a phase transition at Tm = 216°C (Figure 6A). The phase transition peak disappears for proteins denatured with 6M guanidine SCN (Figure 6B).

[0255] Fibers derived from Sf9 have a phase transition at Tm = 242 °C (Figure 6C).

[0256] TEM of SVXE particles reveals a microstructure not seen in typical inclusion bodies (Figure 7A-B). Furthermore, SEM images of porous SVXE particles (Figure 21A-B) reveal fibers composed of nanofibrils (represented by arrows in Figure 21B), resulting in highly porous particles. Upon aggregation, the porosity of the particles clearly decreases (Figure 21C-D).

[0257] 21E shows SEM images of purified fermentation products resulting from expression of different constructs having the amino acid sequence set forth in SEQ ID NO: 10 (MSYYHHHHHHDYDIPTTENLYFQGAMPRKSPFPRPEL). Although the fibers obtained from expression of the different constructs are in the form of particles, these fibers are substantially devoid of nanofibrils and lack a porous structure.

[0258] FTIR spectra (data not shown) of SVXE fibers (represented by Figures 21A-B) and fibers obtained from expression of a different construct having the sequence set forth in SEQ ID NO:10 (represented by Figure 21E) indicate that the SVXE fibers are characterized by a substantially increased amount of beta sheets compared to fibers having the sequence set forth in SEQ ID NO:10.

[0259] Example 2 Polymer reinforcement with bacterially expressed spider silk proteins In an exemplary experiment, polyurethane P490RSJT was toughened with increasing amounts of SVX-E (Table 2). [Table 2]

[0260] The results suggest a dose-dependent response, with Young's modulus increasing with increasing SVX-E%, while UTS, elongation at break%, and toughness decrease (Figure 8A-B).

[0261] Similar experiments were performed using polymer E394POTA reinforced with various concentrations of SVX-E. The results showed a dose-dependent response, with Young's modulus increasing with increasing SVX-E%, while UTS, elongation at break, and toughness decreased (Figure 9A-E).

[0262] Figures 10A-E show comparative graphs of composites made from polyurethane P490RSJT containing 20% ​​SVX or SVX-E. Both SVX and SVX-E stiffened the polymer, as evidenced by an increase in Young's modulus compared to the control. There was no difference between SVX and SVX-E in this regard. Both SVX and SVX-E decreased tensile strength, elongation %, and toughness compared to the control. SVX-E showed a greater effect than SVX.

[0263] Figures 11A-E show comparative graphs of composites made from polyurethane E394POTA containing 20% ​​SVX or SVX-E. Both SVX and SVX-E stiffened the polymer, as evidenced by an increase in Young's modulus compared to the control. There was no difference between SVX and SVX-E in this regard. Both SVX and SVX-E decreased tensile strength, elongation %, and toughness compared to the control. Strain-stress curves (not shown) showed similar behavior as in Figures 8B, 9E, and 10E. SVX-E showed a greater effect than SVX.

[0264] Table 3 shows a comparison of composites made from polyurethane P490RSJT containing 20% ​​SVX or SVX-E, and composites made from polyurethane PU399 containing 5% and 10% SVX or SVX-E. [Table 3]

[0265] Additional polymers were reinforced with SVX-E fibers, and the mechanical properties of the resulting materials were investigated. The results are summarized below (Table 3A) and show a significant increase in Young's modulus (130-1160%, including any range in between) for the SVX-E reinforced polymer compositions. [Table 4]

[0266] Additionally, film formers were reinforced with 10% w / w SVX-E fiber, and the mechanical properties of the resulting materials were investigated. The results are summarized in Figure 14 and demonstrate significant increases (73-145%) in Young's modulus and UTS for the film formers (pullulan and Liftonin Xpress®) reinforced with SVX-E. Furthermore, as shown in Figure 14, significant increases in storage modulus (10-500%, including any range in between) and loss modulus (10-60%, including any range in between) were obtained for the film formers tested (pullulan, Liftonin Xpress®, Intansyl®, SKI Nacture®, TriK Fision®, LiftLiss SB®, and Gosulin Agave®) reinforced with SVX-E.

[0267] Example 3 Hyaluronic acid loading and release Spider silk fibers (SVX) or bacterially expressed spider silk fibers (SVX-E) were washed twice with ethanol and then with water (as described above). 10 mg of hyaluronic acid (HA) was added to 10 mg of SVX dispersed in 1–20 mL of water. The pH was adjusted with HCl or phosphate buffer, and water was added to the desired volume. The mixture was shaken and centrifuged. The supernatant was discarded, and a small sample of the pellet was dried on a glass slide to obtain the HA+SVX-E or HA+SVX pellet. The pellet was then examined by FTIR (Nicolet iS5 FTIR spectrometer, Thermo Fisher Scientific). The remaining pellet was resuspended in water, and the suspension was shaken at 200 rpm at 25 °C for 1–30 min. This process was repeated several times. For each pellet sample analyzed by FTIR, the percentage of HA in the total dry weight was calculated by dividing the intensity of the peaks characteristic of HA by the intensity of the peaks characteristic of the polymer (Table 4). These results confirm the ability of MaSp-based fibers (e.g., SVX or SVX-E) to stably encapsulate additional compounds (e.g., HA), with a w / w ratio of encapsulated compound to fiber (e.g., SVX or SVX-E) of approximately 1:1. [Table 5]

[0268] Example 4 Effect of pH on the formation of SVX-E in E. coli The following example shows that the formation of SVX-E in E. coli depends on the pH of the medium (Figure 15). Detection of SVX-E was performed using a beta-sheet-specific fluorescent dye that binds only to self-aggregated spider silk, but not to soluble spider silk.

[0269] The data also show that SVX-E is expressed only in the presence of lactose (which is an inducer of SVX-E expression) and an expression plasmid for SVX-E.

[0270] FIG. 16 shows stained images taken by confocal microscopy at various time points in cultures grown at pH 7.5 or pH 5.8.

[0271] Example 5 SVX-E expression has a biphasic kinetic pattern The following example demonstrates that SVX-E is produced in a final, insoluble form after a delay following induction of recombinant spider silk protein. Protein synthesis is not induced at a specific time by adding an inducer (e.g., lactose or IPTG), but rather because the inducer is present in the growth medium from the beginning. (If a small amount of glucose is present as the preferred carbon source, once this is consumed, lactose is utilized and recombinant protein synthesis begins.) A method for tracking the kinetics of recombinant protein synthesis is to compare the growth rates of two cultures: one with lactose (expression culture) and one without lactose (control culture). The difference in growth rates (absorbance measured at 600 nm) between the two cultures indicates that the culture with lactose had a slower growth rate than the control culture without lactose. Without being bound by any particular theory, it is known that when bacteria express recombinant proteins, their growth rate slows because they utilize metabolic machinery for protein synthesis rather than growth. Therefore, this is interpreted as an induction of spider silk expression in the slower culture.

[0272] Figure 17 shows that the protein expression time frame lags behind the control cultures. We used a probe specific for beta-sheet crystals, structures formed by beta-sheets stacking on top of each other (pleated beta-sheets), a well-known property of natural and artificial spider silks (such as SVX-E), but not soluble spider silk proteins (spidroins).

[0273] The increase in insoluble SVX-E occurs after the two cultures achieve equal growth rates at approximately 20 hours, at which point the bacteria no longer express SVX-E. Furthermore, the absolute growth curves (Figure 18) show that the formation of insoluble SVX-E begins only after the bacteria reach stationary phase, at which point the recombinant protein is normally no longer expressed.

[0274] Taken together, the results indicate that at pH 5.8, the production of recombinant spider silk has biphasic kinetics, with the formation of soluble spider silk proteins in the first phase and the accumulation of a critical intracellular concentration of soluble proteins in the second phase, leading to the self-aggregation of SVX-E, stabilized by intra- and intermolecular beta-sheets and beta-sheet crystals.

[0275] At pH 7.5 (Figure 19), none of the above occurs: no insoluble SVX is detected (phase 2) and there is no indication of a difference in growth and soluble protein expression in phase 1.

[0276] We observed a similar phenomenon in SF9 cells expressing SVX. SVX, detected by specific staining of the same beta-sheet crystals, appeared 70 hours after the cultures were infected with baculovirus, at which point the cells had reached stationary phase and were no longer dividing. These results again demonstrate the biphasic kinetics of SVX production in insect cells.

[0277] Example 6 Dissolution profiles of SVX and SVX-E in various denaturants Table 5 summarizes the experimental data, showing that SVX and SVX-E exhibit similar dissolution profiles with the well-known denaturants (DA) urea (not shown), guanidine chloride, and guanidine thiocyanate. Table 5 also shows the amount of a particular denaturant (C) required to dissolve 50% w / w of the total mass of any one of the fibers (SVX and SVX-E). 1 / 2 The molar concentrations of DA (DA) are shown in Table 5. As shown in Table 5, dissolution of both fibers (SVX and SVX-E) requires similar concentrations of various DAs. [Table 6]

[0278] The data suggests that both have similar chemical compositions and the difference in dispersibility is due to the smaller size of SVX-E, which results in a higher surface area.

[0279] While the present invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications, and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications, and variations that fall within the spirit and broad scope of the appended claims.

[0280] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference. Furthermore, citation or identification of a reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

1. a synthetic major ampullate spidroin protein (MaSp)-based polymer, said MaSp-based polymer being in the form of particles having an average particle size in the range of 0.8 μm to 1.5 μm; the particles are porous particles characterized by a BET surface area of ​​at least 10 m / g; the MaSp-based polymer comprises a repeat region comprising 10 to 20 repeats, each of the repeats comprising the amino acid sequence set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPPSGPGGYGPGGPGSS) or SEQ ID NO: 3 (AAAAAAAAASGPGGYGPGSQGPPSGPGGYGPGGPGSS); Synthetic MaSp-based polymers.

2. 10. The synthetic MaSp-based polymer of claim 1, wherein the MaSp-based polymer is a water-insoluble polymer.

3. 3. The synthetic MaSp-based polymer of claim 1 or 2, having a DSC pattern showing at least one endothermic peak in the range of 200°C to 280°C.

4. 10. The synthetic MaSp-based polymer of claim 1, wherein the particles are characterized by a BET surface area of ​​at least 80 m2 / g.

5. 10. The synthetic MaSp-based polymer of claim 1, wherein the particle comprises a plurality of nanofibrils characterized by a diameter of 1 nm to 50 nm and a plurality of pores.

6. 6. The synthetic MaSp-based polymer of claim 5, wherein the plurality of pores are visible by SEM.

7. 2. The synthetic MaSp-based polymer of claim 1, wherein the MaSp-based polymer is further bound to an additional compound by non-covalent bonding, physical interaction, or both, and the compound is selected from biologically active agents and functional foods.

8. 8. The synthetic MaSp-based polymer of claim 7, wherein the weight / weight (w / w) ratio of the MaSp-based polymer to the additional compound is from 10:1 to 1:

10.

9. 2. The synthetic MaSp-based polymer of claim 1, wherein each of the repeats comprises the amino acid sequence set forth in SEQ ID NO:

3.

10. 2. The synthetic MaSp-based polymer of claim 1, wherein the MaSp-based polymer further comprises a single N-terminal region comprising the amino acid sequence set forth in SEQ ID NO: 4 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLV).

11. 2. The synthetic MaSp-based polymer of claim 1, wherein the MaSp-based polymer further comprises a single C-terminal region comprising the amino acid sequence set forth in SEQ ID NO: 7 (VAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS).

12. 10. A composition comprising the synthetic MaSp-based polymer of claim 1 bound by non-covalent bonds, physical interactions, or both to an additional polymer that is not a MaSp-based polymer, wherein the w / w ratio of the MaSp-based polymer to the additional polymer is from 1:1 to 1:

100.

13. the weight ratio of the additional polymer to the total weight of the composition is 50% to 95% (w / w); Optionally, the composition is formed into the form of a film, suture, surgical mesh, medical adhesive strip, electrospun mesh, skin graft, fat graft, cosmetic, dermal filler, drug eluting / delivery device, replacement ligament, clothing fabric, bulletproof vest lining, cable, tubing, film, rope, fishing line, tire, sporting goods, or reinforced plastic. The composition of claim 12.

14. 10. A process for producing the synthetic MaSp-based polymer of claim 1, comprising: (i) providing a recombinant bacterium transformed or transfected to express a MaSP-based polymer comprising the amino acid sequence set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS); (ii) providing conditions for expression of the MaSP by the recombinant bacterium; (iii) producing synthetic MaSp-based polymers by isolating the expressed proteins; Including, A process wherein step (ii) comprises culturing the bacterium in a solution having a pH in the range of 5 to 6.

5.

15. 15. The process of claim 14, wherein step (ii) comprises providing an inducer of expression.

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