Composition for molded body

Abstract

To provide a composition for a molded body comprising a recombinant spider silk protein and a plasticizer, a molded body comprising a recombinant spider silk protein and a plasticizer, and a process for preparing the molded body.SOLUTION: Provided is a composition for a molded body comprising a recombinant spider silk protein and a plasticizer, wherein the composition can be induced into a flowable state, and wherein the recombinant spider silk protein is not substantially degraded in the flowable state.SELECTED DRAWING: Figure 11

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 717,622, filed August 10, 2018, the entire contents of which are incorporated herein by reference.

[0002] Field The present disclosure relates to compositions for molded bodies comprising recombinant spider silk proteins and a plasticizer. Additionally, the present disclosure relates to molded bodies comprising recombinant spider silk proteins and a plasticizer, as well as processes for preparing said molded bodies. [Background technology]

[0003] background There is growing interest in biorenewable and biodegradable materials as alternatives to petroleum-based products. To this end, considerable efforts have been made to develop methods for producing materials and fibers from molecules derived from plants and animals. The history of fibers made from renewable proteins dates back to the 1890s, when such fibers were produced using a variety of traditional wet-spinning techniques.

[0004] Wet spinning uses both a solvent and a coagulation bath to produce fibers. This procedure has the disadvantages of using a chemical as the solvent, and the coagulation bath requires extraction from the fiber after the spinning process and must be a closed-loop process to provide a sustainable and reliable process. Melt spinning offers an attractive alternative to wet spinning in that it does not require a solvent or coagulation bath, but melt spinning also requires (i) the production of a homogeneous molten composition from which the polymer can be extruded to form commercial-quality fibers, and (ii) that the polymer not decompose during the melting and extrusion steps. Summary of the Invention

[0005] overview Provided herein, according to some embodiments of the invention, are a molded body composition and a molded body comprising a recombinant spider silk protein and a plasticizer, wherein the composition can be substantially homogeneous after conversion to a molten or flowable state, and wherein the recombinant spider silk protein is not substantially degraded or is degraded in an amount less than 6.0% by weight after being formed into a molded body.

[0006] The present disclosure further provides a process for preparing a shaped body, the process comprising applying pressure and / or shear to a composition comprising a recombinant spider silk protein and a plasticizer to form a substantially homogeneous molten composition, and shaping the homogeneous molten composition to form a shaped body. The substantially homogeneous molten composition is generally in a flowable state and can be extruded, for example, to form fibers.

[0007] According to some embodiments, provided herein is a composition for molding comprising a recombinant spider silk protein and a plasticizer, wherein the composition can be induced to a flowable state, and wherein the recombinant spider silk protein is not substantially degraded in the flowable state.

[0008] In some embodiments, the composition can be induced to a flowable state by the application of shear and pressure. In some embodiments, the composition can be induced to a flowable state by the application of shear and pressure without the application of heat. In some embodiments, the composition can be induced to a flowable state and the recombinant spider silk protein remaining substantially undegraded within the composition can be extruded multiple times.

[0009] In some embodiments, the composition is thermoplastic.

[0010] In some embodiments, the composition may be induced to a flowable state by applying a shear force in the range of 1.5 Nm to 13 Nm. In some embodiments, the composition may be induced to a flowable state by applying a shear force in the range of 2 Nm to 6 Nm. In some embodiments, the composition may be induced to a flowable state by applying a pressure in the range of 1 MPa to 300 MPa. In some embodiments, the composition may be induced to a flowable state by applying a pressure in the range of 5 MPa to 75 MPa.

[0011] In some embodiments, the composition can be induced to a flowable state at less than 120° C., less than 80° C., less than 40° C., or at room temperature. In some embodiments, the composition is substantially homogeneous.

[0012] In some embodiments, the recombinant spider silk protein comprises repeat units. In some embodiments, the recombinant spider silk protein comprises an amino acid residue length in the range of 60 to 100 amino acids, with a repeat unit in the range of 2 to 20. In some embodiments, the molecular weight of the recombinant spider silk protein is in the range of 20 to 2000 kDa.

[0013] In some embodiments, the recombinant spider silk protein comprises at least two repeat units, the repeat units comprising: more than 150 amino acid residues and a molecular weight of at least 10 kDa; an alanine-rich region having 6 or more consecutive amino acids with an alanine content of at least 80%; and a glycine-rich region having 12 or more consecutive amino acids with a glycine content of at least 40% and an alanine content of less than 30%.

[0014] In some embodiments, the plasticizer is selected from a polyol, water, and / or urea. In some embodiments, the polyol comprises glycerol. In some embodiments, the plasticizer comprises water. In some embodiments, the recombinant spider silk protein is present in the recombinant spider silk polypeptide powder, and the weight ratio of the plasticizer to the recombinant silk polypeptide powder ranges from 0.05 to 1.50:1. In some embodiments, the recombinant spider silk protein is present in the recombinant spider silk polypeptide powder, and the weight ratio of the plasticizer to the recombinant silk polypeptide powder ranges from 0.20 to 0.70:1.

[0015] In some embodiments, the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder, and the amount of recombinant spider silk polypeptide powder in the composition ranges from 1 to 90% by weight of the recombinant spider silk protein. In some embodiments, the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder, and the amount of recombinant spider silk polypeptide powder in the composition ranges from 20 to 41% by weight of the recombinant spider silk protein. In some embodiments, the composition comprises glycerol as a plasticizer in the range of 1 to 60% by weight. In some embodiments, the composition comprises glycerol as a plasticizer in the range of 15 to 30% by weight. In some embodiments, the composition comprises water as a plasticizer in the range of 5 to 80% by weight. In some embodiments, the composition comprises water as a plasticizer in the range of 19 to 27% by weight.

[0016] In some embodiments, the recombinant spider silk protein is degraded in an amount of less than 10.0% by weight under flowable conditions. In some embodiments, the recombinant spider silk protein is degraded in an amount of less than 6.0% by weight under flowable conditions. In some embodiments, the recombinant spider silk protein is degraded in an amount of less than 2.0% by weight under flowable conditions. In some embodiments, the degradation of the recombinant spider silk protein is assessed by measuring the amount of full-length recombinant spider silk protein present in the composition before and after flowable conditions are induced. In some embodiments, the amount of full-length recombinant spider silk protein is measured using size exclusion chromatography.

[0017] Also provided herein, according to some embodiments of the present invention, is a molded body comprising a molded body composition comprising a recombinant spider silk protein and a plasticizer, wherein the composition can be induced to a flowable state, and wherein the recombinant spider silk protein is not substantially degraded in the flowable state.

[0018] In some embodiments, the shaped article is a fiber. In some embodiments, the fiber has a strength in the range of 100 Pa to 1.2 GPa. In some embodiments, the fiber has a strength of 5×10 as measured by polarized light microscopy. -5 to about 0.04.

[0019] Also provided herein, according to some embodiments of the present invention, is a process for preparing a shaped body, comprising applying pressure and shear to a composition comprising a recombinant spider silk protein and a plasticizer to convert the composition into a flowable state, and extruding the composition in the flowable state to form a shaped body.

[0020] In some embodiments, extruding the composition to form a shaped body comprises extruding the composition to form fibers. In some embodiments, extruding the composition to form fibers comprises extruding the composition through a spinneret. In some embodiments, extruding the composition to form a shaped body comprises extruding the composition into a mold.

[0021] In some embodiments, the process for preparing a shaped body further comprises (a) applying pressure and shear to the shaped body to convert the shaped body into a composition in a flowable state, and (b) extruding the composition in a flowable state to form a second shaped body. In some embodiments, the process further comprises repeating steps (a) and (b) at least once on the second shaped body.

[0022] In some embodiments, the shear force is between 1.5 and 13 N. * In some embodiments, the pressure is from 1 MPa to 300 MPa. In some embodiments, the shear force and pressure are applied to the composition using a capillary rheometer or a twin-screw extruder. In some embodiments, the screw speed of the twin-screw extruder ranges from 10 to 300 RPM during the application of the pressure and shear force.

[0023] In some embodiments, the device used to apply the shear and pressure includes a mixing chamber coupled to and proximal to the extrusion chamber. In some embodiments, the composition is heated in the mixing chamber. In some embodiments, the composition is heated in the extrusion chamber. In some embodiments, the composition is heated to a temperature less than 120°C. In some embodiments, the composition is heated to a temperature less than 80°C. In some embodiments, the composition is heated to a temperature less than 40°C. In some embodiments, the extrusion chamber tapers proximal to the orifice through which the composition is extruded. In some embodiments, the extrusion chamber is temperature controlled. In some embodiments, the residence time of the composition in the mixing chamber is in the range of 3 to 7 minutes.

[0024] In some embodiments, the extruded body loses less than 15% water compared to the composition before extrusion, hi some embodiments, the extruded body loses less than 10% water compared to the composition before extrusion.

[0025] In some embodiments, the shaped body is a fiber and the fiber is drawn manually. In some embodiments, the shaped body is a fiber and the fiber is drawn in multiple steps.

[0026] In some embodiments, the recombinant spider silk protein is substantially not degraded in the shaped body. In some embodiments, the recombinant spider silk protein is degraded in an amount of less than 10% by weight in the shaped body. In some embodiments, the recombinant spider silk protein is degraded in an amount of less than 6% by weight in the shaped body. In some embodiments, the recombinant spider silk protein is degraded in an amount of less than 2% by weight in the shaped body. In some embodiments, degradation of the recombinant spider silk protein is assessed by measuring the amount of full-length recombinant spider silk protein present in the composition before and after extrusion. In some embodiments, the amount of full-length recombinant spider silk protein is measured using size exclusion chromatography.

[0027] In some embodiments, the molded body has minimal birefringence as measured by polarized light microscopy. [The present invention 1001] A composition for a molded body comprising a recombinant spider silk protein and a plasticizer, The composition can be induced to a flowable state; The composition, wherein the recombinant spider silk protein is not substantially degraded in the flowable state. [The present invention 1002] The composition of the present invention 1001, which can be induced into said flowable state by the application of shear and pressure. [The present invention 1003] The composition of claim 1002, which can be induced to said flowable state by the application of shear and pressure without the application of heat. [The present invention 1004] 1003. The composition of claim 1003, wherein said composition can be induced to said flowable state and said recombinant spider silk protein remaining substantially undegraded within said composition can be extruded multiple times. [The present invention 1005] The composition of the present invention 1001, which is thermoplastic. [The present invention 1006] The composition of the present invention 1002, which can be induced into a flowable state by the application of a shear force in the range of 1.5 Nm to 13 Nm. [The present invention 1007] The composition of the present invention 1002, which can be induced into a flowable state by the application of a shear force in the range of 2 Nm to 6 Nm. [The present invention 1008] The composition of the present invention 1002, which can be induced to a flowable state by applying a pressure in the range of 1 MPa to 300 MPa. [The present invention 1009] The composition of the present invention 1002, which can be induced to a flowable state by applying a pressure in the range of 5 MPa to 75 MPa. [The present invention 1010] Any of the compositions of inventions 1006 to 1009, which can be induced to a flowable state at temperatures below 120°C, below 80°C, below 40°C, or at room temperature. [The present invention 1011] Any of the compositions of claims 1001 to 1010, having a melt flow index of at least 0.5, at least 1, at least 2, or at least 5 when tested according to ASTM D1238 at 95°C under a 2.16 kg load. [The present invention 1012] Any of the compositions of claims 1001 to 1010, having a melt flow index of at least 0.5, at least 1, at least 2, or at least 5 when tested according to ASTM D1238 at 95°C under a 21.6 kg load. [The present invention 1013] 1001. The composition of the present invention, which is substantially homogeneous. [The present invention 1014] 1001. The composition of claim 1001, wherein said recombinant spider silk protein comprises a repeating unit. [The present invention 1015] 1001. The composition of the present invention, wherein said recombinant spider silk protein comprises an amino acid residue length in the range of 60 to 100 amino acids, with a range of 2 to 20 repeat units. [The present invention 1016] 1001. The composition of the present invention, wherein the molecular weight of said recombinant spider silk protein is in the range of 20 to 2000 kDa. [The present invention 1017] The recombinant spider silk protein comprises at least two occurrences of a repeat unit, the repeat unit comprising: having more than 150 amino acid residues and a molecular weight of at least 10 kDa; an alanine-rich region having six or more consecutive amino acids with an alanine content of at least 80%; a glycine-rich region having 12 or more consecutive amino acids, the glycine content being at least 40% and the alanine content being less than 30%; 1001. The composition of the present invention comprising: [The present invention 1018] 1001. The composition of claim 1001, wherein the plasticizer is selected from polyols, water, and / or urea. [The present invention 1019] 1016. The composition of claim 1016, wherein the polyol comprises glycerol. [The present invention 1020] 1001. The composition of claim 10, wherein the plasticizer comprises water. [The present invention 1021] the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder; 1001. The composition of the present invention, wherein the weight ratio of the plasticizer to the recombinant silk polypeptide powder is in the range of 0.05:1 to 4:1. [The present invention 1022] the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder; 1001. The composition of the present invention, wherein the weight ratio of the plasticizer to the recombinant silk polypeptide powder is in the range of 0.20:1 to 0.70:1. [The present invention 1023] the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder; 1001. The composition of claim 1001, wherein the amount of recombinant spider silk polypeptide powder in said composition is in the range of 1-90% by weight of recombinant spider silk protein. [The present invention 1024] the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder; 1001. The composition of claim 1001, wherein the amount of recombinant spider silk polypeptide powder in said composition is in the range of 20-41% by weight of recombinant spider silk protein. [The present invention 1025] 1001. The composition of claim 1001, comprising glycerol in the range of 1 to 90% by weight as a plasticizer. [The present invention 1026] 1001. The composition of claim 1001, comprising glycerol in the range of 15 to 30% by weight as a plasticizer. [The present invention 1027] 1001. The composition of the present invention, comprising water in the range of 5 to 80% by weight as a plasticizer. [The present invention 1028] 1001. The composition of the present invention, comprising water in the range of 19 to 27% by weight as a plasticizer. [The present invention 1029] 1001. The composition of claim 1001, wherein the recombinant spider silk protein is degraded in an amount of less than 10.0% by weight in the flowable state. [The present invention 1030] 1001. The composition of claim 1001, wherein the recombinant spider silk protein is degraded in an amount of less than 6.0% by weight in the flowable state. [The present invention 1031] 1001. The composition of claim 1001, wherein the recombinant spider silk protein is degraded in an amount of less than 2.0% by weight in the flowable state. [The present invention 1032] 1032. Any of the compositions of claims 1029 to 1031, wherein degradation of said recombinant spider silk protein is assessed by measuring the amount of full-length recombinant spider silk protein present in the composition before and after said flowable state is induced. [The present invention 1033] 1032. The composition of claim 1032, wherein the amount of full-length recombinant spider silk protein is measured using size exclusion chromatography. [The present invention 1034] A molded article comprising any one of the compositions of present inventions 1001 to 1033. [This invention 1035] The molded article of the present invention 1034, wherein the molded article is a fiber. [The present invention 1036] The molded article of the present invention 1035, wherein the fibers have a strength in the range of 100 Pa to 1.2 GPa. [This invention 1037] The fibers have a molecular weight of 5×10 as measured by polarized light microscopy. -5 The molded article of the present invention 1035 has a birefringence in the range of about 0.04. [The present invention 1038] 1. A process for preparing a shaped body, comprising: (a) applying pressure and shear forces to a composition comprising a recombinant spider silk protein and a plasticizer to convert said composition into a flowable state; (b) extruding the composition in a flowable state to form a shaped body; The process comprising: [This invention 1039] The process of claim 1038, wherein the step of extruding the composition to form a shaped body comprises extruding the composition to form fibers. [The present invention 1040] The process of claim 1039, wherein extruding the composition to form fibers comprises extruding the composition through a spinneret. [This invention 1041] The process of claim 1038, wherein the step of extruding the composition to form a shaped body comprises extruding the composition into a mold. [The present invention 1042] (a) applying pressure and shear to the compact to convert the compact into a flowable composition; (b) extruding the composition in the flowable state to form a second shaped body; The process of the present invention 1038 further comprises: [This invention 1043] The process of claim 1042, further comprising repeating steps (a) and (b) on said second compact at least once. [This invention 1044] The process of any one of claims 1038 to 1043, wherein the shear force is 1.5 to 13 Nm. [This invention 1045] The process of any one of claims 1038 to 1043, wherein the pressure is from 1 MPa to 300 MPa. [The present invention 1046] The process of any one of claims 1038 to 1045, wherein the shear force and pressure are applied to the composition using a capillary rheometer or a twin-screw extruder. [This invention 1047] 1046. The process of claim 1046, wherein the screw speed of said twin screw extruder during application of said pressure and said shear force is in the range of 10 to 300 RPM. [This invention 1048] The process of any of claims 1038 to 1045, wherein the device used to apply the shear force and pressure comprises a mixing chamber coupled to and proximal to the extrusion chamber. [This invention 1049] The process of claim 1048, wherein said composition is heated in said mixing chamber. [The present invention 1050] The process of claim 1048, wherein the composition is heated in the extrusion chamber. [This invention 1051] The process of any of claims 1049 or 1050, wherein the composition is heated to a temperature of less than 120°C. [This invention 1052] The process of claim 1049 or claim 1050, wherein the composition is heated to a temperature of less than 80°C. [This invention 1053] The process of claim 1049 or claim 1050, wherein the composition is heated to a temperature of less than 40°C. [This invention 1054] The process of any one of claims 1038 to 1053, wherein the extruded body has lost less than 15% of its moisture compared to the composition before extrusion. [This invention 1055] The process of any one of claims 1038 to 1053, wherein the extruded body has lost less than 10% of its moisture compared to the composition before extrusion. [This invention 1056] 1048. The process of claim 1048, wherein said composition has a residence time in said mixing chamber in the range of 3 to 7 minutes. [This invention 1057] The process of claim 1048, wherein the extrusion chamber is tapered proximate to the orifice through which the composition is extruded. [This invention 1058] The process of claim 1048, wherein said extrusion chamber is temperature controlled. [This invention 1059] The process of any one of claims 1048 to 1058, wherein the shaped body is a fiber and the fiber is manually drawn. [The present invention 1060] The process of any one of claims 1048 to 1059, wherein the shaped body is a fiber and the fiber is drawn in multiple steps. [This invention 1061] 1061. The process of any of claims 1048 to 1060, wherein the recombinant spider silk protein is not substantially degraded in the shaped body. [This invention 1062] 106. The process of claim 1061, wherein the recombinant spider silk protein is degraded in an amount of less than 10% by weight in the shaped body. [This invention 1063] 106. The process of claim 1061, wherein the recombinant spider silk protein is degraded in an amount of less than 6% by weight in the shaped body. [This invention 1064] 106. The process of claim 1061, wherein the recombinant spider silk protein is degraded in an amount of less than 2% by weight in the shaped body. [This invention 1065] 1065. The process of any of claims 1061 to 1064, wherein degradation of said recombinant spider silk protein is assessed by measuring the amount of full length recombinant spider silk protein present in said composition before and after extrusion. [The present invention 1066] 1065. The process of claim 1065, wherein size exclusion chromatography is used to measure the amount of full-length recombinant spider silk protein. [This invention 1067] The process of any one of claims 1038 to 1066, wherein the molded article has minimal birefringence as measured by polarizing microscope. [Brief explanation of the drawings]

[0028] The foregoing and other objects, features and advantages will become apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings.

[0029] [Figure 1] 1 shows size exclusion chromatography data for P49W21G30 melt compositions extruded under selected thermal and RPM conditions, according to various embodiments of the present invention. [Figure 2]1 shows size exclusion chromatography data for P65W20G15 melt compositions extruded under selected thermal and RPM conditions, according to various embodiments of the present invention. [Figure 3] 1 shows size exclusion chromatography data for P71W19G10 melt compositions extruded under selected thermal and RPM conditions, according to various embodiments of the present invention. [Figure 4] 1 shows a chart of water loss during extrusion of P49W21G30 melt compositions extruded under selected thermal and RPM conditions, as measured by thermogravimetric analysis (TGA), according to various embodiments of the present invention. The data shows the percent moisture content of the starting pellets before extrusion and the extruded sample under the selected conditions after extrusion. [Figure 5] 1 shows a chart of water loss during extrusion of P65W20G15 melt compositions extruded under selected thermal and RPM conditions, as measured by thermogravimetric analysis (TGA), according to various embodiments of the present invention. The data shows the percent moisture content of the starting pellets before extrusion and the extruded sample under the selected conditions after extrusion. [Figure 6] 1 shows a chart of moisture loss during extrusion of P71W19G10 melt compositions extruded under selected thermal and RPM conditions, as measured by thermogravimetric analysis (TGA), according to various embodiments of the present invention. The data shows the percent moisture content of the starting powder before extrusion and the extruded sample under the selected conditions after extrusion. [Figure 7] Figure 1 shows the beta-sheet content of P49W21G30 samples extruded under selected thermal and RPM conditions as measured by Fourier transform infrared spectroscopy (FTIR). Samples were compared to reference controls of the starting protein powder and starting pellets. [Figure 8] Figure 1 shows the beta-sheet content of P65W20G15 samples extruded under selected thermal and RPM conditions as measured by Fourier transform infrared spectroscopy (FTIR). Samples were compared to reference controls of the starting protein powder and starting pellets. [Figure 9]Figure 1 shows the beta-sheet content of P71W19G10 samples extruded under selected thermal and RPM conditions as measured by Fourier transform infrared spectroscopy (FTIR). Samples were compared to reference controls of the starting protein powder and starting pellets. [Figure 10] Shown are images captured using polarized light microscopy of selected extruded products produced at 20°C and at 10, 100, 200, or 300 RPM. [Figure 11] Shown are images captured using polarized light microscopy of selected extruded products produced at 95°C and 10, 100, 200, or 300 RPM. [Figure 12] 1 shows a chart of glycerol loss during extrusion of P49W21G30 extrudates extruded under selected thermal and RPM conditions as measured by HPLC, according to various embodiments of the present invention. The data shows the glycerol content (%) of the starting powder or pellets for samples before and after extrusion under the selected conditions. [Figure 13] 1 shows a chart of glycerol loss during extrusion of P65W20G15 extrudates extruded under selected thermal and RPM conditions, as measured by HPLC, according to various embodiments of the present invention. The data shows the glycerol content (%) of the starting powder or pellets for the samples before and after extrusion under the selected conditions. [Figure 14] 1 shows a chart of glycerol loss during extrusion of P71W19G10 extrudates extruded under selected thermal and RPM conditions as measured by HPLC, according to various embodiments of the present invention. The data shows the glycerol content (%) of the starting powder or pellets for the samples before and after extrusion under the selected conditions. DETAILED DESCRIPTION OF THE INVENTION

[0030] Detailed Description Details of various embodiments of the present invention are set forth in the following description. Other features, objects, and advantages of the present invention will become apparent from the description. Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. The terms "a" and "an" include plural referents unless the context dictates otherwise. Generally, the terminology used in connection with and techniques relating to biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those commonly used and well known in the art.

[0031] definition Unless otherwise indicated, the following terms shall be understood to have the following meanings:

[0032] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides at least 10 bases in length. Such terms include DNA molecules (e.g., cDNA or genomic DNA or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-natural internucleoside linkages, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially duplexed, branched, hairpinned, circular, or padlocked.

[0033] Unless otherwise indicated, for any sequence described herein in the general format of "SEQ ID NO:," as an example, a "nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid having, at least in part, (i) the sequence set forth in SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 1. The alternative is dictated by the context. For example, if the nucleic acid is being used as a probe, the alternative is dictated by the requirement that the probe be complementary to the desired target.

[0034] "Isolated" RNA, DNA, or mixed polymers are those that are substantially separated from other cellular components that naturally accompany the native polynucleotide in its native host cell, such as naturally associated ribosomes, polymerases, and genomic sequences.

[0035] An "isolated" organic molecule (e.g., silk protein) is one that has been substantially separated from cellular components (membrane lipids, chromosomes, proteins) of the host cell from which it is derived or from the medium used to culture the host cell. The term does not require that the biomolecule be separated from all other chemicals, although certain isolated biomolecules may be purified to near homogeneity.

[0036] The term "recombinant" refers to a biological molecule, e.g., a gene or protein, that is (1) removed from its naturally occurring environment, (2) not associated with all or part of a polynucleotide with which the gene is found in nature, (3) operably linked to a polynucleotide with which it is not linked in nature, or (4) not occurring in nature. The term "recombinant" may be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized in heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0037] An endogenous nucleic acid sequence is considered "recombinant" herein when a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence in the genome of an organism such that expression of the endogenous nucleic acid sequence (or the protein product encoded by the sequence) is altered. In this case, the heterologous sequence is a sequence that does not naturally flank the endogenous nucleic acid sequence, regardless of whether the heterologous sequence is itself endogenous (from the same host cell or its progeny) or exogenous (from a different host cell or its progeny). As an example, a promoter sequence can replace (e.g., by homologous recombination) the native promoter of a gene present in the genome of a host cell, resulting in an altered expression pattern of the gene. The gene would now be considered "recombinant" because it has been separated from at least some of the sequences that naturally flank it.

[0038] A nucleic acid is also considered "recombinant" if it contains any alteration relative to the corresponding nucleic acid in a genome that does not occur in nature. For example, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion, or point mutation introduced artificially, such as by human intervention. "Recombinant nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site, and a nucleic acid construct present as an episome.

[0039] As used herein, the term "peptide" refers to a short polypeptide, e.g., generally less than about 50 amino acids in length, more usually less than about 30 amino acids in length. As used herein, the term encompasses analogs as well as mimetics that mimic structure-function and thereby biological function.

[0040] The term "polypeptide" encompasses both naturally occurring and non-naturally occurring proteins, as well as fragments, variants, derivatives, and analogs thereof. Polypeptides may be monomeric or polymeric. Furthermore, a polypeptide may contain multiple different domains, each with one or more distinct activities.

[0041] The term "isolated protein" or "isolated polypeptide" refers to, in terms of its origin or source of derivation, (1) a protein or polypeptide that is not associated with naturally associated components that accompany it in its natural state; (2) a protein or polypeptide that exists to a degree of purity not found in nature, where purity can be determined with respect to the presence of other cellular material (e.g., free from other proteins from the same species); (3) a protein or polypeptide expressed by cells from a different species; or (4) a protein or polypeptide that does not occur in nature (e.g., it is a fragment of a naturally occurring polypeptide or contains amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a chemically synthesized polypeptide or a polypeptide synthesized in a cellular system different from the cell from which it naturally originates is "isolated" from its naturally associated components. A polypeptide or protein may be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art. When defined in this way, "isolated" does not necessarily require that the protein, polypeptide, peptide, or oligopeptide so described has been physically removed from its natural environment.

[0042] The term "polypeptide fragment" refers to a polypeptide that has a deletion compared to a full-length polypeptide, e.g., a deletion at the amino and / or carboxy terminus. In preferred embodiments, a polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to corresponding positions in the naturally occurring sequence. Fragments are generally at least 5, 6, 7, 8, 9, or 10 amino acids in length, preferably at least 12, 14, 16, or 18 amino acids in length, more preferably at least 20 amino acids in length, more preferably at least 25, 30, 35, 40, or 45 amino acids in length, even more preferably at least 50 or 60 amino acids in length, and even more preferably at least 70 amino acids in length.

[0043] A protein has "homology" or is "homologous" to a second protein if the nucleic acid sequence encoding the protein has a similar sequence to the nucleic acid sequence encoding the second protein. Alternatively, a protein has homology to a second protein if the two proteins have "similar" amino acid sequences. (Thus, the term "homologous proteins" is defined to mean that two proteins have similar amino acid sequences.) As used herein, homology between two amino acid sequence regions (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.

[0044] When using "homologous" with respect to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). Generally, conservative amino acid substitutions do not substantially alter the functional properties of the protein. When two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of homology may be adjusted upward to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (incorporated herein by reference).

[0045] The 20 conventional amino acids and their abbreviations follow convention. nded. 1991). Stereoisomers of the 20 conventional amino acids (e.g., D-amino acids), unnatural amino acids, such as α-,α-disubstituted amino acids, N-alkylamino acids, and other unconventional amino acids may also be suitable components of the polypeptides of the invention. Examples of unconventional amino acids include 4-hydroxyproline, γ-carboxyglutamic acid, ε-N,N,N-trimethyllysine, ε-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminus and the right-hand end corresponds to the carboxy terminus, in accordance with standard usage and convention.

[0046] The following six groups each contain amino acids that are conservative substitutions for one another: 1) serine (S), threonine (T); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), alanine (A), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

[0047] Sequence homology of polypeptides, sometimes referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using homology measures assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, GCG includes programs such as "Gap" and "Bestfit," which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms, or between a wild-type protein and its mutant protein. See, e.g., GCG version 6.1.

[0048] A useful algorithm for comparing a particular polypeptide sequence to a database containing many sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0049] Preferred parameters for BLASTp are expectation: 10 (default); filter: seg (default); gap opening cost: 11 (default); gap extension cost: 1 (default); maximum alignments: 100 (default); string size: 11 (default); number of results displayed: 100 (default); penalty matrix: BLOWSUM62.

[0050] Preferred parameters for BLASTp are: expectation: 10 (default); filter: seg (default); gap opening cost: 11 (default); gap extension cost: 1 (default); maximum alignment: 100 (default); string size: 11 (default); number of results displayed: 100 (default); penalty matrix: BLOWSUM62. The length of polypeptide sequences to be compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at least about 24 residues, typically at least about 28 residues, and preferably greater than about 35 residues. When searching a database containing sequences from multiple different organisms, amino acid sequence comparison is preferred. Database searches using amino acid sequences can be performed using algorithms known in the art other than blastp. For example, polypeptide sequences can be compared using FASTA, a program in GCG version 6.1. FASTA provides alignments and percent sequence identity of the regions of best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990), which is incorporated herein by reference. For example, percent sequence identity between amino acid sequences can be determined using FASTA, provided in GCG version 6.1, which is incorporated herein by reference, with its default parameters (string size of 2 and scoring matrix PAM250).

[0051] Throughout this specification and the claims, the word "comprise" or variations such as "comprises" or "comprising" will be understood to mean the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers.

[0052] As defined herein, the term "molded body" refers to an object produced through a molding process that uses a rigid frame called a mold to shape a liquid or flexible feedstock material, including, but not limited to, extrusion, injection molding, compression molding, blow molding, lamination, matrix molding, rotational molding, spin casting, transfer molding, thermoforming, and the like.

[0053] As defined herein, the term "fiber" refers to an elongated shaped body; generally, fibers have the form of a filament.

[0054] As used herein, the term "melt spinning" refers to a process for forming fibers from a polymer in which the polymer is converted to a molten or flowable state and then cooled and solidified after being extruded through a spinneret.

[0055] The term "drawing" as used herein with respect to fibers refers to the application of a force to a spun fiber along its longitudinal axis during or after extrusion of the fiber. The term "undrawn fiber" refers to a fiber that has been extruded but has not been drawn. The term "draw ratio" is a term of art generally defined as the ratio between the withdrawal rate and the feed rate. At a constant volume, the initial diameter (D i ) and final diameter (D f ) ratio (i.e., D i / D r ) is determined.

[0056] As used herein, the term "glass transition" refers to the transition of a substance or composition from a hard, rigid, or "glassy" state to a more flexible, "rubbery" or "viscous" state.

[0057] As used herein, the term "glass transition temperature" refers to the temperature at which a substance or composition undergoes a glass transition.

[0058] As used herein, the term "melt transition" refers to the transition of a material or composition from a rubbery state to a less ordered liquid phase or flowable state.

[0059] As used herein, the term "melting temperature" refers to the temperature range at which a material undergoes a melting transition.

[0060] As used herein, the term "plasticizer" refers to any molecule that interacts with a polypeptide sequence to prevent the polypeptide sequence from forming tertiary structures and bonds and / or to increase the mobility of the polypeptide sequence.

[0061] As used herein, the term "flowable state" refers to a composition that has substantially the same properties as a liquid (ie, has transitioned from a rubbery state to a liquid state).

[0062] Exemplary methods and materials are described below; however, methods and materials similar or equivalent to those described herein can be used in the practice of the present invention and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and are intended to be non-limiting.

[0063] Overview Provided herein are molded body compositions comprising a recombinant spider silk protein and a plasticizer, which are homogeneous or substantially homogeneous in the molten or flowable state, and in which the recombinant spider silk protein is not substantially degraded (e.g., less than 10% by weight, or often less than 6% by weight) after it is formed into a molded body.

[0064] Recombinant silk proteins This disclosure describes embodiments of the invention that include fibers synthesized from synthetic proteinaceous copolymers (i.e., recombinant polypeptides). Suitable proteinaceous copolymers are described in U.S. Patent Publication Nos. 2016 / 0222174, published August 45, 2016, 2018 / 0111970, published April 26, 2018, and 2018 / 0057548, published March 1, 2018, the entire contents of each of which are incorporated herein by reference.

[0065] In some embodiments, synthetic protein copolymers are generated from silk-like polypeptide sequences. In some embodiments, the silk-like polypeptide sequences are: 1) block copolymer polypeptide compositions produced by mixing and matching repeat domains from silk polypeptide sequences; and / or 2) recombinant expression of block copolymer polypeptides large enough (approximately 40 kDa) to form useful molded body compositions by secretion in industrially scalable microorganisms. Large (approximately 40 kDa to approximately 100 kDa) block copolymer polypeptides engineered with repeat domain fragments of silk, including sequences derived from nearly all of the published amino acid sequences of spider silk polypeptides, can be expressed in the engineered microorganisms described herein. In some embodiments, silk polypeptide sequences are matched and designed to produce highly expressed and secreted polypeptides capable of forming molded bodies.

[0066] In some embodiments, block copolymers are engineered to combine and mix silk polypeptide domains across the silk polypeptide sequence space. In some embodiments, block copolymers are produced by expression and secretion in scalable organisms (e.g., yeast, fungi, and gram-positive bacteria). In some embodiments, block copolymer polypeptides comprise zero or more N-terminal domains (NTDs), one or more repeat domains (REPs), and zero or more C-terminal domains (CTDs). In some aspects of the embodiments, the block copolymer polypeptides are single-chain polypeptides of more than 100 amino acids. In some embodiments, the block copolymer polypeptide comprises a domain that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of a block copolymer polypeptide disclosed in International Publication No. WO / 2015 / 042164, "Methods and Compositions for Synthesizing Improved Silk Fibers," the entire contents of which are incorporated herein by reference.

[0067] Several types of natural spider silk have been identified so far, and the various mechanical properties of naturally spun silk are thought to be closely related to the molecular composition of the silk. See, for example, Garb, JE, et al., Untangling spider silk evolution with spidroin terminal domains, BMC Evol. Biol., 10:243 (2010); Bittencourt, D., et al., Protein families, natural history and biotechnological aspects of spider silk, Genet. Mol. Res., 11:3 (2012); Rising, A., et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell. Mol. Life Sci., 68:2, pp. 169-184 (2011); and Humenik, M., et al., Spider silk: understanding the structure-function relationship of a natural fiber, Prog. Mol. Biol. Transl. Sci., 103, pp. 131-85 (2011). For example:

[0068] Tufted (AcSp) silks tend to be tough, combining moderately high strength with moderately high stretchability. AcSp silks are characterized by large block ("repeat aggregate") sizes, often incorporating polyserine and GPX motifs. Tubular (TuSp, or cylindrical) silks tend to be large in diameter, moderate in strength, and highly stretchable. TuSp silks are characterized by their polyserine and polythreonine content and short polyalanine sequences. Large ampullate (MaSp) silks tend to be high in strength and moderately stretchable. MaSp silks are either of two subtypes, MaSp1 or MaSp2. MaSp1 silks are generally less stretchable than MaSp2 silks and feature polyalanine, GX, and GGX motifs. MaSp2 silks feature polyalanine, GGX, and GPX motifs. MiSp silks tend to have moderate strength and moderate stretchability. MiSp silks are characterized by GGX, GA, and polyA motifs and often contain a spacer element of approximately 100 amino acids. Flagelliform (Flag) silks tend to have very high stretchability and moderate strength. Flag silks are usually characterized by GPG, GGX, and a short spacer motif.

[0069] The properties of each silk species may vary from species to species, and spiders with different lifestyles (e.g., stationary web-weaving spiders versus wandering, feeding spiders) or evolutionarily older spiders may produce silks that differ from the above description (for a description of spider diversity and classification, see Hormiga, G., and Griswold, C.E., Systematics, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, pp. 487-512 (2014); and Blackedge, T.A. et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. Natl. Acad. Sci. USA, 106:13, pp. 5229-5234 (2009)). However, synthetic block copolymer polypeptides with sequence similarity and / or amino acid composition similarity to the repeat domains of natural silk proteins can be used to produce consistent molded bodies on a commercial scale that replicate the properties of corresponding molded bodies made from natural silk polypeptides.

[0070] In some embodiments, a list of putative silk sequences can be compiled by searching GenBank for related terms, such as "spidroin," "fibroin," and "MaSp," and these sequences can be pooled with additional sequences obtained from independent sequencing. These sequences are then translated into amino acids, duplicate entries are filtered, and manually divided into each domain (NTD, REP, CTD). In some embodiments, the candidate amino acid sequences are reverse-translated into DNA sequences optimized for expression in Pichia (Komagataella) pastoris. The DNA sequences are each cloned into an expression vector and transformed into Pichia (Komagataella) pastoris. In some embodiments, various silk domains that demonstrate successful expression and secretion are then assembled combinatorially to construct a body-forming competent silk molecule.

[0071] Silk polypeptides characteristically consist of repeat domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). In one embodiment, both the C-terminal and N-terminal domains are 75-350 amino acids in length. The repeat domains exhibit a hierarchical structure, as shown in Figure 1. The repeat domains contain a series of blocks (also known as repeat units). These blocks are sometimes perfectly repeated and sometimes imperfectly repeated (forming quasi-repeat domains) throughout the repeat domain of silk. The length and composition of the blocks vary between different silk species and between different species. Table 1A lists example block sequences for selected species and silk species; further examples are described in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell Mol. Life Sci., 68:2, pp. 169-184 (2011); and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pp. 2603-2605 (2001). In some cases, blocks may be arranged in a pattern to form larger macrorepeats that appear multiple times (usually 2-8 times) in the repeat domain of the silk sequence. Blocks repeated within the repeat domain or macrorepeat may be separated by spacing elements from macrorepeats repeated within the repeat domain. In some embodiments, the block sequence comprises a glycine-rich region followed by a poly(A) region. In some embodiments, a short (about 1-10) amino acid motif occurs multiple times within a block. For purposes of the present invention, blocks derived from different natural silk polypeptides can be selected without regard to circular permutation (i.e., identified blocks may not align due to circular permutation if they are otherwise similar between silk polypeptides).Thus, for example, a "block" of SGAGG (SEQ ID NO: 3) is, for purposes of the present invention, identical to GSGAG (SEQ ID NO: 4), which is also identical to GGSGA (SEQ ID NO: 5); they are all merely circular permutations of each other. The particular permutation chosen for a given silk sequence may be determined more by convenience than anything else (usually starting with G). Silk sequences obtained from the NCBI database can be divided into blocks and non-repetitive regions.

[0072] (Table 1A) Block sequence samples TIFF2025118794000002.tif93157TIFF2025118794000003.tif235157TIFF2025118794000004.tif231157TIFF2025118794000005.tif162157

[0073] Fiber-forming block copolymer polypeptides derived from block and / or macrorepeat domains according to certain embodiments of the present invention are described in International Publication No. WO / 2015 / 042164, which is incorporated herein by reference. Natural silk sequences, obtained from protein databases such as GenBank or by de novo sequencing, are broken down into domains (N-terminal, repeat, and C-terminal domains). The N-terminal and C-terminal domain sequences selected for post-synthetic assembly into fibers or molded bodies contain native amino acid sequence information and other modifications described herein. The repeat domains are broken down into repeat sequences, which contain representative blocks (usually 1-8, depending on the silk species) that capture key amino acid information, while reducing the size of the DNA encoding the amino acids to easily synthesizable fragments. In some embodiments, properly formed block copolymer polypeptides contain at least one repeat domain containing at least one repeat sequence, optionally flanked by an N-terminal and / or C-terminal domain.

[0074] In some embodiments, the repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence is 150-300 amino acid residues. In some embodiments, the repeat sequence comprises multiple blocks. In some embodiments, the repeat sequence comprises multiple macrorepeats. In some embodiments, the blocks or macrorepeats are divided across the multiple repeat sequences.

[0075] In some embodiments, the repeat sequence must begin with glycine and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine (C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) to meet DNA assembly requirements. In some embodiments, some repeat sequences can be altered relative to the native sequence. In some embodiments, the repeat sequence can be altered, such as by adding serine to the C-terminus of the polypeptide (to avoid termination at F, Y, W, C, H, N, M, or D). In some embodiments, the repeat sequence can be modified by filling in an incomplete block with a homologous sequence from another block. In some embodiments, the repeat sequence can be modified by rearranging the order of the blocks or macrorepeats.

[0076] In some embodiments, unique N-terminal and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain can be created by removing a leading signal sequence, for example, as identified by SignalP (Peterson, TN, et. Al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg. 785-786 (2011)).

[0077] In some embodiments, the N-terminal domain sequence, the repeat sequence, or the C-terminal domain sequence is selected from the group consisting of Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope argentata, Argiope orbiculare. bruennichi, Argiope trifasciata, Atypoides riversi, Avicularia juruensis, Bothriocyrtum californicum, Deinopis spinosa, Diguetia canities, Dolomedes tenebrosus, Euagrus chisoseus, Euprosthenops australis, Gasteracantha mammosa, Hypochilus thorelli, Kukulcania hibernalis hibernalis, Latrodectus hesperus, Megahexura fulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavataclavata, Nephila clavipes, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

[0078] In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to a 3X FLAG nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence is operably linked to another affinity tag, such as 6-8 His residues (SEQ ID NO: 33).

[0079] In some embodiments, the recombinant spider silk polypeptide is based on a recombinant spider silk protein fragment sequence from MaSp2, such as from the Argiope species. In some embodiments, the synthetic fibers comprise protein molecules comprising 2-20 repeat units, each repeat unit having a molecular weight greater than about 20 kDa. Within each repeat unit of the copolymer, there are more than about 60 amino acid residues, often in the range of 60-100 amino acids, organized into several "quasi-repeat units." In some embodiments, the repeat units of the polypeptides described herein have at least 95% sequence identity to the MaSp2 dragline silk protein sequence.

[0080] Repeat units of proteinaceous block copolymers that form fibers with good mechanical properties can be synthesized using portions of silk polypeptides. These polypeptide repeat units contain alanine-rich and glycine-rich regions and are 150 amino acids or longer in length. Some exemplary sequences that can be used as repeats in the proteinaceous block copolymers of the present disclosure are provided in co-owned PCT Publication WO2015 / 042164, the entire contents of which are incorporated herein by reference, and have been demonstrated to be expressed using a Pichia expression system.

[0081] In some embodiments, the spider silk protein comprises at least two occurrences of a repeat unit, the repeat unit having: more than 150 amino acid residues and a molecular weight of at least 10 kDa; an alanine-rich region having 6 or more consecutive amino acids with an alanine content of at least 80%; a glycine-rich region having 12 or more consecutive amino acids with an alanine content of at least 40% and an alanine content of less than 30%; and the fiber comprises at least one property selected from the group consisting of an elastic modulus of greater than 550 cN / tex, an extensibility of at least 10%, and an ultimate tensile strength of at least 15 cN / tex.

[0082] In some embodiments, the recombinant spider silk protein comprises repeat units, each repeat unit having at least 95% sequence identity to a sequence comprising 2 to 20 quasi-repeat units; each quasi-repeat unit having a sequence similar to {GGY-[GPG-X1] n1 -GPS-(A) n2 {(SEQ ID NO: 34)}, wherein for each quasi-repeat unit, X1 is independently selected from the group consisting of SGGQQ (SEQ ID NO: 35), GAGQQ (SEQ ID NO: 36), GQGPY (SEQ ID NO: 37), AGQQ (SEQ ID NO: 38), and SQ; n1 is 4 to 8, and n2 is 6 to 10. The repeat unit is composed of multiple quasi-repeat units.

[0083] In some embodiments, three "long" quasi-repeats are followed by three "short" quasi-repeat units. As noted above, short quasi-repeat units are those where n1 = 4 or 5. Long quasi-repeat units are defined as those where n1 = 6, 7, or 8. In some embodiments, all of the short quasi-repeats have the same X1 motif at the same position within each quasi-repeat unit of the repeat unit. In some embodiments, fewer than three quasi-repeat units out of six quasi-repeat units share the same X1 motif.

[0084] In a further embodiment, the repeat unit is made up of quasi-repeat units in which the same X1 does not occur more than two consecutive times within the repeat unit. In a further embodiment, the repeat unit is made up of quasi-repeat units in which at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 quasi-repeats do not use the same X1 more than two consecutive times within a single quasi-repeat unit of the repeat unit.

[0085] In some embodiments, the recombinant spider silk polypeptide comprises the polypeptide sequence of SEQ ID NO: 1 (i.e., 18B). In some embodiments, the repeat unit is a polypeptide comprising SEQ ID NO: 2. These sequences are shown in Table 1B.

[0086] Table 1B: Exemplary polypeptide sequences of recombinant proteins and repeat units TIFF2025118794000006.tif127160

[0087] In some embodiments, the structure of fibers formed from the described recombinant spider silk polypeptides forms a beta-sheet structure, a beta-turn structure, or an alpha-helical structure. In some embodiments, the secondary, tertiary, and quaternary protein structure of the formed fibers is described as having nanocrystalline beta-sheet regions, amorphous beta-turn regions, amorphous alpha-helical regions, randomly spatially distributed nanocrystalline regions embedded in an amorphous matrix, or randomly oriented nanocrystalline regions embedded in an amorphous matrix. Without wishing to be bound by theory, it is believed that the structural characteristics of proteins within spider silks are related to the mechanical properties of the fibers. The crystalline regions of the fibers are related to the tensile strength of the fibers, while the amorphous regions are related to the extensibility of the fibers. Major saccular (MA) silks tend to be stronger and less extensible than flagelliform silks; similarly, MA silks have a larger volume fraction of crystalline regions compared to flagelliform silks. Furthermore, theoretical models based on the molecular dynamics of crystalline and amorphous regions of spider silk proteins support the assertion that the crystalline regions are related to the tensile strength of the fiber, while the amorphous regions are related to the extensibility of the fiber. Furthermore, theoretical modeling supports the importance of secondary, tertiary, and quaternary structure to the mechanical properties of RPFs. For example, the assembly of nanocrystalline domains in random, parallel, and sequential spatial distributions, as well as the strength of the interaction forces between entangled chains within the amorphous regions and between the amorphous and nanocrystalline regions, both affect the theoretical mechanical properties of the resulting fiber.

[0088] In some embodiments, the molecular weight of the silk protein is between 20 kDa and 2000 kDa, or greater than 20 kDa, or greater than 10 kDa, or greater than 5 kDa, or between 5 and 400 kDa, or between 5 and 300 kDa, or between 5 and 200 kDa, or between 5 and 100 kDa, or between 5 and 50 kDa, or between 5 and 500 kDa, or between 5 and 1000 kDa, or between 5 and 2000 kDa, or between 10 and 400 kDa, or between 10 and 300 kDa, or between 10 and 200 kDa, or Alternatively, it may be in the range of 10 to 100 kDa, or 10 to 50 kDa, or 10 to 500 kDa, or 10 to 1000 kDa, or 10 to 2000 kDa, or 20 to 400 kDa, or 20 to 300 kDa, or 20 to 200 kDa, or 40 to 300 kDa, or 40 to 500 kDa, or 20 to 100 kDa, or 20 to 50 kDa, or 20 to 500 kDa, or 20 to 1000 kDa, or 20 to 2000 kDa.

[0089] Characterization of impurities and degradation of recombinant spider silk polypeptide powder Different recombinant spider silk polypeptides have different physicochemical properties, such as melting temperature and glass transition temperature, based on the strength and stability of the secondary and tertiary structures formed by the protein. In the form of monomers, silk polypeptides form beta-sheet structures. In the presence of other monomers, silk polypeptides form a three-dimensional crystalline lattice of beta-sheet structures. The beta-sheet structures are separated from and interspersed with amorphous regions in the polypeptide sequence.

[0090] The beta-sheet structure is highly stable at high temperatures, with a beta-sheet melting temperature of approximately 257°C measured by fast scanning calorimetry (see Cebe et al., "Beating the Heat-Fast Scanning Melts Silk Beta Sheet Crystals," Nature Scientific Reports 3:1130 (2013)). Because the beta-sheet structure is believed to remain intact above the glass transition temperature of silk polypeptides, we hypothesize that the structural changes observed at the glass transition temperature of recombinant silk polypeptides are due to increased mobility of the amorphous regions between the beta-sheets.

[0091] Plasticizers increase the mobility of amorphous regions and may prevent the formation of beta-sheets, thereby lowering the glass transition temperature and melting temperature of silk proteins. Suitable plasticizers for this purpose include, but are not limited to, water and polyols such as glycerol, triglycerol, hexaglycerol, and decaglycerol. Other suitable plasticizers include, but are not limited to, dimethyl isosorbite; bisamide of dimethylaminopropylamine and adipic acid; 2,2,2-trifluoroethanol; amide of dimethylaminopropylamine and caprylic / capric acid; DEA acetamide, and any combination thereof. Other suitable plasticizers are discussed in Ullsten et al., Chapter 5: Plasticizers for Protein Based Materials Viscoeleastic and Viscoplastic Materials (2016) (available at https: / / www.intechopen.com / books / viscoelastic-and-viscoplastic-materials / plasticizers-for-protein-based-materials) and Vierra et al., Natural-based plasticizers and polymer films: A review, European Polymer Journal 47(3):254-63 (2011), the entire contents of which are incorporated herein by reference.

[0092] The hydrophilic portion of the silk polypeptide can bind to ambient water present in the air as humidity, so that water is almost always present, and the bound ambient water can plasticize the silk polypeptide. In some embodiments, a suitable plasticizer can be glycerol, present alone or in combination with water or other plasticizers. Other suitable plasticizers are described above.

[0093] Additionally, when recombinant spider silk polypeptides are produced by fermentation and recovered as recombinant spider silk polypeptide powder, impurities that act as plasticizers or otherwise inhibit the formation of tertiary structure may be present in the recombinant spider silk polypeptide powder. For example, residual lipids and sugars may function as plasticizers and affect the glass transition temperature of the protein by preventing the formation of tertiary structure.

[0094] A variety of established methods can be used to assess the purity and relative composition of recombinant spider silk polypeptide powders or compositions. Size exclusion chromatography separates molecules based on relative size and can be used to analyze the relative amounts of full-length polymeric and monomeric forms of recombinant spider silk polypeptide, as well as the amounts of high-, low-, and medium-molecular-weight impurities in recombinant spider silk polypeptide powders. Similarly, rapid high-performance liquid chromatography can be used to measure various compounds present in a solution, such as the monomeric form of recombinant spider silk polypeptide. Ion-exchange liquid chromatography can be used to assess the concentrations of various trace molecules in solutions containing impurities such as lipids and sugars. Chromatography and other methods for quantitation of various molecules, such as mass spectrometry, are well established in the art.

[0095] Depending on the embodiment, the recombinant spider silk polypeptide may have a purity calculated based on the amount of recombinant spider silk polypeptide in monomeric form, using the weight percentages of the other components in the recombinant spider silk polypeptide powder. In various cases, the purity may range from 50% to 90% by weight, depending on the type of recombinant spider silk polypeptide and the techniques used to recover, separate, and work up the recombinant spider silk polypeptide powder.

[0096] Both size exclusion chromatography and reverse-phase high performance liquid chromatography are useful for measuring full-length recombinant spider silk polypeptides, thereby determining whether or not the recombinant spider silk polypeptides have been degraded during the processing step by comparing the amount of full-length spider silk in the polypeptide in the composition before and after processing. In various embodiments of the invention, the amount of full-length recombinant spider silk polypeptide in the composition before and after processing may be minimally degraded. The amount of degradation may range from 0.001% to 10% by weight, or from 0.01% to 6% by weight, for example, 10% by weight, or less than 8% or 6% by weight, or less than 5% by weight, less than 3% by weight, or less than 1% by weight.

[0097] Melt rheology, secondary and tertiary structure Rheology is commonly used in fiber spinning to analyze the physicochemical properties of materials, such as polymers, being spun into fibers. Different rheological properties can affect the ability to spin the material into fibers and the mechanical properties of the spun fibers. Rheology can also be used to indirectly study the secondary and tertiary structures formed by recombinant spider silk polypeptides and / or plasticizers under various pressures, temperatures, and conditions. Depending on the embodiment, different rheological properties can be analyzed using shear and / or extensional rheometers via oscillatory and extensional rheology.

[0098] In some embodiments, capillary rheometry is used to characterize the glass and / or melt transitions of compositions comprising a recombinant spider silk polypeptide powder and a plasticizer. These compositions prior to conversion to a molten or flowable state are referred to herein as "recombinant spider silk compositions." Furthermore, once the recombinant spider silk compositions are in a molten or flowable state, they are referred to herein as "recombinant spider silk molten compositions."

[0099] In some embodiments, the melt and / or glass transition of a recombinant spider silk composition can be characterized using a capillary rheometer to extrude the recombinant spider silk composition at different pressure ranges and the "slope" obtained by increasing the shear rate. Depending on the embodiment and case, this slope starts at about 300 m / s and goes up to 1500 m / s. Depending on the embodiment, the pressure can be adjusted between 1 MPa and 125 MPa, often between 6 MPa and 50 MPa.

[0100] In some embodiments, differential scanning calorimetry is used to determine the glass transition temperature and / or melt transition temperature of the recombinant spider silk polypeptide and / or fibers comprising same, hi certain embodiments, modulated differential scanning calorimetry is used to measure the glass transition temperature and / or melt transition temperature.

[0101] Depending on the embodiment and type of recombinant spider silk polypeptide, the glass transition temperature and / or melting transition temperature may have a range of values, however, measurements of glass transition temperatures and / or melting transition temperatures that are much lower than normally observed for recombinant spider silk polypeptides in solid form may indicate the presence of impurities or other plasticizers.

[0102] Additionally, Fourier transform infrared (FTIR) spectroscopy data can be combined with rheological data to provide a direct characterization of the tertiary structure of both the recombinant silk powder and / or compositions comprising it. FTIR can be used to quantify the secondary structure of silk polypeptides and / or compositions comprising silk polypeptides, as described below in the section entitled "Fourier transform infrared (FTIR) spectroscopy."

[0103] Depending on the embodiment, FTIR may be used to quantify the amount of beta-sheet structure present in recombinant spider silk polypeptide powders and / or compositions containing the same. Additionally, in some embodiments, FTIR may be used to quantify impurities, such as sugars and lipids, present in recombinant spider silk polypeptide powders. However, various chaotropes and solubilizing agents used in different protein pretreatment methods may reduce the amount of tertiary structure in recombinant spider silk polypeptide powders or compositions containing them. Therefore, there may be no correspondence between the amount of beta-sheet structure in recombinant spider silk polypeptide powders before and after they are molded or spun into fibers. Similarly, there may be little correspondence between the glass transition temperatures of the same powders before and after they are molded or spun into fibers.

[0104] In some embodiments, rheological data characterizing recombinant spider silk polypeptides may be combined with FTIR to analyze the secondary and tertiary structure formed in the polypeptide. In certain embodiments, rheological data may be captured in conjunction with FTIR spectroscopy. For an exemplary method of combining rheology and FTIR, see Boulet-Audet et al., Silk protein aggregation kinetics revealed by Rheo-IR, Acta Biomaterialia 10:776-784 (2014), the entire contents of which are incorporated herein by reference.

[0105] Fourier transform infrared (FTIR) spectroscopy can be used to evaluate the tertiary structure of proteins present in polypeptide powders and / or fibers. Specifically, FTIR spectroscopy can be used to determine the amount of beta-sheet present in fibers subjected to various spinning and post-treatment conditions. Therefore, FTIR spectroscopy can be used to determine the relative amount of beta-sheet structure based on various techniques. Alternatively, FTIR spectroscopy can be compared to natural insect silk.

[0106] Depending on the embodiment, FTIR spectra at different wavenumbers can be used to characterize the different tertiary structures present in the fibers. In various embodiments, wavenumbers corresponding to the amide I and amide II bands can be used to characterize various protein structures such as turns, beta sheets, alpha helices, and side chains. Wavenumbers corresponding to these structures are well known in the art.

[0107] In most embodiments, FTIR spectroscopy at wavenumbers corresponding to beta sheets is used to assess the amount of beta sheet structure in polypeptide powders and / or fibers. In certain embodiments, FTIR spectroscopy at wavenumbers corresponding to beta sheets is used to assess the amount of beta sheet structure in polypeptide powders and / or fibers. -1 (CH2 Locking (A) n ), 1695~1690cm -1 (Amide I) 1620-1625 cm -1 (Amide I), 1440–1445 cm -1 (asymmetric CH3 bending), and / or 1508 cm -1 FTIR spectra of (amide II) are used to determine the amount of beta-sheet present. Depending on the embodiment, different wavenumbers and ranges can be measured to determine the amount of beta-sheet present. In some embodiments, the FTIR spectrum of amide II is measured between 982 and 949 cm to eliminate interference from the corresponding peak. -1 FTIR spectra at these wavenumbers are used. Exemplary methods for obtaining spectra at these wavenumbers are detailed in Boudet-Audet et al., Identification and classification of silks using infrared spectroscopy, Journal of Experimental Biology, 218:3138-3149 (2015), the entire contents of which are incorporated herein by reference.

[0108] Similarly, various methods for characterizing impurities in recombinant silk powders can be combined with rheological and / or FTIR data to analyze the relationship between the presence of impurities and the formation of secondary and / or tertiary structures.

[0109] Recombinant spider silk molten composition It is an object of the present invention to create a variety of recombinant spider silk compositions that can be converted into a molten or flowable state (i.e., converted into a recombinant spider silk molten composition) according to the methods described herein. In various embodiments, the concentrations of recombinant spider silk polypeptide powder and plasticizer in the composition can vary based on the characteristics of the recombinant spider silk polypeptide powder (e.g., the purity of the recombinant spider silk polypeptide powder), the type of plasticizer used, and the desired properties of the fiber. In some embodiments, the concentrations can be adjusted based on rheological data, such as data from a capillary rheometer.

[0110] In some embodiments, a melt flow indexer is used to determine whether a recombinant spider silk melt composition can be drawn into fibers. Specifically, a melt flow indexer may be used to measure the "melt strength" of a recombinant spider silk melt composition, or the ability to draw out a recombinant spider silk melt composition when extruded. In various embodiments, the concentrations of recombinant spider silk polypeptide and plasticizer may be varied based on the desired melt strength.

[0111] In some embodiments, various agents may be added to the recombinant spider silk composition to modify the rheological properties of the recombinant spider silk composition, such as extensional viscosity, shear viscosity, and linear viscoelasticity. Suitable agents used to modify the extensional viscosity include polyethylene glycol (PEG), Tween (polysorbate), sodium dodecyl sulfate, polyethylene, or any combination thereof. Other suitable agents are known in the art.

[0112] In some embodiments, a second polymer may be added to create a polymer blend or bicomponent fiber with the recombinant spider silk composition. In these cases, it may be useful to include a second polymer with a melting temperature suitable for melting alongside the recombinant spider silk composition itself without degrading the amorphous regions of the recombinant spider silk polypeptide. In various embodiments, polymers suitable for blending with recombinant spider silk polypeptides have melting temperatures (Tm) below 200°C, 180°C, 160°C, 140°C, 120°C, or 100°C. In most cases, the melting temperature of recombinant spider silk polypeptides is above 20°C, 25°C, or 50°C. Exemplary polymers and their melting temperatures are shown in the table below, but are not limited to these.

[0113] (Table 1C) Polymers TIFF2025118794000007.tif78128

[0114] Depending on the embodiment, suitable concentrations of recombinant spider silk polypeptide powder in the recombinant spider silk composition range from: 1-90% by weight, 3-80% by weight, 5-70% by weight, 10-60% by weight, 15-50% by weight, 18-45% by weight, or 20-41% by weight.

[0115] In cases where glycerin is used as a plasticizer, suitable weight concentrations of glycerin in the recombinant spider silk composition range from: 1-60 wt%, 10-60 wt%, 10-50 wt%, 10-40 wt%, 15-40 wt%, 10-30 wt%, or 15-30 wt%.

[0116] In cases where water is used as a plasticizer, suitable weight concentrations of water in the recombinant spider silk composition range from: 5-80 wt%, 15-70 wt%, 20-60 wt%, 25-50 wt%, 19-43 wt%, or 19-27 wt%. When water is used in combination with another plasticizer, water may be present in the ranges of 5-50 wt%, 15-43 wt%, or 19-27 wt%.

[0117] In some embodiments, water may evaporate during the extrusion and / or cooling process, depending on the process and / or mold size used. In some embodiments, water loss after molding may range from 1 to 50%, 3 to 40%, 5 to 30%, 7 to 20%, 8 to 18%, or 10 to 15% by weight, based on the total amount of water. In most cases, the loss is less than 15%, and in some cases, less than 10%, e.g., 1 to 10% by weight. Evaporation may be intentional or may be the result of the process applied. The extent of evaporation can be readily controlled, for example, by selection of the operating temperature, flow rate, and pressure applied, as understood in the art.

[0118] In some embodiments, suitable plasticizers include polyols (e.g., glycerol), water, lactic acid, methyl hydroperoxide, ascorbic acid, 1,4-dihydroxybenzene (1,4 benzenediol) benzene-1,4-diol, phosphoric acid, ethylene glycol, propylene glycol, triethanolamine, acid acetate, propane-1,3-diol, or any combination thereof.

[0119] In various embodiments, the amount of plasticizer can vary according to the purity and relative composition of the recombinant spider silk polypeptide powder, for example, a higher purity powder may contain fewer impurities, such as low molecular weight compounds that can act as plasticizers, and therefore require a larger weight percent of plasticizer to be added.

[0120] In certain embodiments, various ratios (by weight) of plasticizer (e.g., a combination of glycerol and water) to recombinant spider silk polypeptide powder may range from 0.5 or 0.75 to 350% by weight plasticizer:recombinant spider silk polypeptide powder, 1 or 5 to 300% by weight plasticizer:recombinant spider silk polypeptide powder, 10 to 300% by weight plasticizer:recombinant spider silk polypeptide powder, 30 to 250% by weight plasticizer:recombinant spider silk polypeptide powder, 50 to 220% by weight plasticizer:recombinant spider silk protein, 70 to 200% by weight plasticizer:recombinant spider silk polypeptide powder, or 90 to 180% by weight plasticizer:recombinant spider silk polypeptide powder. As used herein, 0.5 to 350% by weight plasticizer:recombinant spider silk polypeptide powder corresponds to a ratio of 0.5:1 to 350:1.

[0121] Without wishing to be bound by theory, in various embodiments of the present invention, inducing a recombinant spider silk composition to a flowable state (e.g., inducing a recombinant spider silk melt composition) may be used as a pretreatment step in any formulation in situations where it is beneficial to include the recombinant spider silk polypeptide in monomeric form. More specifically, inducing a recombinant spider silk melt composition may be used in applications where it is desirable to prevent the monomeric recombinant spider silk polypeptide from aggregating into a crystalline polymeric form, or to control the recombinant spider silk polypeptide's transition to a crystalline polymeric form at a later stage in processing. In certain embodiments, the recombinant spider silk melt composition may be used to prevent the recombinant spider silk polypeptide from aggregating prior to mixing the recombinant spider silk polypeptide with a second polymer. In another specific embodiment, the recombinant spider silk melt composition may be used to create a cosmetic or skin care product base in which the recombinant spider silk polypeptide is present in the base in monomeric form. In this embodiment, the inclusion of recombinant spider silk polypeptides in monomeric form in the matrix allows for controlled aggregation of the monomers into a crystalline polymeric form upon contact with skin or through various other chemical reactions.

[0122] Inducing a molten or flowable state According to some embodiments of the present invention, the recombinant spider silk composition is converted into a molten or flowable state by the application of shear and / or pressure, typically both. Suitable means for producing a combination of shear and pressure include, but are not limited to, single screw extruders, twin screw extruders, melt flow extruders, and capillary rheometers.

[0123] In some embodiments, a twin-screw extruder is used to provide the pressure and shear necessary to convert the recombinant spider silk composition into a molten or flowable composition. In some embodiments, the twin-screw extruder is configured to provide a shear force in the range of 1.5 Newton meters (Nm) to 13 Newton meters, 2 Newton meters to 10 Newton meters, 2 Newton meters to 8 Newton meters, or 2 Newton meters to 6 Newton meters. In some embodiments, the shear force provided by the twin-screw extruder is determined in part by the revolutions per minute (RPM) of the twin-screw extruder. In various embodiments and configurations, the revolutions per minute (RPM) of the twin-screw extruder can range from 10 RPM to 300 RPM. In various embodiments, the twin-screw extruder is configured to provide a pressure, in combination with the shear force, in the range of 1 MPa to 300 MPa.

[0124] In any embodiment, the twin-screw extruder is configured to heat the recombinant spider silk melt composition before and / or after it is converted into the recombinant spider silk composition. In some embodiments, heat is applied to the barrel of the twin-screw extruder (i.e., the cylinder where the twin screws mix the composition). In other embodiments, heat is applied to the portion of the twin-screw extruder proximal to the spinneret (i.e., the orifice through which the extruded recombinant spider silk melt composition passes). Alternatively, without heating, the molten / flowable state is induced entirely by heat resulting from shear forces applied to the recombinant spider silk composition in the twin-screw extruder. For example, in some embodiments, the amount of heat applied to achieve the molten / flowable state is equal to ambient room temperature (e.g., above about 20°C).

[0125] In various embodiments, the recombinant spider silk melt composition is heated to a minimum temperature to minimize or completely prevent degradation of the recombinant spider silk polypeptides. In certain embodiments, the recombinant spider silk melt is heated to a temperature below 120° C., below 100° C., below 80° C., below 60° C., below 40° C., or below 20° C. In most cases, the melt will be at a temperature between 10° C. and 120° C., 10° C. and 100° C., 15° C. and 80° C., 15° C. and 60° C., 18° C. and 40° C., or 20±2° C. during processing.

[0126] In other embodiments, other instruments may be used to provide the necessary pressure and shear to convert the recombinant spider silk composition into a molten or flowable state. As noted above, a capillary rheometer may be used to provide the necessary shear and pressure to convert the recombinant spider silk composition into a flowable or molten state.

[0127] In some embodiments, the recombinant spider silk composition is optionally heated after being brought into a molten or flowable state and / or before the molten or flowable recombinant spider silk melt composition is extruded. Because heating is likely necessary due to the high glass transition temperature of the recombinant spider silk composition, the device used to provide the shear and pressure to convert the recombinant spider silk composition into a molten or flowable state may be directly or indirectly connected to the heated extrusion device. In certain embodiments, a twin-screw cylinder mixer is connected (directly or indirectly) to the heated extrusion device. Depending on the embodiment and configuration of the heated extrusion device, the heated extrusion device may be maintained at a temperature in the range of 20-120°C, 80-110°C, 85-100°C, 85-95°C, and / or 90-95°C.

[0128] The extruded recombinant spider silk molten composition is referred to herein as a recombinant spider silk extrudate. Depending on the intended use of the recombinant spider silk extrudate, the diameter of the spinneret through which the extrudate is extruded may be adjusted. For example, in embodiments in which the recombinant spider silk extrudate is extruded into a mold to form a shaped body, the spinneret may have a diameter greater than 200 mm, greater than 150 mm, greater than 100 mm, or greater than 50 mm, e.g., in the range of 100 mm to 500 mm, 150 mm to 400 mm, or 200 mm to 300 mm. As described below, in some embodiments, the recombinant spider silk extrudate may be processed into pellets, which may be reprocessed by subjecting the pellets to sufficient shear and pressure to convert the spider silk extrudate into a recombinant spider silk molten composition. In embodiments in which the recombinant spider silk extrudate is processed into pellets, the spinneret may have a diameter greater than 2 mm, greater than 1.5 mm, or greater than 1 mm, for example, in the range of 1 mm to 5 mm, 1.5 mm to 4 mm, or 2 mm to 3 mm.

[0129] In embodiments in which recombinant spider silk extrudates are converted into fibers, the spinneret may have an orifice less than 500 μm (e.g., in the range of 10 μm to 500 μm). Depending on the initial denier desired for the extruded fiber, the recombinant spider silk protein melt composition may be extruded through spinnerets with orifices of various sizes. In certain embodiments, the orifice may range from 25 μm to 500 μm, 50 μm to 250 μm, or 75 μm to 125 μm. In some embodiments, the ideal orifice size is based on the final draw ratio of the fiber. For example, a higher initial denier of the extruded fiber may accommodate a higher draw ratio.

[0130] In most embodiments of the present invention, both the recombinant spider silk melt composition and the recombinant spider silk extrudate are substantially homogeneous when examined by optical microscopy, meaning that the material is free of inclusions or precipitates. In some embodiments, optical microscopy can be used to measure birefringence, which can be used as a proxy for aligning the recombinant spider silk into a three-dimensional lattice. Birefringence is an optical property of a material, with a refractive index that depends on the polarization and propagation of light. Specifically, a high degree of axial order, as measured by birefringence, can be associated with high tensile strength. In some embodiments, the recombinant spider silk melt extrudate has minimal birefringence.

[0131] According to the present invention, a homogeneous, flowable state can be induced simply by the application of shear and pressure, optionally with the addition of heat. It has been found that a combination of shear and pressure, without the addition of heat or with the optional addition of heat, provides a recombinant spider silk melt composition and a composition that does not degrade during the processing of recombinant spider silk polypeptides in recombinant spider silk extrudates. This is desirable and beneficial, as maintaining full-length recombinant spider silk polypeptides in the extruded composition results in optimal material properties, such as crystallinity, resulting in a higher quality product. In embodiments of the present invention, recombinant spider silk melt extrudates achieved using shear and pressure (and optionally heat) exhibit minimal or negligible degradation.

[0132] The amount of degradation of recombinant spider silk polypeptides can be measured using various techniques. As described above, the amount of degradation of recombinant spider silk polypeptides can be measured using size exclusion chromatography to determine the amount of full-length recombinant spider silk polypeptide present. In various embodiments, after forming the composition into a shaped body, less than 6.0% by weight of the composition is degraded. In other embodiments, after forming the composition, less than 4.0%, less than 3.0%, less than 2.0%, or less than 1.0% by weight of the composition is degraded (such that the amount of degradation is in the range of 0.001% to 10%, 8%, 6%, 4%, 3%, 2%, or 1%, or 0.01% to 6%, 4%, 3%, 2%, or 1% by weight). In other embodiments, the recombinant spider silk protein in the extrudate and / or molten composition is not substantially degraded.

[0133] Stretching the fiber If the extrudate is used to form fibers, the precursor fibers can be stretched to improve fiber orientation and promote a three-dimensional crystalline structure. The application of a stretching force causes the molecules to align with the fiber axis. When polymer molecules, such as polypeptides, are forced through the holes in the spinneret, some of them become aligned. Fibers can be drawn manually or mechanically. In most cases, manual drawing results in well-aligned fibers with low birefringence and minimal fiber diameter reduction.

[0134] In the present invention, passing the precursor fibers over a uniform hot surface while drawing the fibers can optimize alignment. As used herein, the term "hot surface" refers to a surface that provides both substantially uniform heat and a substantially uniform surface. Using a hot surface as a heat source eliminates the variability observed when using ambient heat sources, resulting in more uniform results and improved scalability for commercial mass fiber production processes. In some embodiments, the hot surface is a metal bar or other metallic surface. In other embodiments, the hot surface can be made of ceramic or other materials. Depending on the embodiment, the hot surface can be curved or otherwise configured to facilitate movement of the fiber over the hot surface.

[0135] In embodiments of the invention, the undrawn extruded fiber may be moved over a hot surface simultaneously with drawing. Depending on the embodiment, the temperature of the hot surface may range from 160-210°C, 180-210°C, 190-210°C, 195-210°C, 195-205°C, or 200-205°C.

[0136] Depending on the embodiment, the undrawn extruded fiber can be subjected to different draw ratios while being drawn on the hot surface. Depending on the embodiment, the draw ratio can range from 2 to 7. In some embodiments, the maximum stable draw ratio can vary depending on the temperature of the hot surface.

[0137] In some embodiments, the temperature of the hot surface is calculated as a function of the glass transition temperature of the undrawn extruded fibers. For example, the temperature of the hot surface can be calculated to be 5°C, 10°C, 15°C, 20°C, or 25°C higher than the glass transition temperature of the recombinant silk protein powder and / or the undrawn extruded fibers. In other words, the temperature of the hot surface is 0, or in the range of 0.1°C to 25°C higher than the glass transition temperature of the recombinant silk protein powder, and is often 0 to 10°C, 15°C, or 20°C higher.

[0138] Depending on the embodiment and the speed at which the fibers pass over the uniform hot surface (referred to herein as the "reel speed"), the hot surface can vary in length (i.e., the size in centimeters of the hot surface onto which the fibers converge), thereby varying the period of time during which the undrawn extruded fibers are subjected to heat and deformation. In most embodiments, the hot bar width is 1 cm or greater. However, in various embodiments, the hot surface width can range from 1 to 50 cm, 1 to 2 cm, 1 to 3 cm, 1 to 5 cm, 5 to 38 cm, or 38 to 50 cm. Depending on the embodiment, the reel speed can range from 1 to 60 meters per minute.

[0139] Depending on the reel speed and the length of the hot surface, the total residence time on the hot surface can vary, and in most embodiments, the total residence time can range from 0.2 seconds to 3 seconds.

[0140] Additionally, the undrawn fiber may be subjected to various forces that provide different draw ratios. In most embodiments, a godet provides the tensile force. In some embodiments, the godet is positioned so that the fiber passing over the hot surface forms an angle with the hot surface. For example, in cases where the hot surface is curved, the godet may be positioned so that the fiber passing over the hot surface forms an angle of 10 to 40 degrees with the hot surface.

[0141] In various embodiments, the deformation rate of the undrawn fiber (i.e., the amount of deformation the fiber undergoes due to heat and drawing) can be varied based on the factors discussed above. The deformation rate can be calculated based on the rate at which the undrawn fiber is fed to the hot surface and the rate at which the fiber is withdrawn from the hot surface. For example, the fiber can be fed to the hot surface at a rate of 1 meter / min and withdrawn from the hot surface at a rate of 5 meters / min. In certain embodiments, the deformation rate is calculated using the following formula, where v1 represents the rate at which the fiber is fed to the hot surface, v2 represents the rate at which the fiber is withdrawn from the hot surface, and L0 is the length involved in the deformation: Formula 1: TIFF2025118794000008.tif12128

[0142] Depending on the embodiment, drawing onto the hot surface can be performed in one step or multiple (i.e., two, three, or four) steps. Parameters such as strain rate, deformation rate, reel speed, hot surface temperature, and hot surface length can be changed or different for each step. Performing drawing in multiple steps can affect the overall strain rate of the fiber and promote the formation of crystalline beta-sheet structure, often improving fiber strength.

[0143] Post-processing the fibers Various post-treatment methods can be employed to improve the molecular alignment of the fibers. Depending on the amount of plasticizer and / or recombinant spider silk present in the fibers, the fibers can be heat-treated (e.g., annealed using steam or heat). In other cases, the fibers can be treated with various solvents to anneal the fibers and improve the crystallinity of proteins (e.g., 18B protein) within the fibers. In some cases, the fibers can be annealed using alcohols such as methanol. In certain embodiments, the fibers can be annealed using alcohol vapor.

[0144] In some cases, treating the fibers or fabrics with one or more conditioners, lubricants, surfactants, emulsifiers, anti-agglomerating agents, or annealing agents before treating the fibers with water changes the hand or drape of the fabric after water treatment. In certain embodiments, cyclopentasiloxane or PDMS is used as a conditioner. In certain embodiments, annealing the fibers or fabrics formed from the fibers with alcohol improves the hand and drape of the water-treated fibers or fabrics.

[0145] Remelting and re-extrusion of the extrudate In some embodiments of the present invention, the process for preparing recombinant spider silk extrudates may further comprise reprocessing a shaped body comprising the recombinant spider silk extrudates (e.g., pellets, fibers, or other shaped articles formed from the recombinant spider silk extrudates). In these embodiments, the recombinant spider silk extrudates are subjected to shear forces and pressure sufficient to convert the recombinant spider silk extrudates into a molten or flowable state.

[0146] Without wishing to be bound by theory, it has been suggested that application of shear and pressure to recombinant spider silk polypeptides in the presence of a plasticizer such as glycerol converts the recombinant spider silk polypeptide into an "open-form recombinant spider silk polypeptide," which unfolds the recombinant spider silk polypeptide and interacts with glycerol. Due to the interaction with glycerol, the "open-form recombinant spider silk polypeptide" exhibits reduced intermolecular and intramolecular beta-sheet interactions. Specifically, the open-form recombinant spider silk polypeptide forms intermolecular interactions that prevent the formation of an irreversible three-dimensional lattice.

[0147] Because there is minimal, if any, degradation of the recombinant spider silk polypeptides during the melting and extrusion process, the recombinant spider silk extrudates can be converted back into a recombinant spider silk molten composition and re-extruded multiple times. In this sense, the composition is "thermoplastic" because it can be heated, cooled, and hardened multiple times without significant degradation of the proteins or composition. In various embodiments, the recombinant spider silk extrudates can be re-melted and re-extruded at least 20 times, at least 10 times, or at least 5 times. In these embodiments, degradation observed over multiple re-melting and re-extrusion steps is as low as 10%. The option to re-extrude without degradation allows for the production of substantially homogeneous compositions and also allows for the repurposing or redesign of products formed from the compositions. For example, bodies of insufficient quality can be re-extruded and re-molded. Recycling of used products is also possible. [Example]

[0148] Example 1: Purity of recombinant 18B polypeptide powder Various lots of recombinant spider silk—18B polypeptide sequence (SEQ ID NO: 1) containing a FLAG tag—were produced in large-scale fermentations, harvested as a powder ("18B powder"), and dried. Reverse-phase high-performance liquid chromatography ("RP-HPLC") was used to measure the weight of 18B polypeptide monomer in the powder. Samples were dissolved using 5 M guanidine thiocyanate (GdSCN) reagent and injected onto an Agilent Poroshell 300SB C3 2.1 x 75 mm 5 μm column to separate components based on hydrophobicity. The detection modality was UV absorbance of the peptide bond at 215 nm (360 nm reference). Sample concentrations of 18B-FLAG monomer were determined by comparison with an 18B-FLAG powder standard, for which 18B-FLAG monomer concentrations had previously been determined using size-exclusion chromatography (SEC-HPLC).

[0149] The sample powder contained 57.964% by mass of 18B monomer.

[0150] Example 2: Production of recombinant silk powder extrudates The recombinant silk powder from Example 1 was mixed using a household spice grinder. Water and glycerol were added to the recombinant silk powder ("18B powder") in the ratios shown in Table 2 below to produce recombinant spider silk compositions with different ratios of protein powder to plasticizer.

[0151] Batches of 10-100 grams of recombinant spider silk composition (i.e., "formulation"), as described in Table 2 below, were mixed using an Xceptional Instruments Twin Screw Extruder (TSE) (item number TT-ZE5-MSMS-3HT), which was used for all TSE experiments. The stainless steel (S316) extruder barrel had three heated zones, each approximately 5 cm long. The screws used were standard twin co-rotating stainless steel (S316) screws, 180 mm long, 9 mm in diameter, and (20:1 L / D ratio). The screw pitch was 9 mm.

[0152] For the P25W05G70, P49W21G30, and P65W20G15 formulations described below, the recombinant spider silk composition was first extruded into pellets, which were then re-extruded and reprocessed in subsequent experiments. To create pellets, the recombinant spider silk composition, containing an 18B / water / glycerol mixture, was introduced into a TSE using a metal funnel and forced into the twin screw using a loading device while the TSE was running at 300 RPM and at a temperature of approximately 90-95°C through all three barrel sections, including the beginning, middle, and end barrel sections. This material (i.e., the recombinant spider silk molten composition) was extruded in the molten state through a 0.5 mm die with an orifice at a 180° angle to the screw axis to form the recombinant spider silk extrudate.

[0153] The 0.5 mm recombinant spider silk extrudates emerged from the mold as continuous elastomeric "noodles" approximately 10 meters in length. 5-10 g amounts of the corresponding extrudate compositions were sequentially placed in a kitchen spice grinder and subjected to a total of six 5-second pulses (total 30 seconds) to produce pellets. The pellets were inspected to confirm that their length was 5 mm or less, with an average pellet length of approximately 2.5 mm.

[0154] For the following P71W19G10 formulation, the 18B / water / glycerol recombinant spider silk mixture was pre-mixed under the conditions described in Example 2 and extruded directly (i.e., without first being extruded as a pellet) to form recombinant spider silk extrudates.

[0155] Table 2. Composition of recombinant spider silk preparations by weight TIFF2025118794000009.tif70128

[0156] Example 3: Production of recombinant silk extrudates with minimal degradation To evaluate degradation under several different conditions, the recombinant spider silk preparation described in Example 2 was subjected to various temperatures during extrusion, as well as various pressures and shear forces. Specifically, the revolutions per minute of the twin-screw extruder pellet was varied to apply variable amounts of torque and shear. The various temperature and RPM combinations used to convert the recombinant spider silk preparation to a molten state and extrude various samples are listed below.

[0157] Pellets obtained by extruding the P49W21G30 and P65W20G15 formulations listed in Table 1 were extruded at various RPMs and temperatures using an Xceptional Instruments TSE. Other parameters for operation of the Xceptional Instruments TSE were the same as those described for Example 2.

[0158] The P71W19G10 formulation was also extruded at various RPMs and temperatures using an Xceptional Instruments TSE, as described in Example 2. Other parameters for operation of the Xceptional Instruments TSE were the same as those described for Example 2.

[0159] Size-exclusion chromatography (SEC) was used to collect data characterizing the relative amounts of high-, low-, and medium-molecular-weight impurities, monomeric 18B, and aggregated 18B. 18B powder was dissolved in 5 M guanidine thiocyanate and injected onto a Yarra SEC-3000 SEC-HPLC column to separate components based on molecular weight. Refractive index was used as the detection modality. 18B aggregates, 18B monomer, low-molecular-weight (1-8 kDa) impurities, medium-molecular-weight (8-50 kDa) impurities, and high-molecular-weight (110-150 kDa) impurities were quantified. Relative compositions were reported as mass and area percent. BSA was used as a common protein standard, assuming that >90% of all proteins exhibit dn / dc values (refractive index response factor) within approximately 7% of each other. Poly(ethylene oxide) was used as the retention time standard, and a BSA calibrator was used as a check standard to ensure consistent performance of the method.

[0160] Tables 3-5 below show various SEC analyses of extrudates produced at various RPMs and temperatures. The fifth column lists either the difference in 18B monomer (area %) reported between the starting pellets and extrudates (P49W21G30 and P65W20G15) or the difference in 18B monomer (area %) reported between the starting powder and extrudate (P71W19G10). Figures 1-3 are detailed below and include graphs corresponding to Tables 3-5, respectively. Minimal decomposition was observed at all temperatures and RPMs tested, demonstrating flexibility in processing conditions and general robustness to processing using extrusion methods.

[0161] Table 3. SEC analysis of P49W21G30 TIFF2025118794000010.tif235170

[0162] Table 4. SEC analysis of P65W20G15 TIFF2025118794000011.tif209170

[0163] Table 5. SEC analysis of P71W19G10 TIFF2025118794000012.tif113170

[0164] Figure 1 shows SEC data for the P49W21G30 samples listed in Table 3 above under extrusion conditions of 20, 40, 60, 80, 95, or 120°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, or 300 RPM. The 18B monomer (black bars), medium molecular weight impurities (gray bars), and low molecular weight impurities (cross-hatched bars) are shown as area %.

[0165] Figure 2 shows SEC data for the P65W20G15 samples listed in Table 4 above under extrusion conditions of 20, 40, 60, 95, or 140°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, or 300 RPM. The 18B monomer (black bars), medium molecular weight impurities (gray bars), and low molecular weight impurities (cross-hatched bars) are shown as area %.

[0166] Figure 3 shows SEC data for the P71W19G10 samples listed in Table 5 above under extrusion conditions of 90 or 120°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, or 300 RPM. The 18B monomer (black bars), medium molecular weight impurities (gray bars), and low molecular weight impurities (cross-hatched bars) are shown as area %.

[0167] Example 4: Thermogravimetric Analysis - P49W21G30 To analyze the water loss during extrusion, the moisture content of the recombinant spider silk composition before extrusion and the recombinant spider silk extrudate after extrusion was analyzed by TGA (thermogravimetric analysis) using a TA brand TGAQ 500 instrument. For the P49W21G30 and P65W20G15 samples, the moisture content of the pellets used in the extrusion experiments described in Example 3 was used as the reference sample for measuring water loss. For the P71W19G10 sample, the moisture content of the recombinant spider silk composition used in the extrusion experiments described in Example 3 was used as the reference sample for measuring water loss.

[0168] For each sample, 10 mg, + / - 1 mg, of powder or pellets containing the above formulation was analyzed. To measure moisture content, samples were run in air rather than nitrogen. The autosampler installed was used to sequentially introduce samples into the TGA oven. Using the TA brand software suite, the temperature was programmed to increase from room temperature at a rate of 20°C / min until it reached 110°C. The samples were then held at this temperature for 45 minutes. The samples were then removed from the oven, and the oven was purged with air for 15 minutes before the next run.

[0169] Tables 6-8 below show various measurements for the reference sample (i.e., starting pellets or powder) and the extruded samples. Figures 4-6 depict graphs of the data contained in Tables 6-8, respectively. From this data, it can be seen that water loss during extrusion is low and well within the tolerances for the extrusion process. Typically, water loss ranges from 2-18%.

[0170] Table 6: Water loss of P49W21G30 TIFF2025118794000013.tif196151

[0171] Table 7: Water loss of P65W20G15 TIFF2025118794000014.tif136150

[0172] Table 8: Water loss of P71W19G10 TIFF2025118794000015.tif77150

[0173] Figure 4 shows TGA data for the samples listed in Table 6 above, produced under extrusion conditions of 20, 40, 95, and 120°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, and 300 RPM. Figure 4 also shows TGA data for a reference sample of the starting pellets used to produce these samples. This data shows the percent moisture of the samples for all treatments, with moisture loss ranging from approximately 1 to 13% when compared to the starting pellets.

[0174] Figure 5 shows TGA data for the samples listed in Table 7 above, produced under extrusion conditions of 20, 40, 60, and 140°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, and 300 RPM. Figure 5 also shows TGA data for a reference sample of the starting pellets used to produce these samples. This data shows the percent moisture content of the samples for all treatments, with moisture loss ranging from approximately 1 to 8% when compared to the starting pellets.

[0175] Figure 6 shows TGA data for the samples listed in Table 8 above, produced under extrusion conditions of 90 and 120°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, and 300 RPM. Figure 5 also shows TGA data for a reference sample of the starting powder used to produce these samples. This data shows the percent moisture content of the samples across all runs, with moisture loss ranging from approximately 1.5 to 4% when compared to the starting powder.

[0176] Example 5: Beta-sheet content analysis using Fourier transform infrared spectroscopy To assess the formation of secondary and tertiary structures in the extrudates, the beta-sheet content was measured by Fourier transform infrared spectroscopy (FTIR). FTIR was performed on the extrudates using a Bruker Alpha spectrometer equipped with a diamond attenuated total reflection accessory in front of a wire grid polarizer, selecting primarily S (vertical) polarized light. Recombinant polypeptide powder and precursor fibers were used as controls. To quantify molecular alignment, three spectra (0 and 90° to the polarizing field) for each orientation were obtained from 4000 to 600 cm. -1 4cm -1 Thirty-two scans were collected at a resolution of 100 s.

[0177] 982~949cm -1 The average absorbance values of the peaks corresponding to the bands were calculated based on the following procedure. -1 Next, the average of the 1350–1315 cm bands corresponding to the isotropic (non-oriented) side chain vibration band was subtracted. -1 The spectra were normalized by dividing the average of the spectra by the mean of the spectra. The metric of beta-sheet content was determined by the peak at 982–949 cm. -1 The absorbance was taken as the average of the integrated absorbance values.

[0178] The beta-sheet content of the recombinant spider silk extrudates (i.e., "sample beta-sheet") was compared to i) the beta-sheet content of the starting recombinant spider silk polypeptide powder used to generate the recombinant spider silk composition (i.e., "reference pre-hydrated powder"), and ii) the beta-sheet content of the starting pellets (P49W21G30 and P65W20G15) (i.e., "reference pellets"). Tables 9-11 below list the measurements for the reference samples and extrudates generated under the conditions shown in the tables. Figures 7-9 provide graphs of the data presented in Tables 9-11. As shown therein, there was no significant change in the beta-sheet content of the material from the starting recombinant silk polypeptide powder to the recombinant spider silk extrudate, indicating that this method allows for the plasticization and movement of amorphous protein domains without disrupting the beta-sheets, as occurs when solvent treatment is used.

[0179] Table 9. Beta-sheet formation in P49W21G30 TIFF2025118794000016.tif204170

[0180] Table 10. Beta-sheet formation in P65W20G15 TIFF2025118794000017.tif176170

[0181] Table 11. Beta-sheet formation in P71W19G10 TIFF2025118794000018.tif92142

[0182] Figure 7 shows FTIR data for the samples listed in Table 9 above produced under extrusion conditions of 20, 40, 60, 80, 95, or 120°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, or 300 RPM. Data was extracted from the 949-982 band, and no clear trends were observed compared to the starting pellets.

[0183] Figure 8 shows FTIR data for samples listed in Table 10 above produced under extrusion conditions of 20, 40, 60, 95, or 140°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, or 300 RPM. Data was extracted from the 949-982 band, and no clear trends were observed compared to the starting pellets.

[0184] Figure 9 shows FTIR data for the samples listed in Table 11 above, produced under extrusion conditions of 90 or 120°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, or 300 RPM. Data was extracted from the 949-982 band to avoid effects due to the presence of water, and no clear trends were observed compared to the starting pellets.

[0185] Example 6: Polarized Light Microscopy Polarized light microscopy (PL) was used to examine the smoothness and uniformity of various extrudates. Optical and polarized light (PL) images were obtained using a Leica DM750P polarized light microscope equipped with a 4X PL objective. This microscope was connected to the complementary PC-based image analysis Leica Application Suite, LAS V4.9. Approximately 20–30 mm long TSE extrudates were carefully positioned along the long axis of a standard microscope slide and aligned horizontally (east–west; i.e., 0°) with the aperture of the microscope. The edge of the sample was first focused, followed by the overall focus of the sample. Samples were first observed under white light, controlled by the illumination control knob, and images were captured with the appropriate scale bar included. In all cases, the auto-brightness function in the LAS V4.9 software was switched off.

[0186] The Analyzer / Bertrand Lens module was then fitted by flipping its lower rocker to the right ("A" position / Analyzer in) while ensuring that its upper rocker was flipped to the left ("O" position / Bertrand Lens out). This setup allows for analysis in "cross-polarization mode," with the polarizer and analyzer optically aligned such that the allowable vibration directions of light passing through them are 90° apart.

[0187] To control for background variations in light intensity, all samples were first displayed and the illumination control knob was lowered until the background brightness was completely black. Each eyepiece was then covered with an eyepiece light shielding accessory to prevent ambient light from passing through to the image capture sequence. Images were captured at 0° and 45° orientations using the LAS V4.9 software package. The microscope's circular rotating stage was used to rotate the glass side to a 45° angle, allowing 45° images to be obtained.

[0188] Figures 10 and 11 are images of exemplary samples captured using polarized light microscopy. They demonstrate that smooth fibers with minimal melt fracture are obtained using the claimed process. Thus, the conditions are clearly favorable for melt flow and extrusion. Additionally, qualitative birefringence, as well as axial alignment, were observed under a number of conditions.

[0189] Figure 10 shows photographs taken for samples P49W21G30-1, P49W21G30-2, P49W21G30-3, and P49W21G30-4, all of which were produced at various RPMs at 20°C. Under these conditions, the extrudates were smooth with minimal melt fracture. Polarized light microscopy showed preferential axial alignment depending on the condition (examine at 45° for differences), with the best axial alignment being obtained at 100 RPM.

[0190] Figure 11 shows photographs obtained for samples P49W21G30-17, P49W21G30-18, P49W21G30-19, and P49W21G30-20, all produced at various RPMs at 95°C. The extrudates exhibited moderate melt fracture / surface imperfections. Polarized light microscopy showed good axial alignment between 10 and 100 RPM. The 100-300 RPM samples exhibited similar characteristics to each other when examined at 0° and 45°.

[0191] Example 7: Metabolite analysis of glycerol content To determine glycerol loss in recombinant spider silk compositions during extrusion, glycerol content was analyzed using a Benson Polymeric 150x7.8mm H+ 7110-0 HPLC column equipped with a Phenomenex Security Guard Carbo H+ Guard Column using a 0.004M sulfuric acid mobile phase. A glycerol calibrant was run to allow for quantification. To measure the amount of glycerol in 18B-based samples, the glycerol content of the compositions was measured before extrusion (i.e., as pellets or powder) and after extrusion. For each sample, 25mg of powder or pellet was dissolved in 1ml of 0.004M sulfuric acid and sonicated for 1 hour. The samples were then vortexed and placed in HPLC vials for subsequent analysis under each condition / treatment.

[0192] Tables 12-14 below show various measurements of extrudates produced under the conditions in the tables below. Figures 12-14 show graphs of the same samples. From these, it can be seen that the glycerol content of the composition is stable over the range of conditions tested, as evidenced by minimal loss in the tests.

[0193] Table 12. Glycerol loss in extrudates - P49W21G30 TIFF2025118794000019.tif218160

[0194] Table 13. Glycerol loss in extrudates - P65W20G15 TIFF2025118794000020.tif188160

[0195] Table 14 - Glycerol loss in extrudate - P71W19G10 TIFF2025118794000021.tif99160*Results within the error range of the test equipment.

[0196] Figure 12 shows metabolite data for samples listed in Table 12 above produced under extrusion conditions of 20, 40, 60, 80, 95, and 120°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, and 300 RPM. Glycerol loss was negligible in all treatments.

[0197] Figure 13 shows metabolite data for the samples listed in Table 13 above, produced under extrusion conditions of 20, 40, 60, 95, and 140°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, and 300 RPM. Glycerol loss was negligible in all treatments.

[0198] Figure 14 shows metabolite data for the samples listed in Table 14 above, produced under extrusion conditions of 90 and 120°C, with extrudates obtained at each temperature using operating parameters of 10, 100, 200, and 300 RPM. Glycerol loss was negligible in all treatments.

[0199] Example 8: Metabolite analysis of glycerol content The P49W21G30 and P25W05G70 silk powder compositions were mixed and subjected to twin-screw extrusion as described in Example 2. The extrudate was chopped into pellets and subjected to melt flow index (MFI) testing. MFI testing was performed on a Goettfert Melt Indexer, Model MI-40, Serial Number 10005563. The barrel diameter was 9.5320 mm, the die length was 8.015 mm, and the orifice diameter was 2.09 mm. A 2-minute preheat period was utilized. Testing was performed in an extrusion plastometer for thermoplastic flow rate according to ASTM D1238 standard test method. Testing was performed at 95°C and with a force of 2.16 kg or 21.6 kg.

[0200] Table 15 shows the melt flow index values obtained for each material composition. n=3 for P49W21G30 and n=6 for P25W05G70, tested at 2.1 and 21.1 kg, respectively. "+ / -" indicates standard deviation among n samples. This data shows that protein / glycerol / water-based pellets have MFI values in a similar range to polypropylene, e.g., (20 g / 10 min). The smaller the protein composition, the higher the flow rate.

[0201] (Table 15) Melt flow index values TIFF2025118794000022.tif17128

[0202] Sequence information SEQUENCE LISTING <110> BOLT THREADS, INC. <120> Composition for a molded body <150> US 62 / 717,622 <151> 2018-08-10 <160> 38 <170> PatentIn version 3.5 <210> 1 <211> 945 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 1 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly 1 5 10 15 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 35 40 45 Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 50 55 60 Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 65 70 75 80 Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln 85 90 95 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 100 105 110 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro 130 135 140 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 145 150 155 160 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly 165 170 175 Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 180 185 190 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 195 200 205 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 210 215 220 Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln 225 230 235 240 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 245 250 255 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 260 265 270 Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 275 280 285 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 290 295 300 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 305 310 315 320 Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly 325 330 335 Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly 340 345 350 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala 355 360 365 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 370 375 380 Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly 385 390 395 400 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser 405 410 415 Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 420 425 430 Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 435 440 445 Gly Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro Gly Gly Gln Gly Pro 450 455 460 Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly 465 470 475 480 Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser 485 490 495 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly 500 505 510 Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly 515 520 525 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala 530 535 540 Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser 545 550 555 560 Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 565 570 575 Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly 580 585 590 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly 595 600 605 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala 610 615 620 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 625 630 635 640 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 645 650 655 Gly Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 660 665 670 Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala 675 680 685 Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala 690 695 700 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr 705 710 715 720 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 725 730 735 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala 740 745 750 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly 755 760 765 Gln Arg Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 770 775 780 Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln 785 790 795 800 Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala 805 810 815 Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 820 825 830 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly 835 840 845 Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr 850 855 860 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 865 870 875 880 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala 885 890 895 Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln 900 905 910 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 915 920 925 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 930 935 940 Ala 945 <210> 2 <211> 315 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly 1 5 10 15 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly 20 25 30 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 35 40 45 Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 50 55 60 Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly 65 70 75 80 Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln 85 90 95 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 100 105 110 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro 130 135 140 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly 145 150 155 160 Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly 165 170 175 Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr 180 185 190 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 195 200 205 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala 210 215 220 Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln 225 230 235 240 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 245 250 255 Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 260 265 270 Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 275 280 285 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 290 295 300 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 305 310 315 <210> 3 <211> 5 <212> PRT <213> Unknown <220> <223> Description of Unknown: Silk polypeptide block sequence <400> 3 Ser Gly Ala Gly Gly 1 5 <210> 4 <211> 5 <212> PRT <213> Unknown <220> <223> Description of Unknown: Silk polypeptide block sequence <400> 4 Gly Ser Gly Ala Gly 1 5 <210> 5 <211> 5 <212> PRT <213> Unknown <220> <223> Description of Unknown: Silk polypeptide block sequence <400> 5 Gly Gly Ser Gly Ala 1 5 <210> 6 <211> 181 <212> PRT <213> Aliatypus gulosus <400> 6 Gly Ala Ala Ser Ser Ser Ser Thr Ile Ile Thr Thr Lys Ser Ala Ser 1 5 10 15 Ala Ser Ala Ala Ala Asp Ala Ser Ala Ala Ala Thr Ala Ser Ala Ala 20 25 30 Ser Arg Ser Ser Ala Asn Ala Ala Ala Ser Ala Phe Ala Gln Ser Phe 35 40 45 Ser Ser Ile Leu Leu Glu Ser Gly Tyr Phe Cys Ser Ile Phe Gly Ser 50 55 60 Ser Ile Ser Ser Ser Tyr Ala Ala Ala Ile Ala Ser Ala Ala Ser Arg 65 70 75 80 Ala Ala Ala Glu Ser Asn Gly Tyr Thr Thr His Ala Tyr Ala Cys Ala 85 90 95 Lys Ala Val Ala Ser Ala Val Glu Arg Val Thr Ser Gly Ala Asp Ala 100 105 110 Tyr Ala Tyr Ala Gln Ala Ile Ser Asp Ala Leu Ser His Ala Leu Leu 115 120 125 Tyr Thr Gly Arg Leu Asn Thr Ala Asn Ala Asn Ser Leu Ala Ser Ala 130 135 140 Phe Ala Tyr Ala Phe Ala Asn Ala Ala Ala Gln Ala Ser Ala Ser Ser 145 150 155 160 Ala Ser Ala Gly Ala Ala Ser Ala Ser Gly Ala Ala Ser Ala Ser Gly 165 170 175 Ala Gly Ser Ala Ser 180 <210> 7 <211> 126 <212> PRT <213> Plectreurys tristis <400> 7 Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala 1 5 10 15 Gly Ser Gly Ala Ser Thr Ser Val Ser Thr Ser Ser Ser Ser Gly Ser 20 25 30 Gly Ala Gly Ala Gly Ala Gly Ser Gly Ala Gly Ser Gly Ala Gly Ala 35 40 45 Gly Ser Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly 50 55 60 Phe Gly Ser Gly Leu Gly Leu Gly Tyr Gly Val Gly Leu Ser Ser Ala 65 70 75 80 Gln Ala Gln Ala Gln Ala Gln Ala Ala Ala Gln Ala Gln Ala Gln Ala 85 90 95 Gln Ala Gln Ala Tyr Ala Ala Ala Gln Ala Gln Ala Gln Ala Gln Ala 100 105 110 Gln Ala Gln Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 115 120 125 <210> 8 <211> 239 <212> PRT <213> Plectreurys tristis <400> 8 Gly Ala Ala Gln Lys Gln Pro Ser Gly Glu Ser Ser Val Ala Thr Ala 1 5 10 15 Ser Ala Ala Ala Thr Ser Val Thr Ser Gly Gly Ala Pro Val Gly Lys 20 25 30 Pro Gly Val Pro Ala Pro Ile Phe Tyr Pro Gln Gly Pro Leu Gln Gln 35 40 45 Gly Pro Ala Pro Gly Pro Ser Asn Val Gln Pro Gly Thr Ser Gln Gln 50 55 60 Gly Pro Ile Gly Gly Val Gly Gly Ser Asn Ala Phe Ser Ser Ser Phe 65 70 75 80 Ala Ser Ala Leu Ser Leu Asn Arg Gly Phe Thr Glu Val Ile Ser Ser 85 90 95 Ala Ser Ala Thr Ala Val Ala Ser Ala Phe Gln Lys Gly Leu Ala Pro 100 105 110 Tyr Gly Thr Ala Phe Ala Leu Ser Ala Ala Ser Ala Ala Ala Asp Ala 115 120 125 Tyr Asn Ser Ile Gly Ser Gly Ala Asn Ala Phe Ala Tyr Ala Gln Ala 130 135 140 Phe Ala Arg Val Leu Tyr Pro Leu Val Gln Gln Tyr Gly Leu Ser Ser 145 150 155 160 Ser Ala Lys Ala Ser Ala Phe Ala Ser Ala Ile Ala Ser Ser Phe Ser 165 170 175 Ser Gly Thr Ser Gly Gln Gly Pro Ser Ile Gly Gln Gln Gln Pro Pro 180 185 190 Val Thr Ile Ser Ala Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala 195 200 205 Val Gly Gly Gly Gln Val Gly Gln Gly Pro Tyr Gly Gly Gln Gln Gln 210 215 220 Ser Thr Ala Ala Ser Ala Ser Ala Ala Ala Ala Thr Ala Thr Ser 225 230 235 <210> 9 <211> 182 <212> PRT <213> Araneus gemmoides <400> 9 Gly Asn Val Gly Tyr Gln Leu Gly Leu Lys Val Ala Asn Ser Leu Gly 1 5 10 15 Leu Gly Asn Ala Gln Ala Leu Ala Ser Ser Leu Ser Gln Ala Val Ser 20 25 30 Ala Val Gly Val Gly Ala Ser Ser Asn Ala Tyr Ala Asn Ala Val Ser 35 40 45 Asn Ala Val Gly Gln Val Leu Ala Gly Gln Gly Ile Leu Asn Ala Ala 50 55 60 Asn Ala Gly Ser Leu Ala Ser Ser Phe Ala Ser Ala Leu Ser Ser Ser 65 70 75 80 Ala Ala Ser Val Ala Ser Gln Ser Ala Ser Gln Ser Gln Ala Ala Ser 85 90 95 Gln Ser Gln Ala Ala Ala Ser Ala Phe Arg Gln Ala Ala Ser Gln Ser 100 105 110 Ala Ser Gln Ser Asp Ser Arg Ala Gly Ser Gln Ser Ser Thr Lys Thr 115 120 125 Thr Ser Thr Ser Thr Ser Gly Ser Gln Ala Asp Ser Arg Ser Ala Ser 130 135 140 Be Be Wing Be Gln Wing Be Wing Be Wing Phe Wing Gln Gln Be Be 145 150 155 160 Ala Ser Leu Ser Ser Ser Ser Ser Phe Ser Ser Ala Phe Ser Ser Ala 165 170 175 Thr Ser Ile Ser Ala Val 180 <210> 10 <211> 180 <212> PRT <213> Argiope aurantia <400> 10 Gly Ser Leu Ala Ser Ser Phe Ala Ser Ala Leu Ser Ala Ser Ala Ala 1 5 10 15 Being Val Ala Being Being Ala Ala Ala Gln Ala Ala Being Gln Being Gln Ala 20 25 30 Ala Ala Ser Ala Phe Ser Arg Ala Ala Ser Gln Ser Ala Ser Gln Ser 35 40 45 Ala Ala Arg Ser Gly Ala Gln Ser Ile Ser Thr Thr Thr Thr Thr Ser 50 55 60 Thr Ala Gly Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ala Ala Ser 65 70 75 80 Gln Ala Ser Ala Ser Ser Phe Ala Arg Ala Ser Ser Ala Ser Leu Ala 85 90 95 Ala Ser Ser Ser Phe Ser Ser Ala Phe Ser Ser Ala Asn Ser Leo Ser 100 105 110 Ala Leu Gly Asn Val Gly Tyr Gln Leu Gly Phe Asn Val Ala Asn Asn 115 120 125 Leu Gly Ile Gly Asn Ala Ala Gly Leu Gly Asn Ala Leu Ser Gln Ala 130 135 140 Val Ser Ser Val Gly Val Gly Ala Ser Ser Ser Thr Tyr Ala Asn Ala 145 150 155 160 Val Ser Asn Ala Val Gly Gln Phe Leu Ala Gly Gln Gly Ile Leu Asn 165 170 175 Ala Ala Asn Ala 180 <210> 11 <211> 199 <212> PRT <213> Spiny Deinopis <400> 11 Gly Ala Ser Ala Ser Ala Tyr Ala Ser Ala Ile Ser Asn Ala Val Gly 1 5 10 15 <h2 style=";text-align:left;direction:ltr">Pro Tyr Leu Tyr Gly Leu Gly Leu Phe Asn Gln Ala Asn Ala Ala Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 20 25 30<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Phe Ala Ser Ser Phe Ala Ser Ala Val Ser Ser Ala Val Ala Ser Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 35 40 45<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ser Ala Ser Ala Ala Ser Ser Ala Tyr Ala Gln Ser Ala Ala Ala Gln<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 50 55 60<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Gln Ala Ala Ser Ser Ala Phe Ser Gln Ala Ala Ala Gln Ser Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 65 70 75 80<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Ala Ala Ser Ala Gly Ala Ser Ala Gly Ala Gly Ala Ser Ala Gly<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 85 90 95<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Gly Ala Val Ala Gly Ala Gly Ala Val Ala Gly Ala Gly Ala Val<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 100 105 110<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Gly Ala Ser Ala Ala Ala Ala Ser Gln Ala Ala Ala Ser Ser Ser<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 115 120 125<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Ser Ala Val Ala Ser Ala Phe Ala Gln Ser Ala Ser Tyr Ala Leu<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 130 135 140<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Ala Ser Ser Ser Ala Phe Ala Asn Ala Phe Ala Ser Ala Thr Ser Ala<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 145 150 155 160<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> Gly Tyr Leu Gly Ser Leu Ala Tyr Gln Leu Gly Leu Thr Thr Ala Tyr<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> 165 170 175<h2 style=";text-align:left;direction:ltr"> Asn Leu Gly Leu Ser Asn Ala Gln Ala Phe Ala Ser Thr Leu Ser Gln 180 185 190 Ala Val Thr Gly Val Gly Leu 195 <210> 12 <211> 171 <212> PRT <213> Nephila clavipes <400> 12 Gly Ala Thr Ala Ala Ser Tyr Gly Asn Ala Leu Ser Thr Ala Ala Ala 1 5 10 15 Gln Phe Phe Ala Thr Ala Gly Leu Leu Asn Ala Gly Asn Ala Ser Ala 20 25 30 Leo Ala Ser Ser Phe Ala Arg Ala Phe Ser Ala Ser Ala Glu Ser Gln 35 40 45 Ser Phe Ala Gln Ser Gln Ala Phe Gln Ala Gln Ser Ala Phe Gln Gln 50 55 60 Ala Ala Ser Arg Ser Ala Ser Gln Ser Ala Ala Glu Ala Gly Ser Thr 65 70 75 80 Ser Ser Ser Thr Thr Thr Thr Thr Ser Ala Ala Arg Ser Gln Ala Ala 85 90 95 Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala Phe Ala Gln Ala Ala 100 105 110 Ser Ser Ser Leu Ala Thr Ser Ser Ala Leu Ser Arg Ala Phe Ser Ser 115 120 125 Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Ser Ile Gly Leu 130 135 140 Ser Ala Ala Arg Ser Leu Gly Ile Ala Asp Ala Ala Gly Leu Ala Gly 145 150 155 160 Val Leu Ala Arg Ala Ala Gly Ala Leu Gly Gln 165 170 <210> 13 <211> 268 <212> PRT <213> Argiope trifasciata <400> 13 Gly Gly Ala Pro Gly Gly Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala 1 5 10 15 Gly Phe Gly Pro Gly Gly Gly Ala Gly Phe Gly Pro Gly Gly Gly Ala 20 25 30 Gly Phe Gly Pro Gly Gly Ala Ala Gly Gly Pro Gly Gly Pro Gly Gly 35 40 45 Pro Gly Gly Pro Gly Gly Ala Gly Gly Tyr Gly Pro Gly Gly Ala Gly 50 55 60 Gly Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Tyr Gly 65 70 75 80 Pro Gly Gly Ala Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly 85 90 95 Ala Gly Pro Gly Gly Ala Gly Gly Glu Gly Pro Val Thr Val Asp Val 100 105 110 Asp Val Thr Val Gly Pro Glu Gly Val Gly Gly Gly Pro Gly Gly Ala 115 120 125 Gly Pro Gly Gly Ala Gly Phe Gly Pro Gly Gly Gly Ala Gly Phe Gly 130 135 140 Pro Gly Gly Ala Pro Gly Ala Pro Gly Gly Pro Gly Gly Pro Gly Gly 145 150 155 160 Pro Gly Gly Pro Gly Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly 165 170 175 Gly Tyr Gly Pro Gly Gly Ala Gly Gly Val Gly Pro Ala Gly Thr Gly 180 185 190 Gly Phe Gly Pro Gly Gly Ala Gly Gly Phe Gly Pro Gly Gly Ala Gly 195 200 205 Gly Phe Gly Pro Gly Gly Ala Gly Gly Phe Gly Pro Ala Gly Ala Gly 210 215 220 Gly Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Phe Gly 225 230 235 240 Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly 245 250 255 Glu Gly Pro Val Thr Val Asp Val Asp Val Ser Val 260 265 <210> 14 <211> 420 <212> PRT <213> Nephila clavipes <400> 14 Gly Val Ser Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly 1 5 10 15 Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro Gly Gly Ala Gly Gly Pro 20 25 30 Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr 35 40 45 Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly 50 55 60 Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro 65 70 75 80 Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly 85 90 95 Gly Tyr Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly 100 105 110 Ser Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Thr 115 120 125 Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly 130 135 140 Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro 145 150 155 160 Gly Gly Phe Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly 165 170 175 Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly 180 185 190 Phe Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Ala Pro Gly Gly Ala 195 200 205 Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly 210 215 220 Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Gly 225 230 235 240 Ala Gly Gly Ala Gly Gly Ser Gly Gly Ala Gly Gly Ser Gly Gly Thr 245 250 255 Thr Ile Ile Glu Asp Leu Asp Ile Thr Ile Asp Gly Ala Asp Gly Pro 260 265 270 Ile Thr Ile Ser Glu Glu Leu Pro Ile Ser Gly Ala Gly Gly Ser Gly 275 280 285 Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro 290 295 300 Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Pro Gly 305 310 315 320 Gly Ser Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Pro Tyr 325 330 335 Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Pro 340 345 350 Gly Gly Ala Tyr Gly Pro Gly Gly Ser Tyr Gly Pro Gly Gly Ser Gly 355 360 365 Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro 370 375 380 Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr 385 390 395 400 Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Gly 405 410 415 Pro Tyr Gly Pro 420 <210> 15 <211> 376 <212> PRT <213> Latrodectus hesperus <400> 15 Gly Ile Asn Val Asp Ser Asp Ile Gly Ser Val Thr Ser Leu Ile Leu 1 5 10 15 Ser Gly Ser Thr Leu Gln Met Thr Ile Pro Ala Gly Gly Asp Asp Leu 20 25 30 Ser Gly Gly Tyr Pro Gly Gly Phe Pro Ala Gly Ala Gln Pro Ser Gly 35 40 45 Gly Ala Pro Val Asp Phe Gly Gly Pro Ser Ala Gly Gly Asp Val Ala 50 55 60 Ala Lys Leu Ala Arg Ser Leu Ala Ser Thr Leu Ala Ser Ser Gly Val 65 70 75 80 Phe Arg Ala Ala Phe Asn Ser Arg Val Ser Thr Pro Val Ala Val Gln 85 90 95 Leu Thr Asp Ala Leu Val Gln Lys Ile Ala Ser Asn Leu Gly Leu Asp 100 105 110 Tyr Ala Thr Ala Ser Lys Leu Arg Lys Ala Ser Gln Ala Val Ser Lys 115 120 125 Val Arg Met Gly Ser Asp Thr Asn Ala Tyr Ala Leu Ala Ile Ser Ser 130 135 140 Ala Leu Ala Glu Val Leu Ser Ser Ser Gly Lys Val Ala Asp Ala Asn 145 150 155 160 Ile Asn Gln Ile Ala Pro Gln Leu Ala Ser Gly Ile Val Leu Gly Val 165 170 175 Ser Thr Thr Ala Pro Gln Phe Gly Val Asp Leu Ser Ser Ile Asn Val 180 185 190 Asn Leu Asp Ile Ser Asn Val Ala Arg Asn Met Gln Ala Ser Ile Gln 195 200 205 Gly Gly Pro Ala Pro Ile Thr Ala Glu Gly Pro Asp Phe Gly Ala Gly 210 215 220 Tyr Pro Gly Gly Ala Pro Thr Asp Leu Ser Gly Leu Asp Met Gly Ala 225 230 235 240 Pro Ser Asp Gly Ser Arg Gly Gly Asp Ala Thr Ala Lys Leu Leu Gln 245 250 255 Ala Leu Val Pro Ala Leu Leu Lys Ser Asp Val Phe Arg Ala Ile Tyr 260 265 270 Lys Arg Gly Thr Arg Lys Gln Val Val Gln Tyr Val Thr Asn Ser Ala 275 280 285 Leu Gln Gln Ala Ala Ser Ser Leu Gly Leu Asp Ala Ser Thr Ile Ser 290 295 300 Gln Leu Gln Thr Lys Ala Thr Gln Ala Leu Ser Ser Val Ser Ala Asp 305 310 315 320 Ser Asp Ser Thr Ala Tyr Ala Lys Ala Phe Gly Leu Ala Ile Ala Gln 325 330 335 Val Leu Gly Thr Ser Gly Gln Val Asn Asp Ala Asn Val Asn Gln Ile 340 345 350 Gly Ala Lys Leu Ala Thr Gly Ile Leu Arg Gly Ser Ser Ala Val Ala 355 360 365 Pro Arg Leu Gly Ile Asp Leu Ser 370 375 <210> 16 <211> 200 <212> PRT <213> Argiope trifasciata <400> 16 Gly Ala Gly Tyr Thr Gly Pro Ser Gly Pro Ser Thr Gly Pro Ser Gly 1 5 10 15 Tyr Pro Gly Pro Leu Gly Gly Gly Ala Pro Phe Gly Gln Ser Gly Phe 20 25 30 Gly Gly Ser Ala Gly Pro Gln Gly Gly Phe Gly Ala Thr Gly Gly Ala 35 40 45 Ser Ala Gly Leu Ile Ser Arg Val Ala Asn Ala Leu Ala Asn Thr Ser 50 55 60 Thr Leu Arg Thr Val Leu Arg Thr Gly Val Ser Gln Gln Ile Ala Ser 65 70 75 80 Ser Val Val Gln Arg Ala Ala Gln Ser Leu Ala Ser Thr Leu Gly Val 85 90 95 Asp Gly Asn Asn Leu Ala Arg Phe Ala Val Gln Ala Val Ser Arg Leu 100 105 110 Pro Ala Gly Ser Asp Thr Ser Ala Tyr Ala Gln Ala Phe Ser Ser Ala 115 120 125 Leu Phe Asn Ala Gly Val Leu Asn Ala Ser Asn Ile Asp Thr Leu Gly 130 135 140 Ser Arg Val Leu Ser Ala Leu Leu Asn Gly Val Ser Ser Ala Ala Gln 145 150 155 160 Gly Leu Gly Ile Asn Val Asp Ser Gly Ser Val Gln Ser Asp Ile Ser 165 170 175 Be Be Be Be Be Phe Leu Be Thr Be Be Be Be Ala Be Tyr Be 180 185 190 Gln Ala Ser Ala Ser Ser Thr Ser 195 200 <210> 17 <211> 357 <212> PRT <213> Uloborus diversus <400> 17 Gly Ala Ser Ala Ala Asp Ile Ala Thr Ala Ile Ala Ala Ser Val Ala 1 5 10 15 Thr Ser Leu Gln Ser Asn Gly Val Leu Thr Ala Ser Asn Val Ser Gln 20 25 30 Leu Ser Asn Gln Leu Ala Ser Tyr Val Ser Ser Gly Leu Ser Ser Thr 35 40 45 Ala Ser Ser Leu Gly Ile Gln Leu Gly Ala Ser Leu Gly Ala Gly Phe 50 55 60 Gly Ala Ser Ala Gly Leu Ser Ala Ser Thr Asp Ile Ser Ser Ser Val 65 70 75 80 Glu Ala Thr Ser Ala Ser Thr Leu Ser Ser Ser Ala Ser Ser Thr Ser 85 90 95 Val Val Ser Ser Ile Asn Ala Gln Leu Val Pro Ala Leu Ala Gln Thr 100 105 110 Ala Val Leu Asn Ala Ala Phe Ser Asn Ile Asn Thr Gln Asn Ala Ile 115 120 125 Arg Ile Ala Glu Leu Leu Thr Gln Gln Val Gly Arg Gln Tyr Gly Leu 130 135 140 Ser Gly Ser Asp Val Ala Thr Ala Ser Ser Gln Ile Arg Ser Ala Leu 145 150 155 160 Tyr Ser Val Gln Gln Gly Ser Ala Ser Ser Ala Tyr Val Ser Ala Ile 165 170 175 Val Gly Pro Leu Ile Thr Ala Leu Ser Ser Arg Gly Val Val Asn Ala 180 185 190 Ser Asn Ser Ser Gln Ile Ala Ser Ser Leu Ala Thr Ala Ile Leu Gln 195 200 205 Phe Thr Ala Asn Val Ala Pro Gln Phe Gly Ile Ser Ile Pro Thr Ser 210 215 220 Ala Val Gln Ser Asp Leu Ser Thr Ile Ser Gln Ser Leu Thr Ala Ile 225 230 235 240 Ser Ser Gln Thr Ser Ser Ser Val Asp Ser Ser Thr Ser Ala Phe Gly 245 250 255 Gly Ile Ser Gly Pro Ser Gly Pro Ser Pro Tyr Gly Pro Gln Pro Ser 260 265 270 Gly Pro Thr Phe Gly Pro Gly Pro Ser Leu Ser Gly Leu Thr Gly Phe 275 280 285 Thr Ala Thr Phe Ala Ser Ser Phe Lys Ser Thr Leu Ala Ser Ser Thr 290 295 300 Gln Phe Gln Leu Ile Ala Gln Ser Asn Leu Asp Val Gln Thr Arg Ser 305 310 315 320 Ser Leu Ile Ser Lys Val Leu Ile Asn Ala Leu Ser Ser Leu Gly Ile 325 330 335 Ser Ala Ser Val Ala Ser Ser Ile Ala Ala Ser Ser Ser Gln Ser Leu 340 345 350 Leu Ser Val Ser Ala 355 <210> 18 <211> 32 <212> PRT <213> Euprosthenops australis <400> 18 Gly Gly Gln Gly Gly Gln Gly Gln Gly Arg Tyr Gly Gln Gly Ala Gly 1 5 10 15 Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala 20 25 30 <210> 19 <211> 42 <212> PRT <213> Tetragnatha kauaiensis <400> 19 Gly Gly Leu Gly Gly Gly Gln Gly Ala Gly Gln Gly Gly Gln Gln Gly 1 5 10 15 Ala Gly Gln Gly Gly Tyr Gly Ser Gly Leu Gly Gly Ala Gly Gln Gly 20 25 30 Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala 35 40 <210> 20 <211> 42 <212> PRT <213> Argiope aurantia <400> 20 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly 1 5 10 15 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Leu Gly Pro Tyr Gly 20 25 30 Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala 35 40 <210> 21 <211> 46 <212> PRT <213> Deinopis spinosa <400> 21 Gly Pro Gly Gly Tyr Gly Gly Pro Gly Gln Gln Gly Pro Gly Gln Gly 1 5 10 15 Gln Tyr Gly Pro Gly Thr Gly Gln Gln Gly Gln Gly Pro Ser Gly Gln 20 25 30 Gln Gly Pro Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala 35 40 45 <210> 22 <211> 42 <212> PRT <213> Nephila clavata <400> 22 Gly Pro Gly Gly Tyr Gly Leu Gly Gln Gln Gly Pro Gly Gln Gln Gly 1 5 10 15 Pro Gly Gln Gln Gly Pro Ala Gly Tyr Gly Pro Ser Gly Leu Ser Gly 20 25 30 Pro Gly Gly Ala Ala Ala Ala Ala Ala Ala 35 40 <210> 23 <211> 174 <212> PRT <213> Deinopis spinosa <400> 23 Gly Ala Gly Tyr Gly Ala Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala 1 5 10 15 Gly Thr Gly Tyr Gly Gly Gly Ala Gly Tyr Gly Thr Gly Ser Gly Ala 20 25 30 Gly Tyr Gly Ala Gly Val Gly Tyr Gly Ala Gly Ala Gly Ala Gly Gly 35 40 45 Gly Ala Gly Ala Gly Ala Gly Gly Gly Thr Gly Ala Gly Ala Gly Gly 50 55 60 Gly Ala Gly Ala Gly Tyr Gly Ala Gly Thr Gly Tyr Gly Ala Gly Ala 65 70 75 80 Gly Ala Gly Gly Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala 85 90 95 Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Tyr Gly Ala Gly Ala 100 105 110 Gly Tyr Gly Ala Gly Ala Gly Ala Gly Gly Val Ala Gly Ala Gly Ala 115 120 125 Ala Gly Gly Ala Gly Ala Ala Gly Gly Ala Gly Ala Ala Gly Gly Ala 130 135 140 Gly Ala Ala Gly Gly Ala Gly Ala Gly Ala Gly Ala Gly Ser Gly Ala 145 150 155 160 Gly Ala Gly Ala Gly Gly Gly Ala Arg Ala Gly Ala Gly Gly 165 170 <210> 24 <211> 149 <212> PRT <213> Latrodectus hesperus <400> 24 Gly Gly Gly Tyr Gly Arg Gly Gln Gly Ala Gly Ala Gly Val Gly Ala 1 5 10 15 Gly Ala Gly Ala Ala Ala Gly Ala Ala Ala Ile Ala Arg Ala Gly Gly 20 25 30 Tyr Gly Gln Gly Ala Gly Gly Tyr Gly Gln Gly Gln Gly Ala Gly Ala 35 40 45 Ala Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Tyr Gly Gln Gly 50 55 60 Ala Gly Gly Tyr Gly Arg Gly Gln Gly Ala Gly Ala Gly Ala Gly Ala 65 70 75 80 Gly Ala Gly Ala Arg Gly Tyr Gly Gln Gly Ala Gly Ala Gly Ala Ala 85 90 95 Ala Gly Ala Ala Ala Ser Ala Gly Ala Gly Gly Tyr Gly Gln Gly Ala 100 105 110 Gly Gly Tyr Gly Gln Gly Gln Gly Ala Gly Ala Ala Ala Gly Ala Ala 115 120 125 Ala Ser Ala Gly Ala Gly Gly Tyr Gly Gln Gly Ala Gly Gly Tyr Gly 130 135 140 Gln Gly Gln Gly Ala 145 <210> 25 <211> 161 <212> PRT <213> Nephila clavipes <400> 25 Gly Ala Gly Ala Gly Gly Ala Gly Tyr Gly Arg Gly Ala Gly Ala Gly 1 5 10 15 Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly 20 25 30 Ala Gly Ala Gly Gly Tyr Gly Gly Gln Gly Gly Tyr Gly Ala Gly Ala 35 40 45 Gly Ala Gly Ala Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Ala 50 55 60 Gly Tyr Ser Arg Gly Gly Arg Ala Gly Ala Ala Gly Ala Gly Ala Gly 65 70 75 80 Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Gly Tyr Gly Gly Gln Gly 85 90 95 Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala 100 105 110 Gly Ser Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala 115 120 125 Ala Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Gly 130 135 140 Tyr Gly Gly Gln Gly Gly Tyr Gly Ala Gly Ala Gly Ala Ala Ala Ala 145 150 155 160 Oh my God <210> 26 <211> 186 <212> PRT <213> Nephilengys cruentata <400> 26 Gly Ala Gly Ala Gly Val Gly Gly Ala Gly Gly Tyr Gly Ser Gly Ala 1 5 10 15 Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ser Gly Ala Ala Ala 20 25 30 Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Thr 35 40 45 Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala 50 55 60 Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala 65 70 75 80 Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala 85 90 95 Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Asp Gly Ala Gly Ala 100 105 110 Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala 115 120 125 Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala 130 135 140 Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala 145 150 155 160 Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala 165 170 175 Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala 180 185 <210> 27 <211> 132 <212> PRT <213> Uloborus diversus <400> 27 Gly Ser Gly Ala Gly Ala Gly Ser Gly Tyr Gly Ala Gly Ala Gly Ala 1 5 10 15 Gly Ala Gly Ser Gly Tyr Gly Ala Gly Ser Ser Ala Ser Ala Gly Ser 20 25 30 Ala Ile Asn Thr Gln Thr Val Thr Ser Ser Thr Thr Thr Ser Ser Gln 35 40 45 Ser Ser Ala Ala Ala Thr Gly Ala Gly Tyr Gly Thr Gly Ala Gly Thr 50 55 60 Gly Ala Ser Ala Gly Ala Ala Ala Ser Gly Ala Gly Ala Gly Tyr Gly 65 70 75 80 Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Arg Ala Ala 85 90 95 Gly Ser Gly Tyr Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ser 100 105 110 Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Gly Ser Gly Tyr Gly Ala 115 120 125 Gly Ala Ala Ala 130 <210> 28 <211> 198 <212> PRT <213> Uloborus diversus <400> 28 Gly Ala Gly Ala Gly Tyr Arg Gly Gln Ala Gly Tyr Ile Gln Gly Ala 1 5 10 15 Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Val Gly Tyr Gly 20 25 30 Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala 35 40 45 Ala Ala Ala Gly Ala Gly Ala Gly Arg Gln Ala Gly Tyr Gly Gln Gly 50 55 60 Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Ala Gly Arg 65 70 75 80 Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala 85 90 95 Ala Gly Ala Asp Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly 100 105 110 Ala Gly Ala Ser Ala Gly Ala Ala Ala Ser Gly Ala Gly Ala Gly Tyr 115 120 125 Gly Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala 130 135 140 Ala Ala Ala Gly Ala Gly Ala Gly Tyr Leu Gly Gln Ala Gly Tyr Gly 145 150 155 160 Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly 165 170 175 Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Thr Gly Ala Ala Ala Ser 180 185 190 Ala Ala Ala Ser Ser Ala 195 <210> 29 <211> 190 <212> PRT <213> Araneus ventricosus <400> 29 Gly Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly 1 5 10 15 Ala Gly Gln Gly Gly Tyr Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala 20 25 30 Ala Ala Ala Gly Gly Ala Gly Gly Ala Gly Arg Gly Gly Leu Gly Ala 35 40 45 Gly Gly Ala Gly Gln Gly Tyr Gly Ala Gly Leu Gly Gly Gln Gly Gly 50 55 60 Ala Gly Gln Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gly Ala 65 70 75 80 Arg Gln Gly Gly Leu Gly Ala Gly Gly Ala Gly Gln Gly Tyr Gly Ala 85 90 95 Gly Leu Gly Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala 100 105 110 Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu 115 120 125 Gly Ser Gln Gly Ala Gly Gln Gly Gly Tyr Gly Ala Gly Gln Gly Gly 130 135 140 Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gly 145 150 155 160 Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Gln Gly Gly Tyr Gly Gly 165 170 175 Arg Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala 180 185 190 <210> 30 <211> 166 <212> PRT <213> Dolomedes tenebrosus <400> 30 Gly Gly Ala Gly Ala Gly Gln Gly Ser Tyr Gly Gly Gln Gly Gly Tyr 1 5 10 15 Gly Gln Gly Gly Ala Gly Ala Ala Thr Ala Thr Ala Ala Ala Ala Gly 20 25 30 Gly Ala Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Gly Leu Gly 35 40 45 Gly Tyr Gly Gln Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala 50 55 60 Ala Ala Gly Gly Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly 65 70 75 80 Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ala Gly Ala Ala Ala Ala 85 90 95 Ala Ala Gly Gly Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly 100 105 110 Gly Tyr Gly Gln Gly Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala 115 120 125 Ala Ser Gly Gly Ser Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly 130 135 140 Gly Leu Gly Gly Tyr Gly Gln Gly Ala Gly Ala Gly Ala Gly Ala Ala 145 150 155 160 Ala Ser Ala Ala Ala Ala 165 <210> 31 <211> 177 <212> PRT <213> Nephilengys cruentata <400> 31 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala 1 5 10 15 Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly 20 25 30 Gly Gln Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala Ala Ser Gly Ala 35 40 45 Gly Gln Gly Gly Tyr Glu Gly Pro Gly Ala Gly Gln Gly Ala Gly Ala 50 55 60 Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu 65 70 75 80 Gly Gly Gln Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala 85 90 95 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala 100 105 110 Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln 115 120 125 Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala 130 135 140 Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gly Gln 145 150 155 160 Gly Gly Tyr Gly Arg Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala 165 170 175 Ala <210> 32 <211> 174 <212> PRT <213> Nephilengys cruentata <400> 32 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala 1 5 10 15 Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly 20 25 30 Gly Gln Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala Ala Ser Gly Ala 35 40 45 Gly Gln Gly Gly Tyr Gly Gly Pro Gly Ala Gly Gln Gly Ala Gly Ala 50 55 60 Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu 65 70 75 80 Gly Gly Gln Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala 85 90 95 Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly 100 105 110 Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly 115 120 125 Leu Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Ala Ala 130 135 140 Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly 145 150 155 160 Gln Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala 165 170 <210> 33 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic His tag <220> <221> MISC_FEATURE <222> (1)..(8) <223> This sequence may encompass 6-8 residues <400> 33 His His His His His His His His 1 5 <210> 34 <211> 1600 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <220> <221> MISC_FEATURE <222> (7)..(11) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (15)..(19) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (23)..(27) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (31)..(35) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (39)..(43) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (47)..(51) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (55)..(59) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (63)..(67) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (4)..(67) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (71)..(80) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (87)..(91) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (95)..(99) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (103)..(107) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (111)..(115) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (119)..(123) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (127)..(131) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (135)..(139) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (143)..(147) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (84)..(147) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (151)..(160) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (167)..(171) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (175)..(179) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (183)..(187) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (191)..(195) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (199)..(203) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (207)..(211) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (215)..(219) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (223)..(227) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (164)..(227) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (231)..(240) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (247)..(251) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (255)..(259) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (263)..(267) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (271)..(275) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (279)..(283) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (287)..(291) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (295)..(299) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (303)..(307) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (244)..(307) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (311)..(320) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (327)..(331) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (335)..(339) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (343)..(347) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (351)..(355) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (359)..(363) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (367)..(371) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (375)..(379) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (383)..(387) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (324)..(387) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (391)..(400) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (407)..(411) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (415)..(419) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (423)..(427) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (431)..(435) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (439)..(443) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (447)..(451) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (455)..(459) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (463)..(467) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (404)..(467) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (471)..(480) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (487)..(491) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (495)..(499) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (503)..(507) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (511)..(515) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (519)..(523) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (527)..(531) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (535)..(539) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (543)..(547) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (484)..(547) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (551)..(560) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (567)..(571) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (575)..(579) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (583)..(587) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (591)..(595) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (599)..(603) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (607)..(611) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (615)..(619) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (623)..(627) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (564)..(627) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (631)..(640) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (647)..(651) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (655)..(659) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (663)..(667) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (671)..(675) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (679)..(683) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (687)..(691) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (695)..(699) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (703)..(707) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (644)..(707) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (711)..(720) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (727)..(731) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (735)..(739) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (743)..(747) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (751)..(755) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (759)..(763) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (767)..(771) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (775)..(779) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (783)..(787) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (724)..(787) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (791)..(800) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (807)..(811) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (815)..(819) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (823)..(827) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (831)..(835) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (839)..(843) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (847)..(851) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (855)..(859) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (863)..(867) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (804)..(867) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (871)..(880) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (887)..(891) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (895)..(899) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (903)..(907) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (911)..(915) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (919)..(923) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (927)..(931) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (935)..(939) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (943)..(947) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (884)..(947) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (951)..(960) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (967)..(971) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (975)..(979) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (983)..(987) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (991)..(995) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (999)..(1003) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1007)..(1011) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1015)..(1019) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1023)..(1027) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (964)..(1027) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1031)..(1040) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (1047)..(1051) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1055)..(1059) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1063)..(1067) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1071)..(1075) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1079)..(1083) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1087)..(1091) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1095)..(1099) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1103)..(1107) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1044)..(1107) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1111)..(1120) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (1127)..(1131) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1135)..(1139) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1143)..(1147) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1151)..(1155) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1159)..(1163) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1167)..(1171) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1175)..(1179) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1183)..(1187) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1124)..(1187) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1191)..(1200) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (1207)..(1211) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1215)..(1219) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1223)..(1227) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1231)..(1235) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1239)..(1243) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1247)..(1251) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1255)..(1259) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1263)..(1267) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1204)..(1267) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1271)..(1280) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (1287)..(1291) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1295)..(1299) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1303)..(1307) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1311)..(1315) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1319)..(1323) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1327)..(1331) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1335)..(1339) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1343)..(1347) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1284)..(1347) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1351)..(1360) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (1367)..(1371) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1375)..(1379) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1383)..(1387) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1391)..(1395) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1399)..(1403) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1407)..(1411) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1415)..(1419) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1423)..(1427) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1364)..(1427) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1431)..(1440) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (1447)..(1451) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1455)..(1459) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1463)..(1467) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1471)..(1475) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1479)..(1483) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1487)..(1491) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1495)..(1499) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1503)..(1507) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1444)..(1507) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1511)..(1520) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (1527)..(1531) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1535)..(1539) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1543)..(1547) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1551)..(1555) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1559)..(1563) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1567)..(1571) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1575)..(1579) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1583)..(1587) <223> This region may encompass "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," wherein some positions may be absent <220> <221> MISC_FEATURE <222> (1524)..(1587) <223> This region may encompass 4-8 repeating "GPG-X1" repeating units, wherein X1 is "SGGQQ," "GAGQQ," "GQGPY," "AGQQ" or "SQ," and some positions may be absent <220> <221> MISC_FEATURE <222> (1591)..(1600) <223> This region may encompass 6-10 residues <220> <221> MISC_FEATURE <222> (1)..(1600) <223> This sequence may encompass 2-20 "GGY-[GPG-X1]n1-GPS-(A)n2" repeating units, wherein X1 is “SGGQQ,” “GAGQQ,” "GQGPY," "AGQQ" or "SQ," n1 is 4-8 and n2 is 6-10 and some Positions may be absent. <400> 34 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 1 5 10 15 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 20 25 30 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 35 40 45 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 50 55 60 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 65 70 75 80 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 85 90 95 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 100 105 110 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 115 120 125 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 130 135 140 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 145 150 155 160 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 165 170 175 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 180 185 190 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 195 200 205 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 210 215 220 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 225 230 235 240 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 245 250 255 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 260 265 270 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 275 280 285 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 290,295,300 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 305 310 315 320 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 325 330 335 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 340 345 350 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 355 360 365 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 370 375 380 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 385 390 395 400 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 405 410 415 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 420 425 430 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 435 440 445 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 450 455 460 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 465 470 475 480 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 485,490,495 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 500 505 510 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 515,520,525 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 530 535 540 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 545 550 555 560 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 565,570,575 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 580,585,590 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 595,600,605 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 610 615 620 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 625 630 635 640 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 645,650,655 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 660,665,670 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 675,680,685 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 690,695,700 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 705 710 715 720 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 725 730 735 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 740,745,750 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 755,760,765 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 770,775,780 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 785,790,795,800 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 805 810 815 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 820 825 830 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 835 840 845 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 850 855 860 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 865 870 875 880 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 885,890,895 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 900 905 910 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 915,920,925 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 930,935,940 Yeah Yeah Yeah Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala 945 950 955 960 Gly Gly Tyr Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly Pro Gly 965,970,975 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 980,985,990 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 995 1000 1005 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1010 1015 1020 Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 1025 1030 1035 No No Gly Gly Tyr Gly Pro Gly Yes Yes Yes Yes Gly Pro 1040 1045 1050 Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 1055 1060 1065 Pro Gly Cheap Cheap Cheap Gly Pro Gly Cheap Cheap Cheap 1070 1075 1080 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1085 1090 1095 Yes Gly Pro Gly Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala 1100 1105 1110 No No No No No No No Gly Gly Tyr Gly Pro Gly Stone 1115 1120 1125 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1130 1135 1140 Yes Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly 1145 1150 1155 Free Free Gly Pro Gly Free Free Gly Pro 1160 1165 1170 Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 1175 1180 1185 Pro Being Wing Wing Wing Wing Wing Wing Wing Wing Wing Gly Gly Tyr 1190 1195 1200 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1205 1210 1215 No Gly Pro Gly No No No No Gly Pro Gly No no 1220 1225 1230 Too Much Gly Pro Gly Too Much Too Much Too Much Gly Pro Gly Too Much 1235 1240 1245 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1250 1255 1260 Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 1265 1270 1275 No No Gly Gly Tyr Gly Pro Gly Yes Yes Yes Yes Gly Pro 1280 1285 1290 Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 1295 1300 1305 Pro Gly Cheap Cheap Cheap Gly Pro Gly Cheap Cheap Cheap 1310 1315 1320 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1325 1330 1335 Yes Gly Pro Gly Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala 1340 1345 1350 No No No No No No No Gly Gly Tyr Gly Pro Gly Stone 1355 1360 1365 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1370 1375 1380 Yes Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly 1385 1390 1395 Free Free Gly Pro Gly Free Free Gly Pro 1400 1405 1410 Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 1415 1420 1425 Pro Being Wing Wing Wing Wing Wing Wing Wing Wing Wing Gly Gly Tyr 1430 1435 1440 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1445 1450 1455 No Gly Pro Gly No No No No Gly Pro Gly No no 1460 1465 1470 Too Much Gly Pro Gly Too Much Too Much Too Much Gly Pro Gly Too Much 1475 1480 1485 Yes Yes Gly Pro Gly Yes Yes Yes Gly Pro Gly Yes 1490 1495 1500 Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 1505 1510 1515 No No Gly Gly Tyr Gly Pro Gly Yes Yes Yes Yes Gly Pro 1520 1525 1530 Gly Yellow Yellow Gly Pro Gly Yellow Gly Yellow Gly 1535 1540 1545 Pro Gly Cheap Cheap Cheap Gly Pro Gly Cheap Cheap Cheap 1550 1555 1560 Gly Pro Gly Yellow Yellow Gly Pro Gly Yellow Yellow Yellow 1565 1570 1575 Yes Gly Pro Gly Yes Yes Yes Yes Gly Pro Ser Ala Ala Ala 1580 1585 1590 No No No No No No 1595 1600 <210> 35 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 35 Ser Gly Gly Gln Gln 1 5 <210> 36 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 36 Gly Ala Gly Gln Gln 1 5 <210> 37 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 37 Gly Gln Gly Pro Tyr 1 5 <210> 38 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 38 Ala Gly Gln Gln 1

Claims

1. A composition for a molded body comprising a recombinant spider silk protein and a plasticizer, The composition can be induced to a flowable state; The composition, wherein the recombinant spider silk protein is not substantially degraded in the flowable state.

2. 10. The composition of claim 1, which can be induced into said flowable state by the application of shear and pressure.

3. 3. The composition of claim 2, which can be induced to said flowable state by the application of shear and pressure without the application of heat.

4. 4. The composition of claim 3, wherein the composition can be induced to the flowable state and the recombinant spider silk protein remaining substantially undegraded within the composition can be extruded multiple times.

5. The composition of claim 1 which is thermoplastic.

6. 3. The composition of claim 2, which can be induced to a flowable state by the application of a shear force in the range of 1.5 Nm to 13 Nm.

7. 3. The composition of claim 2, which can be induced to a flowable state by the application of a shear force in the range of 2 Nm to 6 Nm.

8. 3. The composition of claim 2, which can be induced into a flowable state by application of a pressure in the range of 1 MPa to 300 MPa.

9. 3. The composition of claim 2, which can be induced to a flowable state by application of a pressure in the range of 5 MPa to 75 MPa.

10. The composition of any one of claims 6 to 9, which can be induced to a flowable state at temperatures below 120°C, below 80°C, below 40°C, or at room temperature.

11. 11. The composition of any one of claims 1 to 10, having a melt flow index of at least 0.5, at least 1, at least 2, or at least 5 when tested according to ASTM D1238 at 95°C under a 2.16 kg load.

12. 11. The composition of any one of claims 1 to 10, having a melt flow index of at least 0.5, at least 1, at least 2, or at least 5 when tested according to ASTM D1238 at 95°C under a load of 21.6 kg.

13. The composition of claim 1 which is substantially homogeneous.

14. 10. The composition of claim 1, wherein the recombinant spider silk protein comprises repeating units.

15. 2. The composition of claim 1, wherein the recombinant spider silk protein comprises an amino acid residue length in the range of 60 to 100 amino acids, with a range of 2 to 20 repeat units.

16. 2. The composition of claim 1, wherein the molecular weight of the recombinant spider silk protein is in the range of 20 to 2000 kDa.

17. The recombinant spider silk protein comprises at least two occurrences of a repeat unit, the repeat unit comprising: having more than 150 amino acid residues and a molecular weight of at least 10 kDa; an alanine-rich region having six or more consecutive amino acids with an alanine content of at least 80%; a glycine-rich region having 12 or more consecutive amino acids, the glycine content being at least 40% and the alanine content being less than 30%; The composition of claim 1 comprising:

18. The composition of claim 1 , wherein the plasticizer is selected from polyols, water, and / or urea.

19. The composition of claim 16 , wherein the polyol comprises glycerol.

20. The composition of claim 1 , wherein the plasticizer comprises water.

21. the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder; 10. The composition of claim 1, wherein the weight ratio of plasticizer to recombinant silk polypeptide powder ranges from 0.05:1 to 4:

1.

22. the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder; 10. The composition of claim 1, wherein the weight ratio of plasticizer to recombinant silk polypeptide powder ranges from 0.20:1 to 0.70:

1.

23. the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder; 10. The composition of claim 1, wherein the amount of recombinant spider silk polypeptide powder in the composition ranges from 1 to 90% by weight of recombinant spider silk protein.

24. the recombinant spider silk protein is present in a recombinant spider silk polypeptide powder; 10. The composition of claim 1, wherein the amount of recombinant spider silk polypeptide powder in the composition ranges from 20 to 41% by weight of recombinant spider silk protein.

25. The composition of claim 1, comprising glycerol as a plasticizer in the range of 1 to 90% by weight.

26. The composition of claim 1, comprising glycerol as a plasticizer in the range of 15 to 30% by weight.

27. The composition of claim 1, comprising water as a plasticizer in the range of 5 to 80% by weight.

28. The composition of claim 1, comprising water as a plasticizer in the range of 19 to 27% by weight.

29. 10. The composition of claim 1, wherein the recombinant spider silk protein is degraded in an amount of less than 10.0% by weight in the flowable state.

30. 10. The composition of claim 1, wherein the recombinant spider silk protein is degraded in an amount of less than 6.0% by weight in the flowable state.

31. 10. The composition of claim 1, wherein the recombinant spider silk protein is degraded in an amount of less than 2.0% by weight in the flowable state.

32. 32. The composition of any one of claims 29 to 31, wherein degradation of the recombinant spider silk protein is assessed by measuring the amount of full length recombinant spider silk protein present in the composition before and after the flowable condition is induced.

33. 33. The composition of claim 32, wherein the amount of full-length recombinant spider silk protein is measured using size exclusion chromatography.

34. A molded article comprising the composition according to any one of claims 1 to 33.

35. 35. The molded article of claim 34, wherein the molded article is a fiber.

36. The molded body according to claim 35, wherein the fibers have a strength in the range of 100 Pa to 1.2 GPa.

37. The fibers have a thickness of 5×10 as measured by polarized light microscopy. －５ The molded article according to claim 35, having a birefringence in the range of about 0.

04.

38. 1. A process for preparing a shaped body, comprising: (a) applying pressure and shear forces to a composition comprising a recombinant spider silk protein and a plasticizer to convert said composition into a flowable state; (b) extruding the composition in a flowable state to form a shaped body; The process comprising:

39. 40. The process of claim 38, wherein the step of extruding the composition to form a shaped body comprises extruding the composition to form fibers.

40. 40. The process of claim 39, wherein extruding the composition to form fibers comprises extruding the composition through a spinneret.

41. 40. The process of claim 38, wherein the step of extruding the composition to form a shaped body comprises extruding the composition into a mold.

42. (a) applying pressure and shear forces to the compact to convert the compact into a flowable composition; (b) extruding the composition in the flowable state to form a second compact; 39. The process of claim 38, further comprising:

43. 43. The process of claim 42, further comprising repeating steps (a) and (b) at least once on the second compact.

44. 44. The process of any one of claims 38 to 43, wherein the shear force is from 1.5 to 13 Nm.

45. 44. The process of any one of claims 38 to 43, wherein the pressure is from 1 MPa to 300 MPa.

46. 46. The process of any one of claims 38 to 45, wherein the shear force and pressure are applied to the composition using a capillary rheometer or a twin-screw extruder.

47. 47. The process of claim 46, wherein the screw speed of the twin screw extruder during the application of the pressure and the shear ranges from 10 to 300 RPM.

48. 46. The process of any one of claims 38 to 45, wherein the device used to apply the shear force and pressure comprises a mixing chamber coupled to and proximal to the extrusion chamber.

49. 49. The process of claim 48, wherein the composition is heated in the mixing chamber.

50. 49. The process of claim 48, wherein the composition is heated in the extrusion chamber.

51. 51. The process of claim 49 or claim 50, wherein the composition is heated to a temperature of less than 120°C.

52. 51. The process of claim 49 or claim 50, wherein the composition is heated to a temperature of less than 80°C.

53. 51. The process of claim 49 or claim 50, wherein the composition is heated to a temperature of less than 40°C.

54. 54. The process of any one of claims 38 to 53, wherein the extruded body has lost less than 15% water compared to the composition before extrusion.

55. 54. The process of any one of claims 38 to 53, wherein the extruded body has lost less than 10% water compared to the composition before extrusion.

56. 49. The process of claim 48, wherein the composition has a residence time in the mixing chamber in the range of 3 to 7 minutes.

57. 49. The process of claim 48, wherein the extrusion chamber tapers proximate to the orifice through which the composition is extruded.

58. 49. The process of claim 48, wherein the extrusion chamber is temperature controlled.

59. The process of any one of claims 48 to 58, wherein the shaped body is a fiber and the fiber is manually drawn.

60. 60. The process of any one of claims 48 to 59, wherein the shaped body is a fiber and the fiber is drawn in multiple steps.

61. 61. The process of any one of claims 48 to 60, wherein the recombinant spider silk protein is substantially not degraded in the shaped body.

62. 62. The process of claim 61, wherein the recombinant spider silk protein is degraded in an amount of less than 10% by weight in the shaped body.

63. 62. The process of claim 61, wherein the recombinant spider silk protein is degraded in an amount of less than 6% by weight in the shaped body.

64. 62. The process of claim 61, wherein the recombinant spider silk protein is degraded in an amount of less than 2% by weight in the shaped body.

65. 65. The process of any one of claims 61 to 64, wherein degradation of the recombinant spider silk protein is assessed by measuring the amount of full length recombinant spider silk protein present in the composition before and after extrusion.

66. 66. The process of claim 65, wherein size exclusion chromatography is used to measure the amount of full-length recombinant spider silk protein.

67. The process of any one of claims 38 to 66, wherein the molded body has minimal birefringence as measured by polarized light microscopy.

Images