Prokaryotic expression systems and methods of use thereof

Abstract

Provided are a recombinant bacterium expressing / having the ability to express a MaSp-based protein, a nucleic acid encoding the same, and a method for preparing a synthetic traction spider thread.

Description

[0001] This application is a divisional application. The filing date of the original application is July 5, 2020, the application number is (202080058997.X), and the name is “Prokaryotic expression system and its use method”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit of priority under 35 USC §119(e) to U.S. Provisional Patent Application No. 62 / 870,750, filed on July 4, 2019, entitled “PROKARYOTIC EXPRESSION SYSTEMS AND METHODS OF USE THEREOF,” the contents of which are incorporated herein by reference in their entirety. Technical Field

[0004] The present invention relates to synthetic dragline spider silk polymers. Background Art

[0005] Traction silk is considered in the art to be the silk that orb-web spiders use to build the framework and radius of their webs and as a lifeline when they fall or escape danger. To be able to accomplish these tasks, traction fibers exhibit very high toughness due to a combination of high elasticity and strength, which makes them the toughest fibers, whether natural or artificial. For example, traction silk has a diameter six times that of high-strength steel and is three times stronger than Kevlar, one of the strongest synthetic fibers ever created.

[0006] Dragline silk consists of two major polypeptides, primarily known as major ampullate glandular silk proteins (MaSp) 1 and 2, and also known as ADF-3 and ADF-4 in the cross garden spider (Araneus diadematus). The apparent molecular weight of these proteins ranges from 200-720 kDa, depending on the sample age and analytical conditions. The known glandular silk proteins of dragline silk consist of highly iterative blocks of alternating alanine-rich segments and glycine-rich segments, the alanine-rich segments forming crystalline β-sheets in the fiber and the glycine-rich segments being more flexible and mainly lacking ordered structure. The C-terminal region is not repeated, is highly conserved between species, and adopts an α-helical conformation. The N-terminal region of the dragline silk proteins was also found to be highly conserved between different glandular silk proteins and between different spider species.

[0007] Many attempts have been made to produce spider silk synthetically, such as through genetic engineering using bacteria, yeast, plant and mammalian cells in tissue culture and even transgenic goats.

[0008] U.S. Patent No. 8,461,301 relates to an isolated amino acid sequence comprising multiple repeats of a semisynthetic spider silk protein domain or any functional homologue, variant, derivative, fragment or mutant thereof, etc. Other publications related to dragging 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.

[0009] There is an unmet need for improved compositions and methods for producing synthetic spider silk having mechanical properties similar to natural spider silk. Summary of the invention

[0010] The present invention relates to a recombinant bacterium having the ability to produce MaSp-like proteins organized in a β-pleated crystal structure, a method for producing MaSp-like polymers using the same, and a composition comprising the same.

[0011] According to one aspect, the present invention provides a composition comprising synthetic major ampullate spidroin protein (MaSp) based polymers in the form of particles having a size ranging from 0.5 μm to 1.5 μm.

[0012] In some embodiments, the MaSp-based polymer is a water-insoluble polymer.

[0013] In some embodiments, the DSC graph of the composition exhibits at least one endothermic peak in the range of 200°C to 280°C.

[0014] In some embodiments, the particles are porous particles and are characterized by at least 10 m 2 / g of BET surface area.

[0015] In some embodiments, the particle comprises a plurality of nanofibrils.

[0016] In some embodiments, the composition further comprises an additional compound in contact with the MaSp-class polymer.

[0017] In some embodiments, the compound is selected from the group consisting of a bioactive agent and a nutraceutical.

[0018] In some embodiments, the weight ratio (w / w) of the MaSp-based polymer to the additional compound is between 10:1 and 1:10.

[0019] In some embodiments, the MaSp-based polymer comprises an amino acid sequence as set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS).

[0020] In some embodiments, the MaSp-based polymer comprises an amino acid sequence as set forth in SEQ ID NO: 3 (AAAAAAAASGPGGYGPGSQGPSGPGGYGPGGPGSS).

[0021] In some embodiments, the MaSp-type polymer comprises 10-20 repeating sequences of SEQ ID NO:3.

[0022] In some embodiments, the MaSp-like polymer includes a single N-terminal region selected from the group consisting of: SEQ ID NO: 4 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLV).

[0023] In some embodiments, the MaSp-based polymer includes a single C-terminal region selected from the group consisting of: SEQ ID NO: 7 (VAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDALVQ ALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS).

[0024] In some embodiments, the composition is obtained by expressing MaSp in bacteria.

[0025] In some embodiments, the bacteria is Escherichia coli.

[0026] In another aspect, there is a composition comprising a MaSp-based polymer of the present invention in combination with an additional polymer, wherein the w / w ratio of the MaSp-based polymer to the additional polymer is between 1:1 and 1:100.

[0027] In some embodiments, the w / w concentration of the additional polymer is from 50% to 95% (w / w) of the total composition.

[0028] In some embodiments, the additional polymer is selected from a synthetic polymer, a thermoplastic polymer, a thermosetting polymer, a film former, an epoxy resin, a polyester, a polyamide, a polyol, a polyurethane, a polyethylene, a silicon, a liquid crystal polymer, a maleic anhydride grafted polypropylene, a polyacrylate, a polycarbonate, a polyamide, nylon 4,6, nylon 6, nylon 6,6, nylon 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, polylactic acid (PLA) or a copolymer thereof, polycaprolactone (PCL), xanthan gum, cellulose, collagen, elastin, keratin, cotton, rubber, cellulose, wool, and any combination thereof.

[0029] In some embodiments, the film former is a solid film former.

[0030] In some embodiments, the w / w ratio between the film-forming agent and the MaSp-like fibers is 5:1 to 50:1.

[0031] In some embodiments, the composition is characterized by at least one improved mechanical property compared to the property of another polymer without the MaSp class polymer, wherein the property is selected from: 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.

[0032] According to another aspect, the present invention provides an article comprising the composition described herein, wherein the article is in the form of a film, suture, surgical mesh, medical tape, electrospun mesh, skin graft, fat graft, cosmetic, subcutaneous filler, drug eluting / delivery device, replacement ligament, clothing fabric, bulletproof vest lining, cable, tube, film, rope, fishing line, tire, sports equipment and reinforced plastic.

[0033] According to another aspect, the present invention provides a recombinant bacterium having the ability to express a MaSp-type polymer, the polymer comprising the amino acid sequence set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS).

[0034] According to another aspect, the present invention provides a method comprising: (i) providing a recombinant bacterium as described herein; (ii) providing conditions for the bacteria to express MaSP; and (iii) isolating the expressed protein, thereby producing synthetic traction spider silk.

[0035] In some embodiments, step (ii) comprises providing a solution having a pH in the range of 5 to 6.5.

[0036] In some embodiments, step (ii) comprises providing an expression inducer.

[0037] In some embodiments, step (ii) comprises waiting for a period of time to obtain an insoluble polymer.

[0038] In some embodiments, step (iii) further comprises drying the synthetic traction spider silk.

[0039] In some embodiments, the method further comprises an enrichment step using an additional polymer.

[0040] In some embodiments, the enriching step comprises mixing a solution of synthetic dragline spider silk with a solution of a second polymer.

[0041] From the detailed description given below, other embodiments and the entire scope of application of the present invention will become apparent. However, it should be understood that although the preferred embodiments of the present invention are shown, the detailed description and specific examples are given by way of illustration only, because from the detailed description, various changes and modifications within the spirit and scope of the present invention will be apparent to those skilled in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1A -C is the UV spectrum of the obtained protein ( Figure 1A ), a graph showing measured versus expected amino acid content ( Figure 1B ) and Fourier transform infrared spectroscopy (FTIR) spectra of the resulting protein (SVX-E) ( Figure 1C ).

[0043] Figure 2 is a graph showing the particle size distribution of the protein obtained using the described expression system.

[0044] Figure 3 is the FTIR analysis of the obtained particles.

[0045] Figure 4 FTIR spectra of spider silk protein (SVX-E) expressed in bacteria and spider silk SVX expressed in Sf9 cells.

[0046] Figure 5 FTIR spectra of spider silk expressed in Sf9 cells (SVX) and spider silk protein expressed in bacteria (SVX-E) compared to silk fibers.

[0047] Fig. 6A -C is the differential scanning calorimetry (DSC) curve of spider silk protein expressed in bacteria SVX-E ( Fig. 6A and Figure 6B) and DSC curves of SVX protein ( Figure 6C );

[0048] Fig. 7A -B is the same as SVX from Sf9 ( Fig. 7A ) compared to the transmission electron microscopy image of SVXE ( Figure 7B ).

[0049] Fig. 8A -B is a graph of the dose-dependent effect of SVXE on the mechanical properties of polymer P490RSJT composites. Fig. 8A Represents a bar graph showing the increased stress at 500% modulus (10% to 20% increase) for P490RSJT enriched with 10%, 20% and 30% SVXE compared to the control (original P490RSJT polymer). Figure 8B Shown are stress-strain curves for P490RSJT enriched with 5%, 10%, 20% and 30% SVXE compared to the control (original P490RSJT polymer).

[0050] Fig.9A -E is a graph of the dose-dependent effect of SVXE on the mechanical properties of polymer E394POTA composites. Fig.9A is a bar graph showing the increased Young's modulus (increases from about 50% to about 400%) of E394POTA enriched with 10%, 20%, and 30% SVXE compared to the control (original E394POTA polymer). Fig. 9B -D is a graph showing the UTS of E394POTA enriched with 10%, 20% and 30% SVXE compared to the control (original E394POTA polymer). Fig. 9B ), % elongation at break ( Fig. 9C ) and toughness ( Fig.9D ) of the descending bar graph. Fig.9E Shown are stress-strain curves for E394POTA enriched with 10%, 20% and 30% SVXE compared to the control (pristine E394POTA polymer).

[0051] Fig. 10A -E is a comparison of the mechanical properties of polymer P490RSJT PU composites enriched with 20% SVX, milled SVX, and SVX-E. Fig. 10A is a bar graph showing the increased Young's modulus (between about 150% to about 300% increase) of the enriched P490RSJT PU compared to the control (virgin P490RSJT PU polymer). Fig. 10B-D is a graph showing the UTS of P490RSJT PU enriched with 10%, 20% and 30% SVXE compared to the control (original P490RSJT PU polymer) Fig. 10B ), % elongation at break ( Fig. 10C ) and toughness ( Fig. 10D ) of the descending bar graph. Fig. 10E Shown are stress-strain curves of P490RSJT PU enriched with 20% SVX, milled SVX & SVX-E compared to control (virgin E394POTA polymer).

[0052] Fig.11A -E are graphs showing a comparison of the mechanical properties of polymer E394POTAPU and composites enriched with 20% SVX, ground SVX and SVX-E, respectively.

[0053] Fig.12 is a bar graph showing the sizes 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.

[0054] Fig.13 is a graph showing comparative stress-strain curves of PU samples enriched with 15% w / w SVX-E. The graph shows the stress-strain behavior of PU samples enriched with non-porous or aggregated particles (SVX aggregated) and porous or non-aggregated particles (SVX well dispersed) compared to the control PU sample (solid line).

[0055] Fig.14 is a table summarizing the mechanical properties of film formers enriched with 10% w / w SVX-E.

[0056] Fig.15 is a graph showing the amount of SVX-E in E. coli after 30 hours of cultivation; SVX-E was measured using a specific β-sheet crystal-binding fluorescent dye;

[0057] Fig.16 The SVX-E staining images were obtained at different culture times at pH 5.8 and pH 7.5. The green spots are caused by the specific β-crystals binding to the fluorescent dye entering the cells.

[0058] Fig.17 is a graph of growth rate and staining differences of β-sheet crystals at pH 5.8 over a period of time;

[0059] Fig.18 is a graph of absolute growth at pH 5.8 over a period of time;

[0060] Fig.19 is a graph of growth rate and staining differences of β-sheet crystals at pH 7.5 over time;

[0061] Fig. 20 is a 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) versus the number of washes (X-axis).

[0062] Fig.21A -E is a scanning electron micrograph (SEM) of MaSp-like (SVX) fibers. Fig.21A and Fig. 21B An image representing a porous fiber. Fig. 21C and Fig.21D An image representing a non-porous particle. Fig.21E Fibers resulting from expression of a different sequence (SEQ ID NO: 10) are shown. DETAILED DESCRIPTION

[0063] In some embodiments, provided herein are bacteria expressing compositions comprising proteins of the class of major ampullae silk proteins (MaSp) useful for preparing synthetic dragline spider silk. The present invention further provides articles and composite materials comprising these compositions.

[0064] The present invention is based in part on the surprising discovery that microorganisms such as E. coli are surprisingly effective hosts for producing MaSp proteins. MaSp proteins are not only expressed in E. coli, but also self-assemble inside the bacteria to form functional spider silk. Surprisingly, this self-assembly only occurs when the external pH is <7, but not at neutral pH values ​​of 7-7.5.

[0065] The present invention is further based in part on the surprising discovery that microbially produced MaSp proteins (also used herein as SVX-E) unexpectedly possess superior mechanical properties compared to native dragline spider silk or compared to dragline spider silk proteins produced in other expression systems.

[0066] The present invention is further based in part on the unexpected discovery that SVX-E type fibers are in the form of particles having a size ranging from 0.5 μm to 1.5 μm. Fig.12 ), in contrast to the control MaSp-based polymers, these particles remain stable (e.g., substantially free of aggregates) in aqueous dispersions. In addition, the SVX-E-based fibers are characterized by increased porosity and an extremely large BET surface area (e.g., about 180 m 2 / g).

[0067] Combination of substances

[0068] According to some embodiments, a composition is provided, which includes a synthetic macroamyricularia fibroin (MaSp)-like polymer in the form of particles with a size ranging from 0.5 μm to 1.5 μm. In some embodiments, the MaSp-like polymer is an insoluble polymer. In some embodiments, the DSC graph of the composition exhibits at least one endothermic peak in the range of 200°C to 280°C. In some embodiments, the composition has a thermal conductivity of 1615 cm -1 Up to 1635cm -1 The amide peak is within the range of , as measured by FTIR analysis.

[0069] According to some embodiments, a composition comprising a synthetic MaSp-like polymer is provided, wherein the MaSp-like polymer has at least one characteristic selected from the following:

[0070] a) is an insoluble polymer;

[0071] b) is in the form of particles with a size ranging from 0.5 μm to 1.5 μm;

[0072] c) has at least 10m 2 / g BET surface area;

[0073] d) having a DSC chart exhibiting at least one endothermic peak in the range of 200° C. to 280° C.; and

[0074] e) has a peak at 1615cm -1 and 1638cm -1 The amide peak is within the range of , as measured by FTIR analysis.

[0075] According to some embodiments, a composition is provided, which includes a synthetic MaSp-like polymer in the form of particles. In some embodiments, the size of the particles is in the range of 0.5μm to 1.5μm, 0.7μm to 1.5μm, 0.8μm to 1.5μm, 0.9μm to 1.5μm, 0.5μm to 1μm, 0.7μm to 1μm, 0.8μm to 1μm, 0.9μm to 1μm, 0.5μm to 1.3μm, 0.5μm to 1.2μm, 0.7μm to 1.3μm, 0.7μm to 1.2μm or 0.9μm to 1.2μm, including any range therebetween. In some embodiments, the particle size refers to the average particle size in an aqueous solvent (e.g., an aqueous dispersion) measured by laser diffraction (see the Examples section). In some embodiments, the particle size refers to the dry particle size (e.g., the particle size substantially free of a shell including water molecules).

[0076] In some embodiments, a composition is provided, which includes an insoluble MaSp polymer. In some embodiments, the insoluble MaSp polymer is in the form of particles. In some embodiments, the insoluble MaSp polymer is insoluble in an organic solvent. In some embodiments, the insoluble MaSp polymer is insoluble in an aqueous solution.

[0077] As used herein, the term "insoluble" refers to a material that does not dissolve but can be dispersed to varying degrees when exposed to an excess of solvent. 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 a solvent. In some embodiments, "insoluble" refers to a material that can only be partially dissolved in a 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 includes an aqueous surfactant solution. Surfactants (e.g., ionic surfactants) are well known in the art. In some embodiments, the solvent includes an aqueous urea solution.

[0078] In some embodiments, the disclosed compositions are characterized by a determined differential scanning calorimetry (DSC) graph. In some embodiments, a "DSC graph" refers to the position of a peak. In some embodiments, a "peak" refers to an exothermic peak. Throughout this document, "peak position" or "peak position" refers to a peak along the temperature axis in a thermogram, and in some embodiments, may refer to the peak position at any peak intensity. It will be appreciated by those skilled in the art that the data obtained in a DSC measurement depends in part on the instrument used and the environmental conditions (e.g., humidity) when the measurement is performed.

[0079] In some embodiments, the disclosed compositions are characterized by a DSC plot exhibiting at least one endothermic peak in the range of 200° C. to 280° C. In some embodiments, the disclosed compositions are characterized by a DSC plot 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.

[0080] In some embodiments, the disclosed compositions are characterized by a DSC plot 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 plot of a corresponding composition comprising (MaSp)-like fibers.

[0081] In some embodiments, the disclosed compositions have no DSC peaks in the range of about -100° C. to about 190° C. In some embodiments, the disclosed compounds have no DSC peaks in the range of about -100° C. to about 25° C. In some embodiments, the disclosed compositions are characterized at least in exhibiting a DSC plot with no exothermic peaks in the range of 40° C. to 70° C.

[0082] In some embodiments, the disclosed compositions have no DSC peaks in the range of about -100°C to about -50°C. In some embodiments, the disclosed compounds have no DSC peaks in the range of about -50°C to about 0°C. In some embodiments, the disclosed compounds have no DSC peaks in the range of about -0°C to about -25°C.

[0083] In some embodiments, the disclosed compositions are characterized by having a -1 Up to 1635cm -1 In some embodiments, the disclosed compositions are characterized by having an amide peak at 1620 cm -1 Up to 1635cm -1 、1620cm -1 Up to 1630cm -1 、1621cm -1 Up to 1630cm -1 or 1620cm -1 Up to 1625cm -1 , including any range of amide peaks therebetween, as measured by FTIR analysis.

[0084] In some embodiments, the disclosed compositions do not have -1 Up to 1800cm -1 The peaks are within the range of , as measured by FTIR analysis.

[0085] By one embodiment, the MaSp-like polymers of the present invention are assembled by self-assembly. "Self-assembly" means that the monomers, i.e., the synthetic spider silk proteins of the present invention, spontaneously associate with each other in an energetically favorable manner under normal physiological conditions or at room temperature to produce macromolecular structures having the properties described herein. In addition, the MaSp-like polymers of the present invention are very resilient and, once assembled, can withstand extreme chemical attacks, such as solubilization in a 10% w / w surfactant solution and boiling for at least 1 hour.

[0086] "Tenacity" or "tensile strength" refers to the weight that a filament can bear before breaking. The maximum specific stress produced is usually in a filament, yarn or fabric to break the material by tensile testing. According to a specific embodiment, the tensile strength of the MaSp polymer of the present invention is about 100-3000 MPa (MPa = N / mm 2 ), 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.

[0087] "Toughness" refers to the energy required to break a MaSp polymer. This is the area under the stress-strain curve, sometimes referred to as "fracture energy" or work of fracture. According to a specific embodiment, the toughness of the MaSp polymer of the present invention is about 20-1000 MJ / m 3 , about 50-950MJ / m 3 , about 100-900MJ / m 3 , about 120-850MJ / m 3 , about 150-800MJ / m 3 , about 180-700MJ / m 3 , about 180-750MJ / m 3 , about 250-700MJ / m 3 , about 280-600MJ / m 3 , about 300-580MJ / m 3 , about 310-560MJ / m 3 , about 320-540MJ / m 3 or about 350-520MJ / m 3 , most specifically about 350-520 MJ / m 3 .

[0088] "Elasticity" refers to the property of an object that tends to return to its original size and shape after being deformed. Plasticity, which is deformation without recovery, is the opposite of elasticity. In the molecular configuration of MaSp polymers, recoverable or elastic deformation is possible through the stretching (reorientation) of the interatomic and intermolecular structural bonds. In contrast, the breaking of intermolecular bonds and the reformation of new stable positions results in irreversible or plastic deformation.

[0089] "Elongation" means the increase in length expressed as a percentage or fraction of the initial length.

[0090] "Fineness" refers to the average diameter of a MaSp-based polymer or filament (eg, biofilament), which is typically expressed in micrometers (μm).

[0091] According to some aspects, the MaSp class proteins or MaSp class polymers used interchangeably herein are in the form of fibers. As used herein, "fiber" refers to a thin rope of fibrous material composed of two or more filaments twisted together. "Filament" refers to a slender, elongated, thread-like object or structure of indefinite length, ranging from microscopic lengths to a mile or more in length. Specifically, synthetic spider silk filaments are microscopic and proteinaceous. "Biological filaments" refer to filaments produced by proteins, including recombinantly produced spider silk proteins. In some embodiments, the term "fiber" does not include unstructured aggregates or precipitates.

[0092] In some embodiments, the MaSp fibers include multiple MaSp polymers. In some embodiments, the multiple MaSp polymers include polymers with different chemical compositions and / or different molecular weights (MW). In some embodiments, the multiple MaSp polymers include polymers with different numbers of repeat regions.

[0093] In some embodiments, the MaSp polymer or MaSp fiber is substantially free of additional non-MaSp proteins. In one embodiment, the MaSp polymer or MaSp fiber is substantially free of additional polymers (eg, synthetic polymers, non-MaSp peptides, non-MaSp proteins).

[0094] In some embodiments, the proteinaceous fibers are characterized by the size of at least one dimension (e.g., diameter, length). For example, but not limited to, the diameter of the fiber is between 10 nm-1 μm, between 20-100 nm, or between 10-50 nm.

[0095] In one embodiment, MaSp fibers are composed of monomers. In one embodiment, multiple MaSp polymers are arranged in nanofibrils. In one embodiment, multiple nanofibrils are arranged in or constitute fibers. In one embodiment, the monomers or nanofibrils within the MaSp fibers have a diameter of 4 to 16 nm. In one embodiment, the monomers or nanofibrils within the MaSp fibers have a diameter of 6 to 14 nm. In one embodiment, the monomers or nanofibrils within the MaSp fibers have a diameter of 8 to 12 nm. In some embodiments, MaSp fibers include multiple fibrils (e.g., nanofibrils), as exemplified below ( Fig.21A -B). In some embodiments, the fiber having a mutant amino acid sequence (e.g., an amino acid sequence as set forth in SEQ ID NO: 10 (MSYYHHHHHHDYDIPTTENLYFQGAMPRKSPFPRPEL)) is substantially free of nanofibrils, such as Fig.21E In some embodiments, the fiber having a mutant amino acid sequence (e.g., an amino acid sequence as set forth in SEQ ID NO: 10 (MSYYHHHHHHDYDIPTTENLYFQGAMPRKSPFPRPEL) is in the form of a non-porous particle, such as Fig.21E As shown in the example.

[0096] In some embodiments, the diameter of the nanofibril is, 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 diameter of the nanofibril is 3-7 nm. In one embodiment, the nanofibrils have a diameter of 4-6 nm.

[0097] In one embodiment, the diameter of the MaSp fibers is 70 to 450 nm. In one embodiment, the diameter of the MaSp fibers is 80 to 350 nm. In one embodiment, the diameter of the MaSp fibers is 80 to 300 nm. In one embodiment, the diameter of the MaSp fibers is 150 to 250 nm. In one embodiment, the MaSp fibers or MaSp polymers are arranged as a coil. In one embodiment, a single fiber or a MaSp polymer is arranged as a coil. In one embodiment, the diameter of the coil is 5 to 800 microns. In one embodiment, the diameter of the coil is 5 to 500 microns. In one embodiment, the diameter of the coil is 5 to 30 microns. In one embodiment, the diameter of the coil is 5 to 20 microns. In one embodiment, the length of the MaSp fibers or MaSp polymers is 5 to 800 microns. In one embodiment, the length of the MaSp fibers or MaSp polymers is 30 to 300 microns. In some embodiments, the length of the MaSp-like fibers is between 1-200 μm, between 10-100 μm, between 100 and 500 μm, or between 200-500 μm, including any range therebetween.

[0098] In some embodiments, MaSp fibers include a plurality of pores. In some embodiments, MaSp fibers are in the form of particles, as described herein. In some embodiments, the composition includes a plurality of MaSp fibers. In some embodiments, a plurality of MaSp fibers include fibers having different chemical compositions. In some embodiments, a plurality of MaSp fibers are in the form of particles having different sizes and / or different structures. In some embodiments, a plurality of MaSp fibers are in the form of particles having different porosities (represented by BET surface area).

[0099] In some embodiments, MaSp-like fibers are characterized by a porous structure. In some embodiments, MaSp-like fibers are porous fibers. In some embodiments, the porous structure or porous MaSp-like fibers are characterized by a porosity of at least 30% (e.g., 30 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 50% (e.g., 50 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 60% (e.g., 60 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 70% (e.g., 70 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 80% (e.g., 80 to 99%). In some embodiments, the porous structure is characterized by a porosity of at least 90% (e.g., 90 to 99%). In some embodiments, the porous structure is characterized by a porosity of about 90%.

[0100] As used herein, the term "porosity" refers to the volume percentage of a material (eg, a "sponge-like" material) that is composed of pores. In another embodiment, porosity is measured as the pores within the surface area divided by the total surface area (porous and non-porous).

[0101] In some embodiments, the porous structure of the disclosed fibers allows for efficient absorption of water on the fiber surface. That is, and without being bound by any particular theory, this surprising finding can be explained in light of the disclosed fiber structure and its porosity being very different from the porosity of natural spider silk found in nature.

[0102] In some embodiments, the porous MaSp 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-aggregated particles. In some embodiments, the particles include a plurality of pores (i.e., spaces or cavities) formed by interwoven polymeric chains of MaSp polymers. In some embodiments, the entangled or interwoven MaSp polymers form a matrix. In some embodiments, the cosmetic active ingredient fills at least a portion of the pores within the matrix or within the particle. In some embodiments, the cosmetic active ingredient is encapsulated by a plurality of pores.

[0103] In some embodiments, the MaSp-based fibers are porous MaSp-based fibers characterized by a BET surface area 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 ranges or values ​​therebetween.

[0104] In some embodiments, the porous MaSp-like fibers are characterized by a BET surface area between 10 and 200 m 2 / g, between 10 and 50m 2 / g, between 10 and 20m 2 / g, between 20 and 50m 2 / g, between 50 and 70m 2 / g, between 70 and 100m 2 / g, between 100 and 120m 2 / g, between 120 and 150m 2 / g, between 150 and 170m 2 / g, between 170 and 190m 2 / g, between 150 and 190m 2 / g, between 160 and 190m 2 / g, between 170 and 190m 2 / g, between 100 and 190m 2 / g, between 180 and 190m 2 / g, between 170 and 180m 2 / g, between 180 and 200m 2 / g, between 190 and 200m 2 / g, including any range or value therebetween. In some embodiments, the porous MaSp-like fibers are characterized by a BET surface area between 100 and 200 m 2 / g, between 150 and 200m 2 / g, including any range or value therebetween.

[0105] In some embodiments, the MaSp fibers are in the form of nonporous MaSp fibers or nonporous particles. In some embodiments, the MaSp fibers are in the form of aggregated particles. In some embodiments, the nonporous MaSp fibers are characterized by a BET surface area of ​​at most 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, up to 0.5m 2 / g, up to 0.3m 2 / g, up to 0.1m 2 / g, including any range or value therebetween. In some embodiments, the non-porous MaSp-like fibers or non-porous particles are characterized by a BET surface area between 0.01 and 1 m 2 / g.

[0106] It should be understood that non-porous particles (e.g., particles with a BET surface area less than 10 m 2 Particles characterized by a large surface area (e.g., porous particles) have an increased encapsulation capacity compared to particles with a surface area of ​​100 μg / g. Fig.21A -D shows the comparative SEM images of porous or non-aggregated and non-porous or aggregated particles.

[0107] In some embodiments, the term "porous particles" and the term "non-aggregated particles" are used interchangeably herein. In some embodiments, the term "non-porous particles" and the term "aggregated particles" are used interchangeably herein.

[0108] In some embodiments, the porous particles are stable in the dispersion. In some embodiments, the porous particles are significantly more stable in the dispersion than the non-porous particles. In some embodiments, the dispersion is an aqueous dispersion. In some embodiments, the dried porous particles substantially retain their size when redispersed in an aqueous solution. In some embodiments, the porous particles do not aggregate in the aqueous dispersion. In some embodiments, the porous particles do not aggregate in the aqueous dispersion.

[0109] In some embodiments, the non-porous particles are characterized by a particle size 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, 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 when redispersed in an aqueous solution.

[0110] As the following example illustrates ( Fig.12 ), dry non-porous (or aggregated) particles tend to form aggregates when redispersed in aqueous solution. Such aggregates may be tens of microns in size (up to 100 microns). Without being bound by any particular theory or mechanism, it is speculated that the unusual stability of porous particles in dispersion may be related to increased interaction with aqueous solvents due to increased surface area of ​​the porous particles. Those skilled in the art will appreciate 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., an aqueous dispersion), and / or the loading capacity of the composition (e.g., a composition comprising an active substance bound within or encapsulated by a plurality of particles). Furthermore, it is assumed that porous particles can be formed solely by any of the bacterial expression systems described herein.

[0111] In some embodiments, the size of the porous particles is 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.

[0112] 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 aggregated) particles (see Fig.13 ). In some embodiments, the mechanical properties are as described herein. Without being bound by any particular theory or mechanism, it is speculated that porous particles may be advantageous over non-porous particles in enriching materials of MaSp fibers (e.g., polymers enriched in MaSp fibers as described below). It is speculated that materials enriched in porous (or non-aggregated) particles may have a higher enrichment percentage than non-porous (or aggregated) particles, thereby improving the reinforcement properties of MaSp fibers.

[0113] Encapsulation

[0114] In some embodiments, the composition further comprises an additional compound in contact with the MaSp polymer or MaSp fiber. In some embodiments, the additional compound is bound to the MaSp polymer or MaSp fiber. In some embodiments, the composition comprises an additional compound substantially bound to the porous MaSp fiber via non-covalent bonds, physical interactions, or both. In some embodiments, the cosmetic active ingredient fills at least a portion of the pores within the matrix or within the particle, wherein the matrix is ​​formed by interwoven polymeric chains of the MaSp polymer. In some embodiments, the additional compound is encapsulated by a plurality of pores.

[0115] In some embodiments, the porous particles are characterized by 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.

[0116] In some embodiments, the additional compound is stably encapsulated within a plurality of pores of the particle. In some embodiments, the encapsulated additional compound is characterized by a gradual release profile (e.g., at the application site, in solution, or in a dispersion). In some embodiments, the particle encapsulating the additional compound substantially prevents the rapid release of the additional compound therefrom.

[0117] As used herein, the term "stable encapsulation" refers to the ability of a composition to substantially prevent the release of an active ingredient (e.g., an additional compound) therefrom. As used herein, the term "substantially prevents" refers to the total amount of active ingredient released from the composition, such as upon subsequent washing (as described in the Examples section).

[0118] In some embodiments, substantially includes 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%, at least 99% by weight of the additional compound. In some embodiments, substantially includes 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%, at least 99% by weight of the additional compound is bound to the MaSp-like fibers via non-covalent bonds, via physical interactions, or both. Non-covalent bonds are well known in the art and include, among others, hydrogen bonds, pp stacking, van der Waals interactions, and the like.

[0119] In some embodiments, physical interaction refers to encapsulation (ie, entrapment) of the additional compound within the matrix formed by the MaSp-like polymer. In some embodiments, the matrix is ​​bound to or in contact with the additional compound.

[0120] In some embodiments, the additional compound is selected from the group consisting of a biological agent, a pharmaceutical agent, a nutrient, and a dietary supplement.

[0121] As used herein, the term "biological agent (also referred to as biological material)" refers 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, such as a disease or disorder, or can be used to otherwise enhance physical, psychological or mental health. Therefore, the term "pharmaceutical agent" contemplated in the context of the present invention includes any agent having a therapeutic, diagnostic or preventive effect, i.e., any therapeutic agent, diagnostic agent or preventive agent.

[0122] The agent may be an agent that affects or participates in tissue growth, cell growth, cell differentiation, an agent capable of inducing a biological effect such as an immune response, or an agent that may play any other role in one or more biological processes.

[0123] Non-limiting examples of pharmaceutical agents include, but are not limited to, antimicrobial agents (such as 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, anthelmintics, growth factors, hormones, hormone antagonists, antibodies, adjuvants (e.g., in combination with an immunologically active compound such as an antibody), antioxidants, proteins (such as 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.

[0124] The agent may also be a small molecule compound. The term "small molecule compound" refers to a molecule that can affect a biological process. Small molecules may include any number of therapeutic agents currently known and used, or may be small molecules synthesized in such a molecule library for the purpose of screening biological functions. The molecular weight of a small molecule compound is generally 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.

[0125] As used herein, "nutrients" are chemicals required for the survival and growth of an organism or substances used in the metabolism of an organism that must be taken up 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.

[0126] 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), galacto-oligosaccharides (GOS) or oligomannans (MOS).

[0127] As used herein, the term "dietary supplement" (also known as food supplement or nutritional supplement) refers to a product intended to provide nutrients such as vitamins, minerals, fiber, fatty acids or amino acids that are missing or not consumed in adequate amounts in an individual's diet.

[0128] Non-limiting examples of dietary supplements include, but are not limited to, steroids such as dehydroepiandrosterone (DHEA), pregnenolone or its derivatives, hormones such as melatonin, and other substances such as hydrazine sulfate, caffeine, catechins, soy isoflavones, glucosamine, coenzyme Q 10 or ephedrine alkaloids, such as ephedrine, synephrine, norephedrine, or pseudoephedrine.

[0129] The active agent can be positively charged or negatively charged. The active agent can also be neutrally charged. Preferably, the active agent is positively charged or negatively charged. The terms "positive charge" and "cation" and "negative charge" and "anion" can be used interchangeably.

[0130] In some embodiments, the weight ratio (w / w) of the MaSp-like 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 ranges therebetween.

[0131] Polymer enrichment

[0132] In another aspect, there is a composition comprising a MaSp-like polymer and an additional polymer. In some embodiments, the additional polymer is in contact with the MaSp-like fiber or the MaSp-like polymer. In some embodiments, the additional polymer is bound to the MaSp-like fiber or the MaSp-like polymer.

[0133] In some embodiments, the composition includes an additional polymer combined with the MaSp-like fibers to form a composite material. In some embodiments, contacting includes bonding or adhering, wherein bonding is as described herein.

[0134] 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, wherein substantially as described 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.

[0135] In some embodiments, the composition comprises a MaSp-like polymer of the invention in combination with an additional polymer, wherein the w / w ratio of the MaSp-like polymer to the additional polymer is between 1:1 and 1:100, between 1:1 and 1:5, between 1:1 and 1:3, between 1:3 and 1:5, between 1:5 and 1:10, between 1:5 and 1:7, between 1:7 and 1:10, between 1:10 and 1:2, between 1:12 and 1:15, between 1:15 and 1:20, between 1:20 and 1:30, between 1:30 and 1:40, between 1:40 and 1:50, between 1:50 and 1:70, between 1:70 and 1:100, including any range or value therebetween.

[0136] In some embodiments, other polymer is synthetic polymer. In some embodiments, synthetic polymer is selected from thermoplastic polymer and thermosetting polymer. Non-limiting examples of synthetic polymer include but are not limited to epoxy resin, polyester, polyamide, polyol, polyurethane, polyethylene, silicon, liquid crystal polymer, maleic anhydride grafted polypropylene, polyacrylate, polycarbonate, polyamide, nylon 4,6, nylon 6, nylon 6,6, nylon 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, polylactic acid (PLA) or its copolymer, polycaprolactone (PCL), xanthan gum, cellulose, collagen, elastin, keratin, cotton, rubber, cellulose, wool and film former or its any combination.

[0137] In some embodiments, the additional polymer is a synthetic polymer. In some embodiments, the additional polymer is a film former. In some embodiments, the w / w content of MaSp fibers in the composition is between 0.1 and 20%, between 0.1 and 1%, between 1 and 2%, between 2 and 5%, between 5 and 7%, between 4 and 6%, between 6 and 8%, between 8 and 10%, between 10 and 12%, between 12 and 15%, between 15 and 20%, including any range or value therebetween. In some embodiments, the composition comprising greater than 20% w / w of MaSp is a heterogeneous composition. In some embodiments, the composition comprising greater than 20% w / w of MaSp is characterized by the formation of aggregates.

[0138] In some embodiments, the film former is selected from a liquid film former and / or a solid film former. In some embodiments, the film former is a solid film former.

[0139] In some embodiments, the w / w concentration of the film former in the composition comprising the film former and MaSp-like fibers is between 20 and 95%, between 20 and 30%, between 30 and 40%, between 40 and 50%, between 50 and 60%, between 60 and 70%, between 70 and 80%, between 80 and 85%, between 85 and 90%, between 90 and 95%, including any range or value therebetween.

[0140] Non-limiting examples of film formers include, but are not limited to, polysaccharides such as pullulan, agave-based polysaccharides (Gosulin ); Liftonin Trik Polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyvinyl acetate, polyalkyl acrylate; dextrin, cellulose derivatives such as alkyl cellulose and nitrocellulose, siliconized polysaccharides such as pullulan tris (trimethylsiloxy) silylpropyl carbamate, etc.; polyphenols, gums, acrylic acid-silicone graft copolymers such as alkyl acrylate-polydimethylsiloxane copolymers, silicone resins such as trimethylsiloxysilicic acid or fluorine-modified silicone resins, silicone-modified polynorbornene, fluorocarbon resins, aromatic resins, polymer emulsion resins, terpene resins, polybutene, polyisoprene, alkylated resins, polyvinyl pyrrolidone-modified polymers, rosin-modified resins and polyurethanes or any combination thereof.

[0141] Other non-limiting examples of film formers include, but are not limited to, pullulan tris(trimethylsiloxy)silylpropylcarbamate (e.g., TSPL-30-D5), alkyl acrylate-polydimethylsiloxane copolymers (e.g., KP-543, 545, 549, 550, and 545L), trimethylsiloxysilicic acid (e.g., KF-7312J and X-21-5250), and silicone-modified polynorbornene, or any combination thereof. In some embodiments, the film former is pullulan, Agave polysaccharide (Gosulin )and (cassava-based polysaccharides).

[0142] In some embodiments, the film former 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, a suitable concentration of the film former in the composition provides it with pliability. In some embodiments, a suitable concentration of the film former enables the composition to be applied (e.g., smeared) on the skin of the subject. In some embodiments, a suitable concentration of the film former promotes the film-forming properties of the composition. In some embodiments, the suitable concentration of the film former is as described herein. Exemplary compositions including a film former are provided in the Examples section.

[0143] In some embodiments, the composition includes a MaSp polymer combined with a film former. In some embodiments, the w / w ratio between the film former and the MaSp fibers in the composition 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, 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 the top of a substrate. In some embodiments, a suitable concentration of MaSp polymers in the composition enhances the flexibility of the composition. In some embodiments, a suitable concentration of MaSp polymers is as described herein. In some embodiments, the MaSp-based polymer improves at least one mechanical property (e.g., Fig.14 Exemplary compositions comprising a film former and a MaSp-like polymer are provided in the Examples section.

[0144] In some embodiments, the additional polymer is present in an amount of 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), 30% to 50% (w / w), 30% to 65% (w / w), 30% to 75% (w / w), 30% to 70 ...5% (w / w), 30% to 75 % (w / w), 30% to 45% (w / w), 30% to 40% (w / w), 40% to 70% (w / w), 40% to 65% (w / w), 40% to 50% (w / w), 40% to 45% (w / w), 50% to 70% (w / w), 50% to 80% (w / w), 50% to 90% (w / w), 50% to 95% (w / w), 70% to 80% (w / w), 70% to 90% (w / w) or 70% to 95% (w / w), including any range therebetween.

[0145] In some embodiments, the additional polymer according to the present invention is enriched in an insoluble MaSp-like polymer described herein. In some embodiments, the additional polymer is enriched in 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 an insoluble MaSp-like polymer.

[0146] In some embodiments, the additional polymer is bound to the MaSp-like fiber via non-covalent bonds, via physical interactions, or both. Non-covalent bonds are well known in the art and include hydrogen bonds, pp stacking, van der Waals interactions, etc., among others.

[0147] In some embodiments, physical interaction refers to the additional polymer being entangled or interwoven with the MaSp fibers, being entrapped in a network or matrix formed by the MaSp fibers. In some embodiments, the matrix is ​​formed by a cosmetic active ingredient that fills at least a portion of the pores within the MaSp fibers.

[0148] In some embodiments, the composition is a composite material, wherein the composite material comprises MaSp-like fibers bound to another polymer, wherein the binding is via non-covalent bonds, covalent bonds, physical interactions, or any combination thereof.

[0149] In some embodiments, the additional polymer is in contact with or combined with the fibrils. In some embodiments, the MaSp fibers are incorporated into the additional polymer. In some embodiments, the MaSp fibers are embedded in the additional polymer. In some embodiments, the additional polymer is doped by the MaSp fibers. In some embodiments, the MaSp fibers are encapsulated by the additional polymer.

[0150] In some embodiments, at least a portion of the additional polymer fills 20% to 100% of the volume (e.g., lumen) of the particle. In 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% of the volume of the particle, including any range therebetween.

[0151] In some embodiments, the additional polymer is substantially free of any additional biologically active components. In some embodiments, the additional polymer is substantially free of proteins, such as MaSp-like proteins. In some embodiments, the additional polymer of the invention consists essentially of the polymers listed above.

[0152] In some embodiments, the compositions of the present invention are substantially homogeneous.

[0153] In some embodiments, the composition or composite material is solid. In some embodiments, the composition or composite material is semi-solid. In some embodiments, the composition or composite material is a gel. In some embodiments, the composition (e.g., solid composition) is substantially free of any of solvents, surfactants, carriers, particles, wherein substantially at least 80%, at least 90%, at least 92%, at least 95%, at least 97%, at least 98%, at least 99% by weight of the composition.

[0154] 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-class polymer.

[0155] In some embodiments, the property is selected from: Young's modulus, tensile strength, strain at break, yield point, toughness, work of rupture, impact strength, tear strength, flexural modulus, flexural strain and stress at a specific elongation, and wear. In some embodiments, the mechanical property is selected from storage modulus and loss modulus or any combination thereof.

[0156] In some embodiments, the present invention provides wear-resistant compositions. In some embodiments, the present invention provides compositions with improved wear resistance. As used herein, the term "wear resistance" refers to the ability of a material to stop displacement when exposed to relative motion of hard particles or projections. Wear resistance can be measured by a variety of tests known in the art, such as, for example, Taber wear test, Gardner scrubber test, sand fall (falling sand) test.

[0157] In some embodiments, one or more properties selected from Young's modulus, tensile strength, yield point, abrasion resistance, and elongation stress are increased, 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%.

[0158] In some embodiments, one or more properties selected from Young's modulus, tensile strength, yield point, abrasion resistance, and elongation stress are increased, 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%.

[0159] In some embodiments, the composition is characterized by a Young's modulus in a range from 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.

[0160] In some embodiments, at least two properties selected from Young's modulus, tensile strength, yield point, abrasion resistance, and elongation stress are increased by, for example, 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%.

[0161] In some embodiments, at least three properties selected from Young's modulus, tensile strength, yield point, abrasion resistance, and elongation stress are increased by, for example, 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%.

[0162] In some embodiments, the Young's modulus 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%, at least 50%, at least 100%, at least 200%, or at least 500%.

[0163] 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%.

[0164] 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%.

[0165] In some embodiments, wear resistance is improved 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%.

[0166] In some embodiments, the composition is characterized by structural strength, wherein more than 20% of the structural strength is due to the incorporated MaSp-based polymer. In some embodiments, the composite material is characterized by structural strength, wherein more than 30% of the structural strength is due to the incorporated MaSp-based polymer.

[0167] In some embodiments, the composition is characterized by structural strength, wherein more than 1% of the tensile strength is due to the incorporated MaSp class polymer. In some embodiments, the composite material is characterized by structural strength, wherein more than 5% of the tensile strength is due to the incorporated MaSp class polymer. In some embodiments, the composite material is characterized by structural strength, wherein more than 10% of the tensile strength is due to the incorporated MaSp class polymer. In some embodiments, the composite material is characterized by structural strength, wherein more than 20% of the tensile strength is due to the incorporated MaSp class polymer. In some embodiments, the composite material is characterized by tensile strength, wherein more than 30% of the structural strength is due to the incorporated MaSp class polymer.

[0168] In some embodiments, as used herein, the phrase "structural strength" refers to mechanical properties such as, but not limited to, elastic modulus, tensile stress, elongation (strain), and toughness [eg, a combination of tensile stress and elongation (strain)].

[0169] In some embodiments, the MaSp-based polymer improves at least one mechanical property of a composition comprising a film former, wherein the mechanical property is as described herein. Fig.14 The experimental results of the mechanical properties of exemplary compositions comprising MaSp-type polymers and film formers are summarized in .

[0170] Generation method

[0171] In some embodiments, a method for producing a spider silk (MaSp) polymer of the present invention is provided. 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 techniques known in the art.

[0172] As used herein, a "recombinant nucleic acid" is one in which a nucleic acid molecule encoding a polypeptide of interest has been modified in vitro so that its sequence is not naturally occurring or corresponds to naturally occurring sequences that are not located as they would be in the unmodified genome.

[0173] In one embodiment, the bacteria as described herein are genetically modified bacteria. In one embodiment, the artificial traction spider silk is not endogenously produced in bacteria such as described herein. The artificial traction spider silk of the present invention produced in bacteria as described herein has unexpected and unique properties.

[0174] In some embodiments, the method of the present invention comprises the following steps:

[0175] 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 a regulatory sequence;

[0176] b. transforming a microbial (eg bacterial) host with the expression vector of (a);

[0177] c. providing conditions for the microorganism of (b) to express heterologous proteins; and

[0178] d. Isolation of expressed protein,

[0179] Thereby obtaining the synthetic amino acid sequence of the present invention.

[0180] According to some embodiments, there is provided a method (e.g., of making synthetic traction spider silk) comprising:

[0181] (i) providing a microorganism (eg, a recombinant bacterium) as described herein;

[0182] (ii) providing conditions for the microorganism to express MaSp; and

[0183] (iii) isolating the expressed protein,

[0184] Thus creating synthetic traction silk.

[0185] In some embodiments, step (ii) of providing conditions for bacteria to express MaSp comprises providing a solution having a pH in the range of 5 to 6.5. In some embodiments, step (ii) comprises providing a solution having a pH below 6.5.

[0186] In some embodiments, the step (ii) of providing conditions for bacterial expression of MaSp comprises providing an expression inducer. In some embodiments, the expression inducer comprises lactose. In some embodiments, the expression inducer comprises isopropyl β-D-1-thiogalactoside (IPTG). As used herein, the term "inducer" refers to a compound that induces and / or increases protein expression. In some embodiments, expression is constitutive expression.

[0187] In some embodiments, the step (ii) of providing conditions for bacterial expression of MaSp includes waiting for a period of time to obtain insoluble MaSp-like polymers. In some embodiments, the time period is in the range of 15 hours to 48 hours, 17 hours to 48 hours, 18 hours to 48 hours, 18 hours to 24 hours, 20 hours to 48 hours, 22 hours to 48 hours, 24 hours to 48 hours, 22 hours to 36 hours, 24 hours to 36 hours or 24 hours to 32 hours, including any range therebetween. In some embodiments, waiting for a period of time allows the formation of synthetic traction spider silk in two stages. In some embodiments, soluble glandular silk protein is formed in the first stage. Once the critical intracellular concentration of soluble protein accumulates, SVX-E will self-assemble to form the desired insoluble MaSp polymer.

[0188] In some embodiments, the step (iii) of separating the expressed protein comprises the steps of dissolving the bacteria with a solution of a surfactant between 0.1% and 10% and centrifuging the mixture. In some embodiments, the step (iii) of separating the expressed protein comprises the steps of dissolving the bacteria with a solution of a surfactant between 0.1 and 5% and centrifuging the mixture. In some embodiments, the obtained precipitate is suspended in a 6M urea solution. In some embodiments, after further centrifugation, the precipitate is suspended in a 0.07% surfactant solution. In some embodiments, after resuspension in a surfactant solution and urea, the protein is separated from cell debris according to well-known procedures (e.g., by using a 10-90% w / w monosaccharide or disaccharide solution).

[0189] In some embodiments, the method further comprises an enrichment step with an additional polymer and / or an additional compound. In some embodiments, the additional polymer and the additional compound are as described herein. In some embodiments, the enrichment step comprises mixing a solution of synthetic traction spider silk with a solution of an additional polymer.

[0190] In some embodiments, the enriching step comprises degassing the mixture of the solution comprising the synthetic dragline spider silk and the additional polymer.

[0191] In some embodiments, deaeration is performed by allowing the obtained suspension to stand for a period of time without shaking at a temperature between 20° C. and 50° C. 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.

[0192] In some embodiments, the synthetic dragline spider silk (e.g., MaSp-like polymers or MaSp-like fibers) and the additional polymer are mixed in 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, 2:3.9 to 4:1, 2:4 to 4:1, 2:5. 3, 1:3.9 to 4:3, 1:3.8 to 4:3, 1:3.5 to 4:3, 1:3 to 4:3, 1:2.8 to 4:3, 1:2.5 to 4:3, or 1:2 to 4:3, including any range therebetween.

[0193] In some embodiments, synthetic traction spider silk (e.g., MaSp-like polymers or MaSp-like fibers) and additional polymers are used in a ratio of between 1:1 and 1:100, between 1:1 and 1:5, between 1:1 and 1:3, between 1:3 and 1:5, between 1:5 and 1:10, between 1:5 and 1:7, between 1:7 and 1:10, between 1:10 and 1:2, between 1:12 and 1:15, between 1:15 and 1:20, between 1:20 and 1:30, between 1:30 and 1:40, between 1:40 and 1:50, between 1:50 and 1:70, between 1:70 and 1:100, including any ranges therebetween.

[0194] In some embodiments, synthetic traction spider silk (e.g., MaSp-like polymers or MaSp-like fibers) and the additional compound are used in a ratio 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 ranges therebetween.

[0195] In some embodiments, the polymer is selected from a synthetic polymer, a thermoplastic polymer, a thermosetting polymer, a film former, an epoxy resin, a polyester, a polyamide, a polyol, a polyurethane, a polyethylene, a nylon, a polyacrylate, a polycarbonate, polylactic acid (PLA) or a copolymer thereof, silicon, a liquid crystal polymer, maleic anhydride grafted polypropylene, polycaprolactone (PCL), rubber, cellulose, or any combination thereof.

[0196] In some embodiments, step (iii) further comprises drying the synthetic traction spider silk (e.g., SVX-E class fibers). In some embodiments, drying comprises partial drying of the fibers. In some embodiments, drying is by evaporating at least 30%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% of the solvent (e.g., aqueous solvent) by weight. In some embodiments, drying is by exposing the fibers to a temperature between -180 and 200°C. In some embodiments, drying is by exposing the fibers to electromagnetic radiation in the visible and / or infrared spectrum.

[0197] In some embodiments, drying is by exposing the fiber to a temperature between 40 and 200°C, between 40 and 50°C, between 40 and 60°C, between 60 and 80°C, between 40 and 80°C, between 60 and 100°C, between 100 and 150°C, between 150 and 200°C, including any range or value therebetween.

[0198] In some embodiments, drying is by exposing the fibers to microwave radiation. In some embodiments, drying is performed by convection drying, such as by applying a hot air stream to the fibers. In some embodiments, drying is performed by cold drying, such as by applying a dehumidified air stream to the fibers. In some embodiments, drying is performed by freeze drying. Typically, the drying method selected and the exact drying conditions will depend on, among other things, the chemical and / or physical stability (e.g., thermal stability) of the fibers.

[0199] MaSp fibers

[0200] The terms "macroampule silk protein" and "adenocarcinoma" are used interchangeably throughout the description and include all known macroampule silk proteins, usually abbreviated as "MaSp" or "ADF" in the case of A. crossi. These macroampule silk proteins are generally of two types, type 1 and type 2. In addition, these terms include non-natural proteins that have a high degree of identity and / or similarity to at least the repeat regions of known macroampule silk proteins as disclosed herein. Other suitable spider silk proteins include MaSp2, MiSp, MiSp2, AcSp, FLYS, FLAS and flagelliform.

[0201] As used herein, the terms "repeat region", "repeat sequence" or "repeat" refer to a recombinant protein sequence derived from a repeating unit that occurs naturally multiple times in a spider silk amino acid sequence (e.g., in the MaSp-1 protein). It will be appreciated by those skilled in the art that the primary structure of spider silk proteins is believed to consist primarily of a series of small variations in unit repeats. Unit repeats in naturally occurring proteins are generally different from one another. That is, there is little or no exact replication of unit repeats along the length of the protein. In some embodiments, synthetic spider silks of the present invention are made in which the primary structure of the protein includes multiple exact repeats of a single unit repeat. In other embodiments, the synthetic spider silk of the present invention includes multiple repeats of one unit repeat and multiple repeats of a second unit repeat. This structure is similar to a typical block copolymer. Unit repeats of several different sequences can also be combined to provide a synthetic spider silk protein with properties suitable for a particular application. As used herein, the term "direct repeat" is a tandem repeat (head-to-tail arrangement) with similar repeats. In another embodiment, the repeat used to form the synthetic spider silk of the present invention is a direct repeat. In some embodiments, the repeat is not found in nature (i.e., it is not a naturally occurring amino acid sequence).

[0202] An exemplary sequence comprising a repetitive sequence is DF-4: AAAAAAASGSGGYGPENQGPSGPVAYGPGGP (SEQ ID NO: 1). In some embodiments, the synthetic repetitive sequences of the invention are based on (e.g., have a high percentage identity as defined below) one or more repetitive sequences derived from ADF-4 (SEQ ID NO: 1). As used herein, the term "based on" refers to a sequence having a high percentage homology to a repetitive sequence.

[0203] In some embodiments, each repetitive sequence comprises at most 60 amino acids, at most 55 amino acids, at most 50 amino acids, at most 49 amino acids, at most 48 amino acids, at most 47 amino acids, at most 46 amino acids, at most 45 amino acids, at most 44 amino acids, at most 43 amino acids, at most 42 amino acids, at most 41 amino acids, at most 40 amino acids, at most 39 amino acids, at most 38 amino acids, at most 37 amino acids, at most 36 amino acids, or at most 35 amino acids, wherein the possibilities represent separate embodiments of the present invention. In some embodiments, each repetitive sequence comprises 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, wherein the possibilities represent separate embodiments of the present invention. In some embodiments, each repeat sequence comprises 30 to 40 amino acids, 31 to 39 amino acids, 32 to 38 amino acids, 33 to 37 amino acids, 34 to 36 amino acids, wherein each possibility represents a separate embodiment of the present invention. In other embodiments, each repeat sequence comprises 35 amino acids.

[0204] In some embodiments, the repeat regions independently comprise the amino acid sequence set forth in Formula 1:

[0205] (X1) Z X2GPGGYGPX3X4X5GPX6GX7GGX8GPGGPGX9X 10 ; wherein X1 is independently A or G in each occurrence.

[0206] In some embodiments, at least 50% of (X1) Z is an integer between 5 and 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 It is S or G.

[0207] 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).

[0208] In another embodiment, a homolog of the repeat region of the MaSP1 protein having 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 to SEQ ID NO: 1 is provided.

[0209] In another embodiment, the homolog is 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% homologous to SEQ ID NO:2.

[0210] In another embodiment, the repeat region of the MaSP1 protein has the amino acid sequence set forth in SEQ ID NO:1.

[0211] 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 (MSY YHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVRPLSNLDNAP); SEQ ID NO: 6 (MSYYHHHHHHDYDIPTTENLYFQGAMDPEFKGLRRRAQLVDPPGCRNSARAGSS), or any functional homolog, variant, derivative or fragment thereof. In another embodiment, the homolog of the C-terminal region has at least 70% homology to any one of SEQ ID NOs: 4-6.

[0212] In another embodiment, the MaSP1 protein further comprises a single C-terminal region selected from the group consisting of SEQ ID NO: 7 (VAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPGLSGCDA LVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS); and SEQ ID NO: 8 (GPSGP GAYGPSPSASASVAASRLSSPAASSRVSSAVSSLVSSGPTNGAAVSGALNSLVSQISASNPG LSGCDALVQALLELVSALVAILSSASIGQVNVSSVSQSTQMISQALS), or any functional homologue, variant, derivative, fragment or mutant thereof. In another embodiment, the homologue of the N-terminal region has at least 70% homology to SEQ ID NO: 7-8.

[0213] In some embodiments, the MaSp-based fibers include a protein mixture disclosed according to WO2017025964, which is herein incorporated by reference in its entirety.

[0214] 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. The skilled person is well aware of alternative suitable tags or other fusion partners.

[0215] 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 in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those amino acids encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. "Amino acid analogs" refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., α-carbon compounds bound to hydrogen, carboxyl, amino, and R groups, 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 that is different from the general chemical structure of amino acids but function in a manner similar to naturally occurring amino acids. Amino acids may be represented herein by their commonly known three letter symbols or by one letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Committee.

[0216] "Amino acid sequence" or "peptide sequence" is the order of amino acid residues linked by peptide bonds in peptide and protein chains. The sequence is usually reported from the N-terminus containing free amino groups to the C-terminus containing free carboxyl groups. If the primary structure of a protein is represented, the amino acid sequence is usually referred to as a peptide or protein sequence, but the terms "amino acid sequence" or "peptide sequence" and "protein" must be distinguished because proteins are defined as amino acid sequences that are folded into a specific three-dimensional configuration and are usually post-translationally modified (such as phosphorylation, acetylation, glycosylation, sulfhydryl bond formation, cleavage, etc.).

[0217] As used herein, in the context of synthetic spider silk amino acid sequences or nucleic acid molecules encoding them, as exemplified by the present invention, "isolated" or "substantially purified" means that the amino acid sequence or polynucleotide has been removed from its natural environment or has been changed from its natural state. Therefore, "isolated" does not necessarily reflect the degree to which the amino acid sequence or nucleic acid molecule has been purified. However, it should be understood that such molecules that have been purified to a certain degree are "isolated". If a molecule does not exist in the natural environment, that is, it does not exist in nature, then the molecule is "isolated" regardless of where it exists. For example, amino acid sequences or polynucleotides that do not naturally occur in humans are "isolated", even if they are present in humans.

[0218] The terms "isolated" or "substantially purified" when applied to amino acid sequences or nucleic acids mean that the amino acid sequence or nucleic acid is substantially free of other cellular components with which it is associated in nature. It may be in a homogeneous state, or may be a dry solution or an aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. An amino acid sequence or nucleic acid that is the predominant species present in a preparation is substantially purified.

[0219] In some embodiments, the repetitive sequence is a homologue, variant or derivative of the repetitive region of MaSp1 protein or a fragment thereof. In some embodiments, the repetitive sequence is a homologue, variant or derivative of the repetitive region of ADF-4 protein or a fragment thereof.

[0220] As used herein, the term "functional" in "functional homologues, variants, derivatives or fragments" refers to an amino acid sequence having a biological function or activity identified by a defined functional assay. More specifically, the defined functional assay is the formation of self-assembled fibers in cells expressing the functional homologues, variants, derivatives or fragments.

[0221] An amino acid sequence or a nucleic acid sequence is a homolog of the corresponding amino acid sequence or nucleic acid when 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%.

[0222] In the context of two or more amino acid or nucleic acid sequences, the terms "identical", "substantial identity", "substantial homology" or "percent identity" refer to two or more sequences or subsequences that are identical or have a specified percentage of identical amino acid residues or nucleotides (i.e., about 60% identity over a specified region (e.g., amino acid sequence SEQ ID NO: 2 or 3), 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 when compared and aligned for maximum correspondence over a comparison window or specified region), as measured using the BLAST or BLAST 2.0 sequence comparison algorithm with default parameters as described below, or by manual alignment and visual inspection. Such sequences are then "substantially identical". This definition also relates to or may apply to the supplement of the test sequence. The definition also includes sequences with deletions and / or additions, as well as sequences with substitutions.Preferred algorithms can take gaps etc. into account.

[0223] For sequence comparison, usually a sequence serves as a reference sequence, and the test sequence is compared with the reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Preferably, default program parameters can be used, or alternative parameters can be specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the program parameters.

[0224] It should be understood that the present invention further includes amino acid sequences comprising n repetitions of a variant of any one of SEQ ID NO: 1, 2 or 3. As used herein, the term "variant" or "substantially similar" includes amino acid or nucleotide sequences that differ from a specific identified sequence in which one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20 or 25) amino acid residues or nucleotides are deleted, substituted or added. Variants may be naturally occurring allelic variants or variants of non-natural origin. Variants or substantially similar sequences refer to fragments of amino acid sequences or nucleic acids, characterized by their percent identity of amino acid or nucleotide sequences to the amino acid or nucleotide sequences described herein, as determined by commonly used algorithms used in the art. Preferred amino acid or nucleic acid fragments are those having at least about 40% or 45% sequence identity, preferably about 50% or 55% sequence identity, more preferably about 60% or 65% sequence identity, more preferably about 70% or 75% sequence identity, more preferably about 80% or 85% sequence identity, still more preferably about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity when compared to a reference sequence.

[0225] As used herein, the terms derivative and functional derivative refer to the amino acid sequences of the present invention with any insertion, deletion, substitution and modification.

[0226] It should be understood that the term "insertion" as used herein refers to the addition of any of the following amino acid residues to the sequence of the present invention: 1 to 50 amino acid residues, specifically 20 to 1 amino acid residues, more specifically between 1 and 10 amino acid residues. Most specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 amino acid residues. In addition, the amino acid sequence of the present invention can be extended at its N-terminus and / or C-terminus with various identical or different amino acid residues.

[0227] An amino acid "substitution" is the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, i.e., a conservative amino acid substitution. Amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the residues involved. 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; negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0228] In another embodiment, the repetitive sequence of the invention has 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, or 7 or less amino acid substitutions to the sequence of any one of SEQ ID NO: 2 or 3. In one embodiment, the repetitive 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 to the sequence of any one of SEQ ID NO: 1, 2, or 3.

[0229] With respect to amino acid sequences, the skilled artisan will recognize that alterations, additions or deletions of single amino acids or individual substitutions, deletions or additions of a small percentage of amino acids in an amino acid, nucleic acid, peptide, polypeptide or protein sequence are "conservatively modified variants" wherein the alterations result in the replacement of 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.

[0230] For example, a substitution can be made in which an aliphatic amino acid (G, A, I, L, or V) is replaced by another member of the group, or a substitution, for example, replacing one polar residue with another, such as replacing lysine with arginine, replacing aspartic acid with glutamic acid, or replacing asparagine with glutamine. Each of the following eight groups contains other exemplary amino acids that are conservatively substituted for each other: 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).

[0231] Conservative nucleic acid substitutions are those that result in conservative amino acid substitutions as defined above.

[0232] Variants of the amino acid sequences of the present 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 with the repeating unit represented by any one of SEQ ID NO: 1, 2 or 3.

[0233] The amino acid sequences of the present invention may include fragments of SEQ ID NO. 1. A "fragment" constitutes a portion of an amino acid or DNA sequence of a specific region. An amino acid fragment may include 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 in SEQ ID NO: 1, 2 or 3.

[0234] The mutants of the amino acid sequences of the invention are characterized by one (point mutant) or more, about up to 10, of their amino acids being exchanged with one or more of another amino acid. They are the result of corresponding mutations at the DNA level leading to different codons.

[0235] Further, 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, for example, those in which functional groups such as amino, hydroxyl, sulfhydryl 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 especially acylated by naturally occurring organic or inorganic acids, such as acetic acid, phosphoric acid or sulfuric acid, usually at the N-terminal amino or hydroxyl group, especially tyrosine or serine, respectively. Esters are esters of naturally occurring alcohols, such as methanol or ethanol. Other derivatives are salts, especially pharmaceutically acceptable salts, such as metal salts, such as alkali metal and alkaline earth metal salts, such as sodium, potassium, magnesium, calcium or zinc salts, or ammonium salts formed with ammonia or suitable organic amines such as lower alkylamines, such as triethylamine, hydroxy-lower alkylamines, such as 2-hydroxyethylamine, etc.

[0236] It should be understood that while the present invention generally relates to synthetic spider silk proteins derived from Dracaena crusae dragline, or any fragments or portions thereof, many other spider species may be used to derive synthetic spider silk in a similar manner. More preferably, the traction 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 Tent Spider (Cyrtophora beccarii), Bird-dropping Spider (Celaenia excavata), Black-and-White Spiny Spider (Gasteracantha kuhlii), Black-and-yellow Garden Spider (Argiopeaurantia), Bolas Spider (Ordgarius furcatus), Bolas Spiders Magnificent Spider (Ordgarius magnificus), Brown Sailor Spider (Brown Sailor SailorSpider (Neoscona nautical), Brown-Legged Spider (Neoscona ufofemorata), Capped Black-Headed Spider (Zygiella calyptrate), Common Garden Spider (Parawixia dehaani), Common Orb Weaver (Neoscona oxancensis), Crab-like Spiny OrbWeaver (Gasteracantha cancriformis (elipsoides)), Curved Spiny Spider (Gasteracantha arcuata), Cyrtophora moluccensis, Cyrtophora parnasia, Dolophonesconifera, Dolophones turrigera, Doria Spiny Spider (Gasteracantha doriae), Double-Spotted Spiny Spider (Gasteracantha mammosa), Double-Tailed Tent Spider (Cyrtophora exanthematica), Aculeperia ceropegia, Eriophora pustulosa, Flat Anepsion (Anepsiondepressium), Four-spined Jewel Spider (Gasteracanthaquadrispinosa), Garden Orb Web Spider (Eriophora transmarina), Giant Lichen Orbweaver (Araneus bicentenarius), Golden Web Spider (Nephilamaculata), Hasselt's Spiny Spider (Gasteracantha hasseltii), Tegenaria atrica, Heurodesturrita, Island Orb Web Spider (Island Orb Web Spider) Cyclosa Spider (Cyclosa insulana), Jewel or Stinging Spider (Astracantha minax), Kidney Garden Spider (Araneusmitificus), 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 (RedTent Spider (Cyrtophora unicolor), Russian Tent Spider (Cyrtophora hirta), Saint Andrew's Cross Spider (Argiope keyserlingi), Scarlet Acusilas (Acusilas coccineus), Silver Argiope (Argiope argentata), Spinybacked Orbweaver (Gasteracanthacancriformis), Spotted Orbweaver (Neoscona domiciliorum), St. Andrews Cross (Argiope aetheria), St. Andrew's Cross Spider (Argiope Keyserlingi), Tree-Stump Spider (Poltysillepidus), Triangular Spider (Arkys clavatus), Triangular Spider (Arkys lancearius), Two-spined Spider (Poecilopachysaustralasia), Nephila species such as Nephila clavipes, Nephila senegalensis, Nephilamadagascariensis and many more.

[0237] Furthermore, synthetic spider silk can be enhanced not only by choosing different spider species to derive from, but also by using various compounds besides proteins. Pyrrolidine is hygroscopic and helps keep the thread moist. It occurs in particularly high concentrations in glue threads. Potassium hydrogen phosphate releases protons in aqueous solution, resulting in a pH of about 4, making the silk acidic, thus protecting it from fungi and bacteria that would otherwise digest the proteins. Potassium nitrate is thought to prevent proteins from denaturing in acidic environments.

[0238] In some embodiments, the bacterial system of the present invention can utilize a variety of expression vectors, which are advantageously selected according to the intended use of the protein to be expressed. In one embodiment, a large amount of protein is required. In one embodiment, a vector that guides the expression of a high-level protein product is desired, and the protein product may be a fusion with a hydrophobic signal sequence, and the product of the vector guides the expression into the periplasm of the bacterium or in a culture medium that is easy to purify the protein product. In one embodiment, a certain fusion protein is engineered with a specific cleavage site to help recover the polypeptide. In one embodiment, vectors suitable for such operations include but are not limited to pET series expression vectors.

[0239] "Nucleic acid" refers to molecules that can be single-stranded or double-stranded and consist of monomers (nucleotides) containing sugars, phosphates, and 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.

[0240] Due to the degenerate nature of the genetic code, it is obvious that a variety of different nucleic acid sequences can be used to encode the amino acid sequences of the present invention. It should be understood that the codons included in the nucleic acid sequences of the present invention can be optimized for expression in bacterial host cells.

[0241] The term "codon-optimized" refers to genes or nucleic acid molecule coding regions for transforming various hosts, and refers to codons that change genes or nucleic acid molecule coding regions to reflect typical codons that use host organisms without changing polypeptides encoded by DNA. In the context of the present invention, genes and DNA coding regions are codon-optimized for optimal expression in host bacterial cells.

[0242] As used herein, the term "expression" is intended to mean transcription and translation of a gene product from a gene encoding a gene product sequence. In expression, the DNA strand encoding the gene product sequence is first transcribed into a complementary RNA (usually a messenger RNA), and then if the gene product is a protein, the transcribed messenger RNA is translated into the above-mentioned gene product.

[0243] 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.

[0244] As used herein, "vector", "expression vector" or "plasmid" as referred to herein is an extrachromosomal element that usually carries exogenous gene(s), which is not part of the central metabolism of the bacterial cell, and is usually in the form of a circular double-stranded DNA molecule. It can be any of a variety of nucleic acids, into which the desired sequence is inserted by restriction and ligation for transport between different genetic environments or expression in host cells. Although RNA vectors can also be used, vectors are usually composed of DNA. Vectors include, but are not limited to, plasmids and phagemids. A cloning vector is a vector that can replicate in a host cell, and is also characterized by one or more endonuclease restriction sites at which the vector can be cut in a determined manner and to which the desired DNA sequence can be ligated so that the new recombinant vector retains the ability to replicate in the host cell. In the case of a plasmid, replication of the desired sequence may occur multiple times as the number of copies of the plasmid increases in the host bacterium, or each host replicates only once, before the host reproduces by mitosis. In the case of a phage, replication can occur actively during the lysis phase or passively during the lysogeny phase. An expression vector is a vector 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. The vector may also contain one or more marker sequences suitable for identification and selection of cells transformed or transfected with the vector. As used herein, "transformation" or "transfection" is the acquisition of new genes in cells by incorporation of nucleic acids. Markers include, for example, genes encoding proteins that increase or decrease 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 significantly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques. Preferred vectors are those that are capable of autonomous replication and expression of structural gene products present in the DNA fragments to which they are operably linked, i.e., those that express synthetic spider silk proteins.

[0245] As described above, the expression vector of the present invention is operably linked to a promoter. The terms "promoter" and "promoter region" refer to a DNA sequence that is usually located upstream (5' to) of the protein coding sequence of a structural gene and controls the expression of the coding region by providing recognition for RNA polymerase and / or other factors required for transcription to start at the correct site. The promoter sequence is necessary, but not always sufficient to drive gene expression. The term "suitable promoter" refers to any prokaryotic promoter that is capable of driving expression of a synthetic spider silk variant gene.

[0246] The promoters that can be used to drive the expression of heterologous DNA fragments in bacteria are numerous and familiar to those skilled in the art. In fact, any bacterial promoter that can drive the gene encoding the silk variant protein is suitable for use in the present invention.

[0247] A coding sequence and a regulatory sequence are "operably linked" or "operably joined" when they are covalently linked in a manner that places the expression or transcription of the coding sequence under the influence or control of the regulatory sequence. A regulatory sequence is operably linked to a gene if the regulatory sequence is positioned relative to the gene so that the regulatory sequence is able to have a measurable effect on the amount of gene product produced. Two DNA sequences are operably linked if translation of the coding sequence into a functional protein is desired, if induction of the promoter in the 5' regulatory sequence results in transcription of the coding sequence, and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a 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 will be operably linked to a coding sequence if it is able to affect the transcription of the DNA sequence so that the resulting transcript can be translated into the desired protein or polypeptide.

[0248] The exact nature of the regulatory sequences required for gene expression may vary from species to species or cell type to cell type, but should generally include 5' non-transcribed and 5' non-translated sequences involved in transcription and translation initiation, such as TATA boxes, capping sequences, CAAT sequences, respectively, as necessary. In particular, such 5' non-transcribed regulatory sequences will include a promoter region including a promoter sequence for transcriptional control of an operably linked gene. Regulatory sequences may also include enhancer sequences or upstream activation sequences as desired.

[0249] "Regulation" and "regulate" refer to the regulation of gene expression controlled by DNA sequence elements located primarily, but not exclusively, upstream (5') of the transcription start site of the gene. Regulation may result in full or no response to a stimulus, or it may result in a change in the level of gene expression.

[0250] In another aspect, the present invention provides a host cell transformed with an expression vector according to the present invention.

[0251] "Cell", "host cell" or "recombinant host cell" are terms used interchangeably herein. It should be understood that such terms refer not only to the specific subject cell, but also to the progeny or potential progeny of such cells. Because certain modifications may occur in the progeny due to mutation or environmental influences, such progeny may not actually be the same as the parent cell, but are still included within the scope of the terms used herein.

[0252] 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 selection markers are intended to facilitate the selection of transformants. In addition, the presence of selection markers such as drug resistance markers can be used to prevent contaminating microorganisms from breeding in the culture medium. By culturing cells under conditions where an induction phenotype is required for survival, such pure cultures of transformed host cells can be obtained.

[0253] The 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 the genotype of a cell is changed due to the cellular uptake of exogenous DNA or RNA, and, for example, the transformed cells express 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.

[0254] In some embodiments, the spidroin proteins of the present invention are not post-translationally modified.

[0255] 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 a regulatory sequence.

[0256] General terms

[0257] As used herein, the term "about" refers to ± 10%.

[0258] The word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any implementation described as "exemplary" is not necessarily to be construed as preferred or advantageous over other implementations and / or to exclude the incorporation of features of other implementations. The word "optionally" is used herein to mean "provided in some implementations and not provided in other implementations." Any particular implementation of the present invention may include multiple "optional" features, unless such features conflict.

[0259] 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 and reference to "a peptide" includes reference to one or more peptides known to those skilled in the art, and so forth.

[0260] Additionally, the use of "or" means "and / or" unless otherwise stated. Similarly, "comprise," "comprises," "comprising," "include," "includes," and "including" are interchangeable and are not intended to be limiting. It should be further understood that where the description of various embodiments uses the term "comprising," those skilled in the art will understand that in some specific cases, the embodiments may alternatively be described using the language "consisting essentially of" or "consisting of."

[0261] Throughout this application, various embodiments of the present invention may be presented in a range format. It should be understood that the description of the range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present invention. Therefore, it should be considered that the description of the range has specifically disclosed all possible subranges and each numerical value within the 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 single numbers within the range, such as 1, 2, 3, 4, 5 and 6. This applies regardless of the breadth of the range.

[0262] In those cases where a convention similar to "at least one of A, B, and C, etc." is used, such construction is generally intended to mean what one skilled in the art would understand the convention to mean (e.g., "a system having at least one of A, B, and C" would include, but is not limited to, systems of only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). In those cases where a convention similar to "at least one of A, B, or C, etc." is used, such construction is generally intended to mean what one skilled in the art would understand the convention to mean (e.g., "a system having at least one of A, B, or C" would include, but is not limited to, systems of only A, only B, only C, A and B together, A and C together, B and C together, and / or A, B, and C together, etc.). One skilled in the art would further understand that virtually any transitional word and / or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibility of including one, either, or both of these terms. For example, the phrase "A or B" would be understood to include the possibility of "A" or "B" or "A and B."

[0263] 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 the present disclosure 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.

[0264] Example

[0265] Experimental steps

[0266] Synthesis of sequences encoding a single repeating unit of the dragline spider silk protein: A 35 amino acid long sequence (GenBank entry U47856) was designed representing the average consensus sequence of the 15 repeats constituting the repeating region of the (MaSp) class of polymers. The average consensus peptide sequence is: SGPGGYGPGSQGPSGPGGYGPGGPGSSAAAAAAAAAAAA (SEQ ID NO: 11) encoded by the 105 DNA base pair sequence: 5′-TCTGGTCCTGGAGGTTATGGCCCAG GAAGCCAAGGACCATCTGGTCCAGGAGGATATGGTCCAGGCGGACCTGGCTCTAGTG CAGCAGCTGCCGCAGCAGCTGCA-3′ (SEQ ID NO: 9). The above synthetic DNA was obtained in the pPCR-ScriptAmpSK(+) plasmid.

[0267] Bacterial growth

[0268] Bacteria expressing pET24R were inoculated into 3 mL of LB medium starter containing chloramphenicol and kanamycin, grown to OD -0.6 (measured in 100 μL in a 96-well plate), and inoculated into growth medium.

[0269] The bacteria were grown at 37°C with shaking, and OD600 and β-sheet-specific staining were performed after about 20 hours. Significant β-sheet staining appeared after 24 hours.

[0270] Lysis and purification

[0271] The bacteria were centrifuged and resuspended in deionized water. Then, a surfactant solution was added and the resulting suspension was shaken overnight at 37°C. After centrifugation, the pellet was resuspended in 6M urea. After centrifugation, the pellet was resuspended and washed several times with a surfactant solution.

[0272] 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 residual amount was calculated.

[0273] UV spectroscopy

[0274] Spider silk (MaSp)-like polymers expressed in bacteria SVXE were dissolved by heating in 8M LiBr, diluted 1:10 with 6M urea, and UV spectra were measured at 240-350 nm in a disposable UV cuvette using an Ultrospec 2100 spectrophotometer. 8M LiBr diluted 1:10 with 6M urea was used as a control.

[0275] dyeing

[0276] The β-fold-specific stain was dissolved in dimethyl sulfoxide (DMSO) at 0.8 mg / mL and stored in the dark at room temperature. 1-30% Triton X-100 was added to the E. coli suspension in the culture medium, and the bacteria were centrifuged. The bacterial pellet was resuspended in PBS and 1-50 μL / mL of β-fold-specific stain was added. The suspension was incubated at room temperature for 30 minutes, then centrifuged and the bacterial pellet was resuspended in the same volume of PBS. If necessary, the stained pellet was stored at 4°C. Fluorescence was read in a 96-well plate of a Cytation fluorometer. At the same time, OD600 was measured and fluorescence was normalized to OD600 values. To stain the final preparation of SVX-E, the MaSp polymer was resuspended in PBS. The remaining steps are the same as for staining bacteria.

[0277] Differential Scanning Calorimetry (DSC)

[0278] Samples of SVX-E or Sf9-derivatized SVX were washed with water to remove the remaining surfactant and the suspension was dried. Capsules were weighed approximately 5 mg and subjected to scanning calorimetry at 10° / min over the range of 25-300°C on a Star calorimeter (Mettler Toledo). In parallel, a sample of SVX-E was dissolved in 6 M guanidine thiocyanate, dialyzed into 6 M urea, and then dialyzed into deionized water (in water, partially precipitated). The denatured protein was dried and subjected to scanning calorimetry under the same conditions.

[0279] TEM

[0280] Spider silk fibers SVX or spider silk polymers expressed in bacterial SVX-E suspension 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 2 minutes. The analysis was performed in TEM 120 KeV.

[0281] SEM

[0282] Scanning electron microscopy (SEM) of various samples was performed according to standard protocols.

[0283] Dynamic Light Scattering (DLS)

[0284] The SVX or SVX-E suspension was diluted 1:100 with 0.07% surfactant solution and the solution was filtered through a 0.22 μm filter. DLS analysis was performed using a Malvern Zetasizer Nano instrument.

[0285] Mass spectrometry analysis

[0286] SVX or SVX-E suspensions were pelleted by centrifugation, solubilized with 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), potential disulfide bonds were reduced with DTT and blocked with iodoacetamide, and then the proteins in two separate tubes were cleaved with trypsin or chemical trypsin.

[0287] The sequence of the resulting peptides was analyzed by Q Exactive mass spectrometer and compared to the expected SVX sequence and to databases of baculovirus proteins of SVX and Spodoptera frugiperda (sf9 origin) and E. coli proteins of SVX-E.

[0288] Amino acid analysis

[0289] For amino acid analysis, SVX or SVXE samples were hydrolyzed with 6 M HCl at 110 °C for 18-24 h.

[0290] FTIR

[0291] 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 exact position of the amide I peak was used to assess the secondary structure. -1 The peak position at is characteristic of amyloid-like β-sheets.

[0292] A general protocol for enriching polyurethanes with spider silk polymers

[0293] 1. Dispersions in organic solvents: Different amounts (Table 1) of spider silk polymers in aqueous suspension were centrifuged and resuspended in DDW (double distilled water). The suspension was allowed to stabilize for 3 minutes. It was then centrifuged and resuspended in ethanol. It was then centrifuged and resuspended twice in sufficient dry ether solvent (to stabilize the suspension). The suspension was then filtered through a 40 μm filter. It was then centrifuged and resuspended in dry ether solvent. The suspension was placed on a rotary shaker for 10-200 minutes (room temperature).

[0294] In the case of cellulose, powdered cellulose is directly dispersed in a sufficient amount of ethereal solvent without prior centrifugation and resuspension steps in water and ethanol.

[0295] 2. Dissolution of polymers: Different amounts of polyurethane (Table 1) were dissolved in sufficient ether solvent in a transparent glass vial with a cork screw (oscillator: 37° C., 200 rpm, 1-24 hours).

[0296] 3. Mixing the spider silk suspension with the polymer solution: The spider silk polymer suspension was centrifuged and resuspended in a sufficient amount of ether solvent. Then, it was sonicated in a sonicator. The spider silk polymer suspension was tested for size, fracture and aggregation using an optical microscope. The suspension was poured into the polymer solution. The polymer and spider silk polymer suspension were thoroughly mixed until homogeneity was achieved, and then placed in a rotary shaker (room temperature, 50 rpm) for 60 minutes.

[0297] 4. Degassing: Let the suspension stand at 20-60°C for 0.5-5 hours without shaking (degassing). If any bubbles are visible, continue degassing longer in a closed container.

[0298] 5. Casting: The size, fracture and aggregation state of the spider silk polymer suspension were tested using an optical microscope. The contents of the flask (18.2 g) were completely cast in a glass Petri dish with a diameter of 9 cm for 14 hours. The Petri dish was covered with cardboard and placed under a plastic box with a hood. The enriched polyurethane sheet was then removed from the Petri dish and placed in a vacuum at 80°C for 90 minutes. The enriched polyurethane sheet was kept at room temperature for 2 days and then cut into 60×7 mm strips.

[0299] 6. Mechanical properties measurements: The composites were analyzed in a Lloyd LS5 universal testing machine. The strips were pulled at a tension rate (mm / min) of 50 mm / min. A 50N load cell was used for accurate Young's modulus evaluation and a 5kN load cell was used for elongation and (ultimate tensile strength) UTS.

[0300] Table 1.

[0301]

[0302] SVX - spider silk fibers; SVX-E - spider silk polymers expressed in bacteria.

[0303] Dissolution profiles of SVX and SVX-E in various denaturants

[0304] Stock solutions of 8M guanidine thiocyanate, 7.5M guanidine hydrochloride, and 6M urea were prepared in DDW and appropriate compounds were dispensed in 80 μL volumes in 96-well plates at different concentrations. 20 μL of SVX (6.3 mg / mL) or SVXE (10 mg / mL) was added, mixed thoroughly, and the OD600 of each well was measured. There was no difference in the results if the same wells were measured repeatedly after 2 hours or overnight.

[0305] Example 1

[0306] Protein expression in bacteria

[0307] The bacteria expressed lysozyme, which destroys the cell wall. Bacteria expressing only lysozyme were used as controls. The medium pH was 5.8.

[0308] β-sheet staining was used to track the formation of insoluble aggregates. After purification, the product was a protein. The average yield (before optimization) was 105 mg / L culture medium.

[0309] After solubilization in 6 M GuaSCN and dialysis into urea, the UV spectrum was characteristic of tyrosine-containing proteins, with a maximum at 276 nm ( Figure 1A ).

[0310] The amino acid content corresponds to the expected sequence ( Figure 1B ).

[0311] FTIR spectrum shows the characteristic peaks of amide bonds: amide I and amide II ( Figure 1C ).

[0312] The resulting product was a particle containing β-sheets. Dynamic light scattering (DLS) was used and the particle size was determined to be 1.04 ± 0.2 μm ( Figure 2 ), and FTIR analysis of the particles confirmed the β-sheet structure ( Figure 3 ).

[0313] The amide I peak of spider silk protein (SVXE) expressed in Escherichia coli is at 1622 cm -1 The maximum is at , similar to spider silk protein (SVX) from Sf9. This is a wavelength characteristic of β-sheets. SVX solubilized with HFIP (resulting in α-helical conformation) or native α-helical protein (BSA) shows a shifted amide I peak.

[0314] The FTIR spectrum of the spidroin expressed in sf9 cells (SVX) is similar to that of the spidroin expressed in E. coli (SVXE) ( Figure 4 ).

[0315] When compared to worm silk, the spectrum of worm silk shows unique peaks that are not present in the spectra of SVX and SVX-E ( Figure 5 ).

[0316] Differential scanning calorimetry (DSC) of the particles confirmed the crystalline structure.

[0317] The particles have a phase transition at Tm = 216 °C ( Fig. 6A The phase transition peak disappears in the protein denatured with 6M guanidine SCN ( Figure 6B ).

[0318] The fiber from Sf9 has a phase transition at Tm = 242 °C ( Figure 6C ).

[0319] TEM of SVXE particles revealed a fine structure that was not seen in common inclusion bodies ( Fig. 7A -B). In addition, the SEM images of porous SVXE particles ( Fig.21A -B) represents fibers composed of nanofibrils (composed of Fig. 21B ), resulting in very porous particles. When aggregation occurs, the porosity of the particles decreases significantly ( Fig. 21C -D).

[0320] Fig.21E SEM images showing purified fermentation products resulting from expression of different constructs having the amino acid sequence as set forth in SEQ ID NO: 10 (MSYYHHHHHHDYDIPTTENLYFQGAMPRKSP FPRPEL). Although, the fibers obtained from expression of different constructs were in granular form, these fibers were substantially free of nanofibrils and lacked any porous structure.

[0321] SVXE fibers (e.g. Fig.21A -B) and fibers obtained from the expression of different constructs having the sequence as set forth in SEQ ID NO: 10 (e.g. Fig.21E FTIR spectra of SVXE fibers (data not shown) indicated that the SVXE fibers were characterized by a significantly increased amount of beta sheets compared to fibers having the sequence as set forth in SEQ ID NO:10.

[0322] Example 2

[0323] Polymer enrichment of spider silk proteins expressed in bacteria

[0324] In an exemplary experiment, polyurethane P490RSJT was enriched with increasing amounts of SVX-E (Table 2).

[0325] Table 2.

[0326]

[0327] The results showed a dose-dependent response, where Young's modulus increased with increasing SVX-E%, while UTS, % elongation at break, and toughness decreased ( Fig. 8A -B).

[0328] Similar experiments were performed with polymer E394POTA enriched with various concentrations of SVX-E. The results showed a dose-dependent response, where Young's modulus increased with increasing %SVX-E, while UTS, % elongation at break, and toughness decreased ( Fig.9A -E).

[0329] Fig. 10A -E presents a graph comparing composites made from polyurethane P490RSJT containing 20% ​​SVX or SVX-E. Both SVX and SVX-E make the polymer harder, as evidenced by the increased Young's modulus compared to the control. In this regard, there is no difference between SVX and SVX-E. Both SVX and SVX-E reduce tensile strength, % elongation, and toughness compared to the control. SVX-E shows a greater effect than SVX.

[0330] Fig.11A -E presents a graph comparing composites made from polyurethane E394POTA containing 20% ​​SVX or SVX-E. Both SVX and SVX-E made the polymer stiffer, as evidenced by the increased Young's modulus compared to the control. In this regard, there was no difference between SVX and SVX-E. Both SVX and SVX-E reduced tensile strength, % elongation, and toughness compared to the control. Figure 8B , 9E The strain-stress curves (not shown) show similar behavior for 10E and SVX-E, respectively. SVX-E shows a greater effect than SVX.

[0331] Table 3 describes a comparison of composites made from polyurethane P490RSJT containing 20% ​​SVX or SVX-E, and a comparison of composites made from polyurethane PU399 containing 5% & 10% SVX or SVX-E.

[0332] Table 3.

[0333]

[0334]

[0335] Additional polymers have been enriched with SVX-E fibers, and the mechanical properties of the resulting materials have been studied. The results are summarized below (Table 3A), showing that the Young's modulus of the SVX-E-enriched polymer compositions is significantly increased (between 130% and 1160%, including any range therebetween).

[0336] Table 3A.

[0337]

[0338] Furthermore, the film former has been enriched with 10% w / w SVX-E fibers and the mechanical properties of the resulting material were investigated. The results are summarized in Fig.14 In the experiment, SVX-E-rich film formers (Pullulan and Liftonin ) significantly increased (73% to 145%). Fig.14 As shown in FIG, the SVX-E-rich film formers tested (Pullulan, Liftonin SKI TriK LiftLiss Gosulin ) has a significant increase in storage modulus (between 10 and 500%, including any ranges therebetween) and a significant increase in loss modulus (between 10 and 60%, including any ranges therebetween).

[0339] Example 3

[0340] Loading and release of hyaluronic acid

[0341] Spider silk fibers (SVX) or spider silk fibers expressed in bacteria (SVX-E) were washed twice with ethanol and then washed 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 reach the desired volume. The mixture was shaken and centrifuged. The supernatant was discarded and a small amount of precipitated sample was dried on a slide to produce HA+SVX-E or HA+SVX precipitation. The precipitation was then tested by FTIR (Nicolet iS5FTIR spectrometer, Thermo Fisher Scientific). The remaining precipitation was resuspended in water and the suspension was vibrated at 200 rpm for 1-30 minutes at 25°C. This process was repeated several times. For each precipitated sample analyzed by FTIR, the proportion of HA in the total dry weight was calculated by dividing the peak intensity specific to HA by the peak intensity specific to the polymer (Table 4). These results demonstrate the ability of MaSp-based fibers (eg, SVX or SVX-E) to stably encapsulate additional compounds (eg, HA) in an approximately 1:1 w / w ratio of encapsulated compound to fiber (eg, SVX or SVX-E).

[0342] Table 4.

[0343] mixture HA peak Polymer peak HA+SVX or HA+SVX-E <![CDATA[1040cm -1 ]]> <![CDATA[1640cm -1 ]]> HA+ Silk <![CDATA[1040cm -1 ]]> <![CDATA[1550cm -1 ]]>

[0344] Example 4

[0345] Effect of pH on the Formation of SVX-E in Escherichia coli

[0346] The following examples show that the formation of SVX-E in E. coli depends on the medium pH ( Fig.15 Detection of SVX-E was accomplished using a β-sheet-specific fluorescent dye that binds to self-assembled silk but not to soluble silk.

[0347] 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 SVX-E expression plasmid.

[0348] Fig.16 Shown are images of staining taken with confocal microscopy at different time points in cultures grown at pH 7.5 or pH 5.8.

[0349] Example 5

[0350] SVX-E expression has a biphasic kinetic pattern

[0351] The following example shows the delay after induction of the recombinant spider silk protein, SVX-E is produced in its final insoluble form. Since protein synthesis is not induced at a specific time by adding an inducer (such as lactose or IPTG), but the inducer is present in the growth medium from the beginning (in the case of small amounts of glucose as the preferred carbon source, once it is consumed, lactose is utilized and recombinant protein synthesis begins). The method to follow the kinetics of recombinant protein synthesis is to compare the growth rate of two cultures, one containing lactose (expression culture), and the other without lactose (control culture). The difference between the growth rates of the two cultures (absorbance measured at 600nm) shows that the growth rate of the culture containing lactose is slower than that of the control culture without lactose. Without being bound by any particular theory, this is interpreted as inducing the expression of spider silk in the slower culture, because it is well known that when bacteria express recombinant proteins their growth rate slows down, which is due to the utilization of metabolic mechanisms for protein synthesis rather than growth.

[0352] Fig.17 The protein expression time window is the time that the expression cultures lag behind the control cultures. The inventors used specific probes for β-sheet crystals, which are structures formed as β-sheets stacked on top of each other (pleated β-sheets) - a well-known property of natural and artificial spider silk (such as SVX-E), but not soluble spidroins.

[0353] The increase in insoluble SVX-E occurred after both cultures reached equal growth rates at approximately 20 h, at which point the bacteria no longer expressed SVX-E. Fig.18 ), it can be seen that the formation of insoluble SVX-E only begins after the bacteria reach stationary phase, when the recombinant protein is usually no longer expressed.

[0354] Taken together, the results suggest that at pH 5.8, the production of recombinant spider silk has a biphasic kinetics, where in the first phase, soluble spider silk proteins are formed, while in the second phase, once a critical intracellular concentration of soluble protein has accumulated, self-assembly of SVX-E occurs, which is stabilized by intra- and intermolecular β-sheets and β-sheet crystals.

[0355] At pH 7.5 ( Fig.19 ), none of the above occurred. No insoluble SVX was detected (stage 2), and no differential growth and soluble protein expression was demonstrated in stage 1.

[0356] The inventors have observed a similar phenomenon in SF9 cells expressing SVX. The appearance of SVX was detected by the same β-sheet crystal-specific staining 70 hours after the culture was infected with baculovirus, at which stage the cells had reached stationary phase and they were not dividing. These results also again indicate the biphasic kinetics of SVX production in insect cells.

[0357] Example 6

[0358] Dissolution curves of SVX and SVX-E in various denaturants

[0359] Table 5 summarizes the experimental data showing that SVX and SVX-E have similar dissolution profiles in the well-known denaturants (DA) urea (not shown), guanidine chloride and guanidine thiocyanate. Table 5 shows the specific denaturants (DA) required to dissolve 50% w / w of the total amount of either fiber (SVX and SVX-E). 1 / 2 As shown in Table 5, similar concentrations of various DA were required for dissolution of both fibers (SVX and SVX-E).

[0360] Table 5.

[0361]

[0362] The data suggest that both have similar chemical compositions and that the difference in dispersibility is due to the smaller size of SVX-E, which results in a higher surface area.

[0363] Although the present invention has been described in conjunction with its specific embodiments, it is apparent that many substitutions, modifications and variations will be apparent to those skilled in the art. Therefore, it is intended to include all such substitutions, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0364] All publications, patents and patent applications mentioned in this specification are incorporated by reference in their entirety into this specification to the same extent as each individual publication, patent or patent application is specifically and individually incorporated herein by reference. In addition, the reference or identification of any reference in this application should 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 composition comprising a synthetic macroampulla protein (MaSp) based polymer in the form of particles having a size ranging from 0.5 μm to 1.5 μm.

2. The composition according to claim 1, wherein the MaSp-type polymer is a water-insoluble polymer. 3 . The composition according to claim 1 , which has a DSC chart showing at least one endothermic peak in the range of 200° C. to 280° C.

4. The composition according to any one of claims 1 to 3, wherein the particles are porous particles and are characterized by at least 10 m 2 / g of BET surface area.

5. The composition of any one of claims 1 to 4, wherein the particles comprise a plurality of nanofibrils.

6. The composition according to any one of claims 1 to 5, further comprising an additional compound in contact with the MaSp-like polymer.

7. The composition of claim 6, wherein the compound is selected from the group consisting of bioactive agents and nutraceuticals.

8. The composition according to any one of claims 6 and 7, wherein the weight ratio (w / w) of the MaSp-like polymer to the additional compound is between 10:1 and 1:

10.

9. The composition according to any one of claims 1 to 8, wherein the MaSp-based polymer comprises an amino acid sequence as set forth in SEQ ID NO: 2 (SGPGGYGPGSQGPSGPGGYGPGGPGSS).

10. The composition according to any one of claims 1 to 8, wherein the MaSp-based polymer comprises an amino acid sequence as set forth in SEQ ID NO: 3 (AAAAAAAASGPGGYGPGSQGPSGPGGYGPGGPGSS).

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