Alkali purification method of spider silk protein

Abstract

To provide a method for purifying recombinant silk polypeptide sustainably and by an environment-friendly process.SOLUTION: A method for isolating a recombinant spider silk protein from a host cell culture solution includes: a) a step of obtaining a cell culture solution, in which the cell culture solution contains a host cell and a growth medium, and the host cell expresses a recombinant spider silk protein; b) a step of collecting a part of the cell culture solution containing the recombinant spider silk protein; c) a step of incubating a part of the cell culture solution in an aqueous solution under an alkaline condition, by which the recombinant spider silk protein is solubilized in the aqueous solution; and d) a step of isolating the recombinant spider silk protein from the aqueous solution, by which an isolated recombinant spider silk protein sample is produced.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Related Applications This application claims the benefit and priority of U.S. Provisional Application No. 62 / 772,588, filed Nov. 28, 2018, which is hereby incorporated by reference in its entirety.

[0002] Sequence Listing This application includes a sequence listing submitted via EFS-Web, which is hereby incorporated by reference herein in its entirety. The ASCII copy created in XX month of 20XX is named XXXXXUS_sequencelisting.txt and is X,XXX,XXX bytes in size.

Background Art

[0003] Background of the Invention Spider silk polypeptides are large (greater than 150 kDa, greater than 1000 amino acids) polypeptides that can be broken down into three domains: an N-terminal non-repetitive domain (NTD), a repetitive domain (REP), and a C-terminal non-repetitive domain (CTD). The NTD and CTD are relatively small (about 150 and about 100 amino acids, respectively) and are well studied and thought to confer water stability, pH sensitivity, and molecular alignment upon aggregation to the polypeptide. The NTD also has a strongly predicted secretion tag, which is often removed during heterologous expression. The repetitive region contains about 90% of the native polypeptide and folds into crystalline and amorphous regions, respectively, that impart strength and flexibility to the silk fiber.

[0004] Silk polypeptides are derived from a variety of sources, including bees, flies, spiders, mites, and other arthropods. Some organisms produce multiple silk fibers with unique sequences, structural components, and mechanical properties. For example, the orb-weaving spider has six unique glands that produce different silk polypeptide sequences that polymerize into fibers tailored to fit the environment or the ecological niche of its life cycle. The fibers are named after the gland from which they are derived, and the polypeptides are labeled with the gland abbreviation (e.g., "Ma") and "Sp" for spidroin (short for the spider fibroin). In the orb-weaver, these types include major ampullate (MaSp, also called dragline silk), minor ampullate (MiSp), flagelliform (Flag), aciniform (AcSp), tubuliform (TuSp), and pyriform (PySp). This combination of polypeptide sequences across fiber types, domains, and variations among different genera and species of organisms results in a vast array of potential properties that can be exploited by the commercial production of recombinant fibers. To date, the overwhelming majority of research on recombinant silk has focused on major ampullate (MaSp).

[0005] Currently, recombinant silk fibers are not commercially available, and with few exceptions, are not produced in microorganisms other than Escherichia coli and other Gram-negative prokaryotes. Most of the recombinant silk produced to date consists, in some cases, of either polymerized short silk sequence motifs or fragments of endogenous repetitive domains, optionally combined with NTD and / or CTD. This has led to the production of small-scale recombinant silk polypeptides (milligrams at the laboratory scale, kilograms at the bioprocess scale) using intracellular expression and purification by chromatography or bulk precipitation. These methods do not provide a commercially feasible scalability that can compete with the prices of existing technical and textile fibers. Additional production hosts that have been utilized to produce silk polypeptides include transgenic goats, transgenic silkworms, and plants. These hosts have not yet enabled commercial-scale production of silk, probably due to slow modification cycles and insufficient scalability.

[0006] Furthermore, recombinant silk polypeptides form undesirable insoluble aggregates during production and purification. Methods for redissolving the peptides during purification often degrade the protein, resulting in poor yields and fibers with low tensile strength and poor handfeel. Additionally, standard protein solubilization methods require the use of chaotropes such as urea, guanidine hydrochloride, or guanidine thiocyanate, which must be properly collected and disposed of after protein isolation. Therefore, there is a need for methods to purify these polypeptides in a sustainable and environmentally friendly process. SUMMARY OF THE INVENTION

[0007] In one aspect, provided herein is a method for isolating a recombinant spider silk protein from a host cell culture solution, the method comprising: obtaining a cell culture solution, wherein the cell culture solution comprises a host cell and a growth medium, and the host cell expresses a recombinant spider silk protein; collecting a portion of the cell culture solution containing the recombinant spider silk protein; incubating the portion of the cell culture solution in an aqueous solution under alkaline conditions, thereby solubilizing the recombinant spider silk protein in the aqueous solution; and isolating the recombinant spider silk protein from the aqueous solution, thereby producing an isolated recombinant spider silk protein sample.

[0008] In some embodiments, the alkaline conditions include an alkaline pH of 9 to 14. In one embodiment, the alkaline pH is 11 to 12.

[0009] In some embodiments, the isolated recombinant spider silk protein is a full-length recombinant spider silk protein. In one embodiment, the isolated recombinant spider silk protein sample comprises at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% full-length recombinant spider silk protein compared to the isolated total recombinant spider silk protein. In one embodiment, the percentage of full-length recombinant spider silk protein is measured using Western blot. In another embodiment, the percentage of full-length recombinant spider silk protein is measured using size exclusion chromatography.

[0010] In some embodiments, the purity of the isolated recombinant spider silk protein is 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%. In some embodiments, the yield of the isolated recombinant spider silk protein is at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100% compared to the recombinant spider silk isolated by the urea or guanidine thiocyanate method.

[0011] In some embodiments, the step of isolating the recombinant spider silk protein includes precipitating the recombinant spider silk protein by changing the alkaline conditions of the aqueous solution. In one embodiment, changing the alkaline conditions includes adjusting the alkaline pH of a portion of the cell culture to a lowered pH of 4-10. In one embodiment, the lowered pH is pH 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the lowered pH is pH 6-7.

[0012] In some embodiments, adjusting the alkaline pH includes adding an acid to the aqueous solution. In one embodiment, the acid is H2SO4.

[0013] In some embodiments, a portion of the cell culture includes the supernatant, whole cell broth, or cell pellet. In some embodiments, the step of collecting the portion of the cell culture includes removing the host cells from the growth medium and reconstituting the host cells in the aqueous solution.

[0014] In some embodiments, the step of collecting the portion of the cell culture includes lysing the host cells. In various embodiments, lysing includes heat treatment, shear disruption, physical homogenization, sonication, or chemical homogenization.

[0015] In some embodiments, the portion of the cell culture medium includes the host cells and the growth medium derived from the cell culture medium.

[0016] In various embodiments, the aqueous solution includes a diluted growth medium.

[0017] In some embodiments, the step of incubating the portion of the cell culture medium under alkaline conditions is carried out for 10 to 120 minutes. In some embodiments, the step of incubating the portion of the cell culture medium under alkaline conditions is carried out for at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 105 minutes, or at least 120 minutes. In some embodiments, the step of incubating the portion of the cell culture medium under alkaline conditions is carried out for 15 to 30 minutes.

[0018] In various embodiments, the step of incubating the portion of the cell culture medium under alkaline conditions further includes stirring a portion of the cell culture medium.

[0019] In various embodiments, the method further includes removing insoluble biomass from the aqueous solution under alkaline conditions. In some embodiments, removing the insoluble biomass includes filtration, centrifugation, gravitational sedimentation, adsorption, dialysis, or phase separation. In some embodiments, the filtration is ultrafiltration, microfiltration, or diafiltration. In some embodiments, removing the insoluble biomass is repeated at least once.

[0020] In various embodiments, the method further includes removing impurities before or after isolating the recombinant spider silk protein. In some embodiments, removing impurities includes filtration, centrifugation, gravitational sedimentation, adsorption, dialysis, or phase separation. In various embodiments, the filtration is ultrafiltration, microfiltration, or diafiltration. In some embodiments, the centrifugation is ultracentrifugation or diafiltration. In one embodiment, the adsorption is charcoal adsorption. In some embodiments, removing impurities is repeated at least once.

[0021] In various embodiments, the method further includes concentrating the isolated recombinant spider silk protein to produce a concentrated spider silk protein. In some embodiments, concentrating includes precipitation, filtration, ultrafiltration, centrifugation, dialysis, evaporation, or lyophilization.

[0022] In various embodiments, the method further includes drying the isolated recombinant spider silk protein.

[0023] In various embodiments, the method further includes generating silk fibers from the isolated recombinant spider silk. In one embodiment, the silk fibers include a tensile strength of at least 19 cN / tex.

[0024] In some embodiments, the recombinant spider silk protein is 18B or P0.

[0025] In some embodiments, the cell culture fluid includes fungal cells, bacterial cells, or yeast cells.

[0026] In some embodiments, the yeast cells are Pichia pastoris cells.

[0027] In another aspect, a method for isolating a recombinant spider silk protein, the method comprising: obtaining a cell culture solution, the cell culture solution comprising a host cell and a growth medium, the host cell expressing a recombinant spider silk protein; collecting a portion of the cell culture solution containing the recombinant spider silk protein; incubating the portion of the cell culture solution in an aqueous solution under alkaline conditions, thereby solubilizing the recombinant spider silk protein in the aqueous solution; adjusting the aqueous solution to a non-alkaline pH, thereby precipitating the solubilized recombinant spider silk protein; and isolating the recombinant spider silk protein from the portion of the cell culture solution, thereby producing an isolated recombinant spider silk protein, is provided herein.

[0028] In another aspect, a composition comprising a recombinant spider silk protein produced by any one of the disclosed methods is provided herein.

[0029] In some embodiments, the recombinant spider silk comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% full-length recombinant spider silk.

[0030] In another aspect, a silk fiber comprising a recombinant spider silk protein produced by any one of the disclosed methods is provided herein.

[0031] In some embodiments, the silk fiber comprises a tensile strength of at least 19 cN / tex.

[0032] In another aspect, a composition comprising a cell culture solution containing a growth medium and a host cell containing a recombinant spider silk protein in an alkaline buffer solution is provided herein.

[0033] In one embodiment, the alkaline buffer solution has a pH of 9 to 14. In another embodiment, the pH is 11 to 12.

[0034] In some embodiments, the spider silk protein is 18B or P0. In some embodiments, the cell culture medium contains fungal cells, bacterial cells, or yeast cells. In one embodiment, the bacterial cells are Escherichia coli (E. coli) cells. In one embodiment, the yeast cells are Pichia pastoris cells. [The present invention 1001] A method for isolating a recombinant spider silk protein from a host cell culture medium, the method comprising the following steps: a. Obtaining a cell culture medium, wherein the cell culture medium contains a host cell and a growth medium, and the host cell expresses a recombinant spider silk protein; b. Collecting a portion of the cell culture medium containing the recombinant spider silk protein; c. Incubating the portion of the cell culture medium in an aqueous solution under alkaline conditions, thereby solubilizing the recombinant spider silk protein in the aqueous solution; d. Isolating the recombinant spider silk protein from the aqueous solution, thereby producing an isolated recombinant spider silk protein sample. [The present invention 1002] The method of the present invention 1001, wherein the alkaline conditions include an alkaline pH of 9 to 14. [The present invention 1003] The method of the present invention 1002, wherein the alkaline pH is 11 to 12. [The present invention 1004] The method according to any one of the preceding inventions, wherein the isolated recombinant spider silk protein is a full-length recombinant spider silk protein. [The present invention 1005] The method of the present invention 1004, wherein the isolated recombinant spider silk protein sample comprises at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the full-length recombinant spider silk protein as compared to the isolated total recombinant spider silk protein. [The present invention 1006] The method of the present invention 1005, wherein the percentage of the full-length recombinant spider silk protein is measured using Western blot. [The present invention 1007] The method of the present invention 1005, wherein the percentage of the full-length recombinant spider silk protein is measured using size exclusion chromatography. [The present invention 1008] The method of any of the prior inventions, wherein the purity of the isolated recombinant spider silk protein is 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 09-95%, or 95-100%. [The present invention 1009] The method of any of the prior inventions, wherein the yield of the isolated recombinant spider silk protein is at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 09-95%, or 95-100% as compared to the recombinant spider silk isolated by the urea or guanidine thiocyanate method. [The present invention 1010] The method of any of the prior inventions, wherein the step of isolating the recombinant spider silk protein comprises precipitating the recombinant spider silk protein by changing the alkaline conditions of the aqueous solution. [The present invention 1011] The method of the present invention 1010, wherein changing the alkaline condition includes adjusting the alkaline pH of the part of the cell culture solution to a lowered pH of 4 to 10. [The present invention 1012] The method of the present invention 1011, wherein the lowered pH is pH 4, 5, 6, 7, 8, 9, or 10. [The present invention 1013] The method of the present invention 1011, wherein the lowered pH is pH 6 to 7. [The present invention 1014] The method according to any one of the present inventions 1010 to 1013, wherein adjusting the alkaline pH includes adding an acid to the aqueous solution. [The present invention 1015] The method of the present invention 1014, wherein the acid is H2SO4. [The present invention 1016] The method according to any one of the preceding present inventions, wherein the part of the cell culture solution includes a supernatant, a whole cell broth, or a cell pellet. [The present invention 1017] The method according to any one of the preceding present inventions, wherein the step of collecting the part of the cell culture solution includes removing the host cell from the growth medium and reconstituting the host cell in the aqueous solution. [The present invention 1018] The method according to any one of the preceding present inventions, wherein the step of collecting the part of the cell culture solution includes lysing the host cell. [The present invention 1019] The method of the present invention 1018, wherein lysing includes heat treatment, shear disruption, physical homogenization, sonication, or chemical homogenization. [The present invention 1020] The method according to any one of the preceding present inventions, wherein the part of the cell culture solution includes the host cell and the growth medium derived from the cell culture solution. [The present invention 1021] The method according to any one of the preceding present inventions, wherein the aqueous solution includes a diluted growth medium. [The present invention 1022] Any method of the prior invention, wherein the step of incubating a portion of the cell culture under alkaline conditions is carried out for 10 to 120 minutes. [Inventive Concept 1023] The method of Inventive Concept 1022, wherein the step of incubating a portion of the cell culture under alkaline conditions is carried out for at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 105 minutes, or at least 120 minutes. [Inventive Concept 1024] The method of Inventive Concept 1022, wherein the step of incubating a portion of the cell culture under alkaline conditions is carried out for 15 to 30 minutes. [Inventive Concept 1025] Any method of the prior invention, wherein the step of incubating a portion of the cell culture under alkaline conditions further comprises stirring the portion of the cell culture. [Inventive Concept 1026] Any method of the prior invention, further comprising removing insoluble biomass from the aqueous solution under alkaline conditions. [Inventive Concept 1027] The method of Inventive Concept 1026, wherein removing the insoluble biomass comprises filtration, centrifugation, gravitational sedimentation, adsorption, dialysis, or phase separation. [Inventive Concept 1028] The method of Inventive Concept 1027, wherein the filtration is ultrafiltration, microfiltration, or diafiltration. [Inventive Concept 1029] Any method of Inventive Concepts 1026 to 1028, wherein removing the insoluble biomass is repeated at least once. [Inventive Concept 1030] Any method of the prior invention, further comprising removing impurities before or after isolating the recombinant spider silk protein. [Inventive Concept 1031] The method of Inventive Concept 1030, wherein removing the impurities comprises filtration, centrifugation, gravitational sedimentation, adsorption, dialysis, or phase separation. [The present invention 1032] The method of the present invention 1031, wherein the filtration is ultrafiltration, microfiltration, or diafiltration. [The present invention 1033] The method of the present invention 1031, wherein the centrifugation is ultracentrifugation or diafiltration centrifugation. [The present invention 1034] The method of the present invention 1031, wherein the adsorption is carbon adsorption. [The present invention 1035] The method according to any one of the present inventions 1031 to 1034, wherein removing impurities is repeated at least once. [The present invention 1036] The method according to any one of the preceding present inventions, further comprising concentrating the isolated recombinant spider silk protein to produce a concentrated spider silk protein. [The present invention 1037] The method of the present invention 1036, wherein concentrating includes precipitation, filtration, ultrafiltration, centrifugation, dialysis, evaporation, or lyophilization. [The present invention 1038] The method according to any one of the preceding present inventions, further comprising drying the isolated recombinant spider silk protein. [The present invention 1039] The method according to any one of the preceding present inventions, further comprising producing silk fibers from the isolated recombinant spider silk. [The present invention 1040] The method of the present invention 1039, wherein the silk fiber has a tensile strength of at least 19 cN / tex. [The present invention 1041] The method according to any one of the preceding present inventions, wherein the recombinant spider silk protein is 18B or P0. [The present invention 1042] The method according to any one of the preceding present inventions, wherein the cell culture solution contains fungal cells, bacterial cells, or yeast cells. [The present invention 1043] The method according to any one of the preceding present inventions, wherein the yeast cells are Pichia pastoris cells. [The present invention 1044] A method for isolating a recombinant spider silk protein, the method comprising the following steps: a. A step of obtaining a cell culture solution, wherein the cell culture solution contains a host cell and a growth medium, and the host cell expresses a recombinant spider silk protein; b. A step of collecting a part of the cell culture solution containing the recombinant spider silk protein; c. A step of incubating the part of the cell culture solution in an aqueous solution under alkaline conditions, thereby solubilizing the recombinant spider silk protein in the aqueous solution; d. A step of adjusting the aqueous solution to a non-alkaline pH, thereby precipitating the solubilized recombinant spider silk protein; e. A step of isolating the recombinant spider silk protein from the part of the cell culture solution, thereby producing an isolated recombinant spider silk protein. [The present invention 1045] A composition comprising a recombinant spider silk protein produced by the method according to any one of the preceding inventions. [The present invention 1046] The composition of the present invention 1045, wherein the recombinant spider silk contains at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of full-length recombinant spider silk. [The present invention 1047] Silk fibers comprising a recombinant spider silk protein produced by the method according to any one of the present inventions 1001 to 1044. [The present invention 1048] The silk fibers of the present invention 1047, wherein the silk fibers have a tensile strength of at least 19 cN / tex. [The present invention 1049] A composition comprising a cell culture solution containing a growth medium and a host cell containing a recombinant spider silk protein in an alkaline buffer solution. [The present invention 1050] The composition according to any one of the present inventions 1045 to 1049, wherein the alkaline buffer solution has a pH of 9 to 14. [The present invention 1051] The composition of the present invention 1050, wherein the pH is 11 to 12. [The present invention 1052] The composition according to any one of the present inventions 1049 to 1051, wherein the spider silk protein is 18B or P0. [The present invention 1053] The composition according to any one of the present inventions 1049 to 1052, wherein the cell culture solution contains fungal cells, bacterial cells, or yeast cells. [The present invention 1054] The composition of the present invention 1053, wherein the bacterial cells are Escherichia coli (E. coli) cells. [The present invention 1055] The composition of the present invention 1053, wherein the yeast cells are Pichia pastoris cells.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0036] Definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms shall include pluralities and plural terms shall include singulars. Generally, the nomenclature used in connection with biochemistry, enzymology, molecular biology and cell biology, microbiology, genetics, and polypeptide and nucleic acid chemistry, and hybridization described herein, and the techniques thereof are well known and commonly employed in the art.

[0037] The methods and techniques of the present invention, unless otherwise indicated, are generally carried out according to conventional methods well known in the art and as described in various general and more specific references cited and discussed throughout this specification. For example, see: Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1989), Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates, (1992, and Supplements to 2002), Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1990), Taylor and Drickamer, Introduction to Glycobiology, Oxford Univ. Press, (2003), Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold, N.J., Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press, (1976), Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press, (1976), Essentials of Glycobiology, Cold Spring Harbor Laboratory Press, (1999).

[0038] All publications, patent applications, and other references mentioned in this specification are hereby incorporated by reference in their entirety.

[0039] The following terms shall be understood to have the following meanings unless otherwise indicated.

[0040] As used herein, the terms "fermenting" and "fermentation" describe culturing a host cell under conditions for producing a desired product, including but not limited to conditions under which the host cell grows.

[0041] As used herein, the term "fermentation broth" means an aqueous medium used to culture a host cell during fermentation.

[0042] As used herein, the term "inoculum" means an amount of host cells added to a fermentation broth to initiate fermentation.

[0043] As used herein, the term "clarifying" means a method of removing host cell biomass, such as whole cells, lysed cells, membranes, lipids, organelles, nuclei, non-spider silk proteins, or any other undesirable cell part or product, or any other undesirable part of a cell culture. Clarifying can also mean removing impurities from a partially purified or isolated spider silk composition. Impurities can include, but are not limited to, non-spider silk proteins, degraded spider silk proteins, large aggregates of proteins, chemicals used during the purification and isolation process, or any other undesirable substance.

[0044] As used herein, the term "purity" means the amount of an isolated full-length recombinant spider silk protein as a fraction of all isolated components, such as a partially or degraded, isolated recombinant spider silk protein, lipid, protein, membrane, or other molecule in a sample, such as an extracted sample.

[0045] As used herein, the term "yield" means the amount of full-length recombinant spider silk protein isolated from cell culture medium as compared to the amount of full-length silk protein or total silk protein in the control sample. The percentage can be referenced to the total amount of full-length spider silk protein in the cell lysate, crude alkaline extraction solution, partially purified or filtered alkaline extraction solution, purified solution subjected to the alkaline extraction method, or purified solution subjected to a control extraction method such as urea or GdSCN, as described herein.

[0046] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides that is 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 that contain non-natural nucleotide analogs, non-natural internucleoside linkages, or both. The nucleic acid may be in any morphological conformation. For example, the nucleic acid may be single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin-shaped, circular, or padlocked conformation.

[0047] Unless otherwise indicated, by way of example for any sequence described herein in the general form of "SEQ ID NO:", "a nucleic acid comprising SEQ ID NO:1" refers to a nucleic acid that has, at least in part, (i) the sequence set forth in SEQ ID NO:1 or (ii) a sequence complementary to the sequence set forth in SEQ ID NO:1. The alternative is determined by the context. For example, when the nucleic acid is used as a probe, the alternative is determined by the requirement that the probe must be complementary to the desired target.

[0048] "Isolated" RNA, DNA, or mixed polymer refers to that which is substantially separated from other cellular components that are naturally associated with the endogenous polynucleotide within its natural host cell, such as ribosomes, polymerases, and genomic sequences that are naturally associated.

[0049] The term "recombinant" refers to a biomolecule, e.g., a gene or polypeptide, that (1) has been removed from its natural occurring environment, (2) is not associated with all or a portion of the polynucleotide in which the gene is found in nature, (3) is operably linked to a polynucleotide that is not linked in nature, or (4) does not occur in nature. The term "recombinant" may be used with respect to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as polypeptides and / or mRNAs encoded by such nucleic acids.

[0050] As used herein, an endogenous nucleic acid sequence of a biological genome (or a polypeptide product encoded by such sequence) is considered "recombinant" herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence such that the expression of the endogenous nucleic acid sequence is altered. In this case, the heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, regardless of whether the heterologous sequence is endogenous (from the same host cell or its progeny) or exogenous (from a different host cell or its progeny). By way of example, a promoter sequence can replace the native promoter of a gene present in the genome of a host cell (e.g., by homologous recombination) such that the expression pattern of the gene is altered. Here, the gene would be considered "recombinant" as it is separated from at least some of the sequences of the sequences that are naturally adjacent to it. In one embodiment, the heterologous nucleic acid molecule is not endogenous to the organism. In a further embodiment, the heterologous nucleic acid molecule is a plasmid or molecule that has been integrated into the host chromosome by homologous or random integration.

[0051] A nucleic acid is also considered "recombinant" if it contains any modification that does not occur naturally with respect to the corresponding nucleic acid in the genome. For example, an endogenous coding sequence is considered "recombinant" if it contains an insertion, deletion, or point mutation that has been artificially introduced, for example, by human intervention. "Recombinant nucleic acids" also include nucleic acids integrated into non-homologous sites within the host cell chromosome and nucleic acid constructs that exist as episomes.

[0052] In a nucleic acid sequence, the term "sequence identity (%)" means a quantitative value of the alignment of residues in two sequences when aligned to maximize the degree of match. The length for comparing sequence identity is at least about 9 nucleotides, usually at least about 20 nucleotides, more generally at least about 24 nucleotides, generally at least about 28 nucleotides, more generally at least about 32 nucleotides, and preferably over a region of at least about 36 or more nucleotides. There are numerous different algorithms known in the art that can be used to measure nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs included in the Wisconsin Package version 10.0 (Genetics Computer Group (GCG), Madison, Wis). FASTA provides the alignment and sequence identity (%) of the most overlapping region between the query sequence and the search sequence. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated herein by reference in its entirety). For example, the sequence identity (%) between nucleic acid sequences can be determined using FASTA with its default parameters (string size is 6 and the factor for the score matrix is NOPAM), as provided by GCG version 6.1, which is incorporated herein by reference, or by using Gap with its default parameters.Alternatively, the sequences can be compared using the BLAST computer programs (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)), particularly blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0053] The terms "substantial homology" or "substantial similarity" when referring to a nucleic acid or fragment thereof indicate that the nucleotide sequence identity, as measured by any of the well-known sequence identity algorithms such as FASTA, BLAST or Gap as described above, is at least about 76%, 80%, 85%, preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98% or 99% when optimally aligned with another nucleic acid (or its complementary strand) using appropriate insertions or deletions of nucleotides.

[0054] Nucleic acids (also referred to as polynucleotides) can include RNA, cDNA, both the sense and antisense strands of genomic DNA, as well as the synthetic forms and mixed polymers described above. They may be chemically or biochemically modified, or contain non-natural or derivatized nucleotide bases, as will be readily understood by those skilled in the art. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridines, psoralens, etc.), chelating agents, alkylating agents, and modifications of the linkage (e.g., alpha anomeric form nucleic acids, etc.). Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a specified sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which the phosphodiester linkages in the molecular backbone are replaced with peptide linkages. Other modifications can include, for example, analogs that contain modifications such as cross-linking moieties or other structures in the ribose ring, such as those found in "locked" nucleic acids.

[0055] The term "mutated", when applied to a nucleic acid sequence, means that a nucleotide within the nucleic acid sequence has been inserted, deleted or changed as compared to a reference nucleic acid sequence. A single change (point mutation) may be made at a locus, or an insertion, deletion or change may be made at a single locus for multiple nucleotides. Further, one or more changes may be made at any number of loci within a nucleic acid sequence. The nucleic acid sequence may be mutated by any method known in the art, including "error-prone PCR" (a method of performing PCR under conditions of low DNA polymerase replication fidelity such that a high rate of point mutations is obtained over the full length of the PCR product, see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)), and "oligonucleotide-directed mutagenesis" (a method that allows for the generation of site-specific mutations in any cloned DNA segment of interest, see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)), but is not limited thereto.

[0056] As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid linked thereto. One type of vector is a "plasmid", which generally refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated, but also includes linear double-stranded molecules, such as those resulting from amplification by polymerase chain reaction (PCR), or those resulting from treatment of circular plasmids with restriction enzymes. Other vectors include cosmids, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another type of vector is a viral vector, in which additional DNA segments may be ligated within the viral genome (detailed below). Certain vectors are capable of self-replication in the host cell into which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors may integrate into the genome of the host cell when introduced into the host cell and are thus replicated with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0057] As used herein, the term "expression system" includes a vehicle or vector for expressing a gene in a host cell, as well as a vehicle or vector that results in stable integration of the gene into the host chromosome.

[0058] "Operatively linked" or "operably linked" expression control sequences refer to a linkage in which the expression control sequence is adjacent to the gene of interest and controls such gene of interest, as well as expression control sequences that act in trans or act remotely to control the gene of interest.

[0059] As used herein, the term "expression control sequence" refers to a polynucleotide sequence necessary to affect the expression of a coding sequence to which it is operably linked. An expression control sequence is a sequence that controls the transcription, post-transcriptional events and translation of a nucleic acid sequence. Expression control sequences include appropriate sequences for transcription initiation, transcription termination, promoters and enhancers; efficient RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance polypeptide stability; and, if necessary, sequences that enhance the secretion of polypeptides. The nature of such control sequences varies depending on the host organism, and in prokaryotes, such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence. The term "control sequence" is intended to include at least all components whose presence is essential for expression, and may also include additional components that are convenient if present, such as leader sequences and fusion partner sequences.

[0060] As used herein, the term "promoter" means a DNA region to which RNA polymerase binds to initiate gene transcription and the position in the 5' direction of the start position of mRNA transcription.

[0061] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such term is intended to refer not only to a particular target cell, but also to the progeny of such cell. Since such progeny may in fact be not identical to the parent cell due to mutations or environmental influences that may occur during passage, they are still included within the scope of the term "host cell" as used herein. A recombinant host cell may be an isolated cell or a cell line grown in culture, or a cell present in a living tissue or organism.

[0062] The term "polypeptide" encompasses both naturally occurring proteins and non-naturally occurring proteins, as well as fragments, variants, derivatives, and analogs thereof. A polypeptide may be a monomer or a polymer. Further, a polypeptide may contain a number of different domains, each having one or more distinct activities.

[0063] As used herein, the term "molecule" means any compound, including but not limited to small molecules, peptides, polypeptides, sugars, nucleotides, nucleic acids, polynucleotides, lipids, etc., and such compounds can be natural or synthetic.

[0064] As used herein, the term "block" or "repeating unit" means, optionally with moderate variation, a subsequence of the native silk polypeptide found in the native silk polypeptide and functioning as a basic repeating unit in the silk polypeptide sequence, which is larger than about 12 amino acids of the native silk polypeptide. A block may contain very short "motifs", but does not necessarily have to. As used herein, "motif" means an amino acid sequence of approximately 2 to 10 amino acids that appears in multiple blocks. For example, a motif may be composed of the amino acid sequences GGA, GPG, or AAAAA (SEQ ID NO: 41). The sequence of multiple blocks is a "block copolymer".

[0065] As used herein, the term "repetitive domain" means an array selected from a set of contiguous (except for known silk spacer elements and not disrupted by an essential non-repetitive domain) repetitive segments within a silk polypeptide. Native silk sequences generally contain one repetitive domain. In some embodiments of the invention, there is one repetitive domain per silk molecule. As used herein, "macrorepeat" is a naturally occurring repetitive amino acid sequence that includes two or more blocks. In one embodiment, the macrorepeat is repeated at least twice within a repetitive domain. In a further embodiment, two repeats are insufficient. As used herein, "quasi-repeat" is an amino acid sequence that includes two or more blocks such that the blocks are similar but not identical within the amino acid sequence.

[0066] As used herein, the term "repetitive sequence" or "R" means a repetitive amino acid sequence. In one embodiment, the repetitive sequence includes a macrorepeat or a fragment of a macrorepeat. In another embodiment, the repetitive sequence includes blocks. In a further embodiment, a single block is split across two repetitive sequences.

[0067] It should be noted that as used in this specification and the appended claims, the singular forms "a", "an", and "the" include the plural referents unless the context clearly dictates otherwise.

[0068] Any range disclosed herein includes both ends of the range. For example, a range of 2-5% includes 2% and 5%, and any number or fraction of a number therebetween, such as 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, and 4.75%.

[0069] Recombinant spider silk composition Several natural spider silks have been identified to date. The various mechanical properties of naturally spun spider silk are thought to be closely related to the molecular composition of the silk. See, for example, Garb, J.E., 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, pg. 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, pg. 131-85 (2011). For example: Grape-like (AcSp) silk fibers tend to be highly tough as a result of a combination of moderately high strength and moderately high extensibility. AcSp silk fibers are often characterized by a large block ( "aggregate repeat") size in which polycelling and GPX motifs are incorporated. Tubular (TuSp or cylindrical) silk fibers tend to be large in diameter and have moderate strength and high extensibility. TuSp silk fibers are characterized by their polycelling and polythreonine content, and short polyalanine sequences. Flagon-like (MaSp) silk fibers tend to have high strength and moderate extensibility. MaSp silk fibers are either of two subtypes, MaSp1 and MaSp2. MaSp1 silk fibers are generally less extensible than MaSp2 silk fibers and are characterized by polyalanine, GX, and GGX motifs. MaSp2 silk fibers are characterized by polyalanine, GGX, and GPX motifs. Vial-like (MiSp) silk fibers tend to have moderate strength and moderate extensibility. MiSp silk fibers are characterized by GGX, GA, and polyA motifs and often contain a spacer element consisting of about 100 amino acids. Flagelliform (Flag) silk fibers tend to have very high extensibility and moderate strength. Flag silk fibers are usually characterized by GPG, GGX, and short spacer motifs.

[0070] The properties of each silk fiber type may vary from species to species, and spiders with different lifestyles (e.g., sedentary web-building spiders and wandering hunting spiders) or more evolutionarily ancient spiders may produce silk that differs from the above description (for descriptions of spider diversity and classification, see Hormiga, G., and Griswold, C.E., Systematics, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, pg.487-512(2014), and Blackedge, T.A. et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. Natl. Acad. Sci. U.S.A., 106:13, pg. 5229-5234(2009)). However, it is possible to produce consistent silk-like fibers that reproduce the properties corresponding to natural silk fibers on a commercial scale using synthetic block copolymer polypeptides having sequence similarity and / or amino acid composition similarity to the repetitive domains of natural silk proteins.

[0071] Silk nucleotide and peptide sequences A list of putative silk sequences can be collected by, for example, searching for related terms such as "spidroin", "fibroin", "MaSp" in GenBank, and those sequences can be pooled together with additional sequences obtained by independent sequencing efforts. The sequences are then translated into amino acids to filter out duplicate entries 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 microbial expression in, for example, Pichia (Komagataella) pastoris or Escherichia coli. The DNA sequences are each cloned into an expression vector and transformed into a microorganism such as Pichia (Komagataella) pastoris or Escherichia coli. In some embodiments, the various silk domains that have shown successful expression and secretion are then assembled in a combinatorial fashion to construct silk molecules capable of fiber formation.

[0072] Silk polypeptides are characteristically composed of a repetitive domain (REP) flanked by non-repetitive regions (e.g., C-terminal domain and N-terminal domain). The repetitive domain exhibits a hierarchical structure. The repetitive domain contains a series of blocks (also called repeat units). The blocks are repeated, sometimes completely and sometimes incompletely (constituting a quasi-repetitive domain), throughout the silk repetitive domain. The length and composition of the blocks vary between different silk species and between different species. Table 1 is a list of examples of block sequences for selected species and silk species, and 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, pg 169-184 (2011), and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pg. 2603-2605 (2001). In some cases, the blocks may be arranged in a normal pattern to form larger macro-repeats that occur multiple times (usually 2-8 times) in the repetitive domain of the silk sequence. The blocks repeated within the repetitive domain or macro-repeat may be separated from the macro-repeat repeated within the repetitive domain by spacer elements. The block sequence may include a glycine-rich region followed by a poly-A region. Short (about 1-10) amino acid motifs may occur multiple times within the block. A subset of the commonly observed motifs is shown in FIG. 1. For the purposes of the present invention, blocks derived from different natural silk polypeptides can be selected regardless of circular permutation (i.e., the identified blocks may not be aligned for circular permutation if they are similar between silk polypeptides in other respects).Thus, for example, a "block" such as SGAGG (SEQ ID NO: 42) is identical for the purposes of the present invention to GSGAG (SEQ ID NO: 43) and also to GGSGA (SEQ ID NO: 44), and they are all merely circular permutations of each other. The particular permutation selected for a given silk sequence may be determined by convenience more than anything else (usually starting with G). Silk sequences obtained from the NCBI database can be divided into blocks and non-repetitive regions.

[0073] (Table 1) Samples of block sequences TIFF2025108573000002.tif137170TIFF2025108573000003.tif232170TIFF2025108573000004.tif241170TIFF2025108573000005.tif108170

[0074] Fiber-forming block copolymer polypeptides derived from blocks and / or macro-repeat domains according to certain embodiments of the present invention are described in International Publication No. WO / 2015 / 042164, which is incorporated by reference. Natural silk sequences obtained from protein databases such as GenBank, or natural silk sequences obtained by newly performed sequencing, are disrupted by domains (N-terminal domain, repetitive domain, and C-terminal domain). Examples of the sequences of the N-terminal domain and the C-terminal domain selected for assembly after synthesis to construct fibers include natural amino acid sequence information and other modifications described herein. The repetitive domain is decomposed into repetitive sequences, which contain representative blocks (usually 1 to 8, depending on the type of silk) for obtaining important amino acid information, while reducing the size of the DNA encoding the amino acids to fragments that can be easily synthesized. In some embodiments, a properly formed block copolymer polypeptide includes at least one repetitive domain including at least one repetitive sequence, and optionally, an N-terminal domain and / or a C-terminal domain are adjacent thereto.

[0075] In some embodiments, the repetitive domain comprises at least one repetitive sequence. In some embodiments, the repetitive sequence is 150 to 300 amino acid residues. In some embodiments, the repetitive sequence comprises a plurality of blocks. In some embodiments, the repetitive sequence comprises a plurality of macro repeats. In some embodiments, the blocks or macro repeats are split across the plurality of repetitive sequences.

[0076] In some embodiments, the repetitive sequence is initiated with glycine and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine (C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) in order to meet DNA assembly requirements. In some embodiments, some repetitive sequences can be varied compared to the native sequence. In some embodiments, the repetitive sequence can be varied, for example, by addition of 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 repetitive sequence can be modified by filling in incomplete blocks with homologous sequences from another block. In some embodiments, the repetitive sequence can be modified by rearranging the order of blocks or macro repeats.

[0077] In some embodiments, the non-repetitive N-terminal and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain can be due to removal of a leading signal sequence, such as that identified by SignalP (Peterson, T.N., et al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg. 785-786 (2011)).

[0078] In some embodiments, the N-terminal domain sequence, repeat sequence, or C-terminal domain sequence is Agelenopsis aperta, Aliatypus gulosus, Aphonopelma seemanni, Aptostichus sp. AS217, Aptostichus sp. AS220, Araneus diadematus, Araneus gemmoides, Araneus ventricosus, Argiope amoena, Argiope argentata, Argiope 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, Latrodectus hesperus, Megahexura fulva, Metepeira grandiosa, Nephila antipodiana, Nephila clavata, Nephila clavipesIt may be derived from Argiope aurantia, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

[0079] 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 other affinity tags such as 6-8 His residues (SEQ ID NO: 45).

[0080] Secretion signal The amount of protein secreted from cells varies widely from protein to protein and, in its initial state, is partially determined by the secretion signal operably linked to the protein. The number of secretion signals is known in the art, and some of them are commonly used for the production of secreted recombinant proteins, including microbial secretion signals from Pichia pastoris and Saccharomyces cerevisiae. Among these, the secretion signal of the α mating factor (αMF) of Saccharomyces cerevisiae, consisting of the N-terminal 19 amino acid signal peptide (also referred to herein as preαMF(sc)), followed by a 70 amino acid leader peptide (also referred to as proαMF(sc)), is prominent. Inclusion of proαMF(sc) in the secretion signal of αMF of Saccharomyces cerevisiae (also referred to herein as preαMF(sc) / proαMF(sc)) has been proven to be essential for achieving high secretion yields of proteins. Addition of proαMF(sc), or a functional variant thereof, to a signal peptide other than preαMF(sc) has been discovered as a means of achieving secretion of recombinant proteins, but varying degrees of effectiveness have been shown, with secretion increasing for certain recombinant proteins in certain recombinant host cells, but having no effect or decreasing secretion for other recombinant proteins.

[0081] By using multiple different secretion signals, the secretion yield of recombinant proteins produced within host cells such as P. pastoris can be improved, as described in U.S. Application No. 15 / 724,196. Recombinant host cells containing the same number of polynucleotide sequences encoding a recombinant protein operably linked to at least two different secretion signals produce a recombinant protein with a higher secretion yield compared to recombinant host cells containing polynucleotide sequences encoding a recombinant protein operably linked to only one secretion signal (e.g., preαMF(sc) / proαMF(sc)). Without being bound by theory, by using at least two different secretion signals, the recombinant host cell can participate in different cellular secretion pathways to achieve efficient secretion of the recombinant protein, and thus it may be possible to prevent oversaturation of any one secretion pathway.

[0082] At least one of the different secretion signals is a functional variant that includes a signal peptide selected from Table 2 or 3, or has at least 80% amino acid sequence identity to a signal peptide selected from Table 2 or 3. In some embodiments, the functional variant is a signal peptide selected from Table 2 or 3 that includes one or two substituted amino acids. In some such embodiments, the functional variant has at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to a signal peptide selected from Table 2 or 3. In some embodiments, the signal peptide mediates the translocation of the initial recombinant protein into the ER after translation (i.e., protein synthesis proceeds with the initial recombinant protein present in the cell cytosol prior to translocation into the ER). In other embodiments, the signal peptide mediates the translocation of the initial recombinant protein into the ER during translation (i.e., protein synthesis and translocation into the ER occur simultaneously). The advantage of using a signal peptide that mediates co-translational translocation into the ER is that the structure that prevents the translocation of the recombinant protein, which is prone to rapid folding, into the ER, and thus secretion, is disrupted.

[0083] (Table 2) Secretion signal TIFF2025108573000006.tif55167

[0084] (Table 3) Recombinant secretion signal TIFF2025108573000007.tif101165

[0085] Expression vector The expression vector of the present invention can be prepared according to the teachings of this specification in view of the techniques known in the art. Sequences, such as vector sequences or sequences encoding transgenes, can be obtained commercially from companies such as Integrated DNA Technologies, Coralville, IA or Atum, Menlo Park, CA. Expression vectors that direct high-level expression of chimeric silk polypeptides are exemplified herein.

[0086] Another standard source of the polynucleotides used in the present invention is polynucleotides isolated from organisms (e.g., bacteria), cells, or selected tissues. Nucleic acids from the selected source can be isolated by standard procedures, which typically include sequential phenol and phenol / chloroform extractions followed by ethanol precipitation. After precipitation, the polynucleotides can be treated with restriction endonucleases that cleave the nucleic acid molecules into fragments. Fragments of the selected size can be separated by a number of techniques, including agarose or polyacrylamide gel electrophoresis, or pulsed field gel electrophoresis (Care et al. (1984) Nuc. Acid Res. 12:5647-5664; Chu et al. (1986) Science 234:1582; Smith et al. (1987) Methods in Enzymology 151:461), to yield starting materials of the appropriate size for cloning.

[0087] Another method for obtaining the nucleotide components of an expression vector or construct is PCR. General procedures for PCR are taught in MacPherson et al., PCR: A PRACTICAL APPROACH, (IRL Press at Oxford University Press, (1991)). The PCR conditions for each amplification reaction may be determined empirically. A number of parameters affect the success of the reaction. In particular, these parameters include the annealing temperature and time, the extension time, the Mg2+ and ATP concentrations, the pH, and the relative concentrations of the primers, template, and deoxyribonucleotides. Exemplary primers are described below in the Examples. After amplification, the resulting fragments can be detected by agarose gel electrophoresis followed by visualization by ethidium bromide staining and ultraviolet irradiation.

[0088] Another method for obtaining a polynucleotide is by enzymatic degradation. For example, a nucleotide sequence can be generated by digestion of a suitable vector with a suitable recognition restriction enzyme. Restriction fragments can be blunt-ended by treatment with the large fragment of Escherichia coli DNA polymerase I (Klenow) in the presence of the four deoxynucleotide triphosphates (dNTPs) using standard techniques.

[0089] The polynucleotide is inserted into a suitable backbone, such as a plasmid, using techniques well known in the art. For example, under suitable conditions, the insert and vector DNA can be contacted with a restriction enzyme to create complementarity or blunt ends on each molecule capable of base pairing with each other and ligated with a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the polynucleotide. These synthetic linkers can contain nucleic acid sequences corresponding to specific restriction enzyme cleavage sites within the vector DNA. Other methods are known and available in the art. Various sources can be used for the polynucleotide that is a component.

[0090] In some embodiments, an expression vector containing an R, N, or C array is transformed into a host organism for expression and secretion. In some embodiments, the expression vector contains a secretion signal. In some embodiments, the expression vector contains a stop signal. In some embodiments, the expression vector is designed to integrate into the host cell genome and includes a homology region to the target genome, a promoter, a secretion signal, a tag (e.g., FLAG tag), a stop / polyA signal, a selection marker for Pichia, a selection marker for E. coli, an origin of replication for E. coli, and a restriction enzyme cleavage site for releasing the fragment of interest.

[0091] Host cell transformant Provided are nucleic acid molecules or vectors that express spider silk polypeptides, and host cells transformed with these progeny. These cells can also carry the nucleic acid sequences of the invention on the vector, and the vector does not necessarily have to be a free replication vector. In other embodiments of the invention, the nucleic acid is integrated into the genome of the host cell.

[0092] In some embodiments, the microorganisms or host cells that enable large-scale production of the block copolymer polypeptides of the invention include 1) the ability to produce large (>40 kDa) polypeptides, 2) the ability to secrete extracellular polypeptides and avoid high-cost downstream intracellular purification, 3) resistance to contaminants (such as viral and bacterial contaminants) on a large scale, and / or 4) the existing knowledge regarding the growth and processing of organisms being a large (1 - 2000 m3) bioreactor.

[0093] Various host organisms can be modified / transformed to include a block copolymer polypeptide expression system. Organisms preferred for the expression of recombinant silk polypeptides include yeast, fungi, Gram-negative bacteria, and Gram-positive bacteria. In certain embodiments, the host organism is Arxula adeninivorans, Aspergillus aculeatus, Aspergillus awamori, Aspergillus ficuum, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus tubigensis, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus anthracis, Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus methanolicus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Candida boydinii,boidinii), Chrysosporium lucknowense, Escherichia coli, Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis, Kluyveromyces marxianus, Myceliopthora thermophila, Neurospora crassa, Ogataea polymorpha, Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum, Penicillium emersonii, Penicillium funiculosum, Penicillium griseoroseum, Penicillium purpurogenum, Penicillium roqueforti, Phanerochaete chrysosporium, Pichia angusta, Pichia methanolica, Pichia (Komagataella) pastoris, Pichia polymorpha, Pichia stipitis, Rhizomucor miehei, Rhizomucor pusillus, Rhizopus arrhizus, Streptomyces lividans, Saccharomyces cerevisiae, Schwanniomyces occidentalis (Schwanniomycesis Aspergillus niger, Aspergillus oryzae, Trichoderma viride, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma gamsii, Trichoderma atroviride, Trichoderma occidentalis, Trichoderma harzianum, Trichoderma reesei, or Yarrowia lipolytica.

[0094] In a preferred embodiment, the method provides for culturing the host cell for direct product secretion for easy recovery without the need to extract the biomass. In some embodiments, the block copolymer polypeptide is secreted directly into the culture medium for collection and processing.

[0095] Modified host cell strain Any suitable host cell strain can be used to produce a recombinant protein. The methylotrophic yeast Pichia pastoris is widely used for the production of recombinant proteins. P. pastoris grows to high cell densities, provides tightly regulated methanol-inducible transgene expression, and efficiently secretes recombinant proteins into synthetic media. However, during the culturing of P. pastoris strains, the recombinantly expressed protein can be degraded before it can be collected, resulting in a mixture of proteins containing fragments of the recombinantly expressed protein and full-length recombinant protein with reduced yields. Another widely used cell strain for recombinant protein production is the bacterium Escherichia coli.

[0096] In some embodiments, the modified strains with reduced protease activity described herein recombinantly express a silk-like polypeptide sequence. In some embodiments, the silk-like polypeptide sequence is 1) a block copolymer polypeptide composition generated by mixing and matching repetitive domains derived from silk polypeptide sequences, and / or 2) a recombinantly expressed block copolymer polypeptide that is large enough (about 40 kDa) to form useful fibers by secretion from industrially expandable microorganisms. Block copolymer polypeptides that are large (about 40 kDa to about 100 kDa) and that have been genetically engineered with silk repetitive domain fragments, including sequences derived from almost all of the published amino acid sequences of spider silk polypeptides, can be expressed in the modified microorganisms described herein. In some embodiments, the silk polypeptide sequences are matched and designed to produce highly expressed and highly secreted polypeptides with fiber-forming ability. In some embodiments, the degradation of the silk-like polypeptide is suppressed by knockout of the protease gene or reduction of protease activity in the host modified strain.

[0097] In some embodiments, to attenuate protease activity in Pichia pastoris, the genes encoding these enzymes are inactivated or mutagenized to reduce or eliminate their activity. This can be done through mutations or insertions into the gene itself or through modification of gene regulatory elements. This is achievable by standard yeast genetic techniques. An example of such a technique is gene replacement by double homologous recombination, in which homologous regions flanking the gene to be inactivated are cloned into a vector flanking a selectable marker gene (such as an antibiotic resistance gene or a gene that complements the auxotrophy of the yeast strain).

[0098] Alternatively, the homologous region can be PCR amplified and ligated to a selectable marker gene by overlap PCR. Thereafter, such a DNA fragment is transformed into Pichia pastoris by a method known in the art, for example, electroporation. Thereafter, transformants grown under selective conditions are analyzed for gene disruption events by standard techniques, such as PCR of genomic DNA or Southern blotting. In an alternative experiment, gene inactivation can be achieved by single homologous recombination, in which case, for example, the 5' end of the ORF of the gene is cloned onto a promoterless vector containing a selectable marker gene as well. When such a vector is linearized by digestion with a restriction enzyme that cleaves the vector only with the target gene homologous fragment, such a vector is transformed into Pichia pastoris. Integration into the target gene locus is confirmed by PCR of genomic DNA or Southern blotting. In this method, replication of the gene fragment cloned on the vector is achieved within the genome, thereby resulting in two copies of the target locus, i.e., a first copy that expresses an inactive protein truncated (even if expressed) due to an incomplete ORF, and a second copy that has no promoter to induce transcription.

[0099] Alternatively, transposon mutagenesis is used to inactivate the target gene. A library of such mutants can be screened for insertion events within the target gene by PCR.

[0100] The functional phenotypes (i.e., deficiencies) of strains subjected to genetic manipulation / knockout can be evaluated using techniques known in the art. For example, deficiencies in protease activity of genetically engineered strains can be confirmed using any of a variety of methods known in the art, particularly hydrolysis activity assays of chromogenic protease substrates, band shifts of substrate proteins against selected proteases, etc.

[0101] The attenuation of protease activity described herein can be achieved by mechanisms other than knockout mutations. For example, by changing the nucleic acid sequence, placing the gene under the control of a less active promoter, downregulating, changing the amino acid sequence by expressing interfering RNA, ribozyme or antisense sequences targeting the target gene, or by any other technique known in the art. Furthermore, the desired protease can be attenuated. In preferred strains, the protease activities of the proteases encoded by PAS_chr4_0584 (YPS1-1) and PAS_chr3_1157 (YPS1-2) are attenuated by any of the above methods. In some embodiments, the present invention is directed to methylotrophic yeast strains, particularly Pichia pastoris strains, in which the YPS1-1 and YPS1-2 genes are inactivated. In some embodiments, additional genes encoding proteases may be knocked out according to the methods provided herein to further reduce the protease activity of the desired protein product expressed by the strain.

[0102] In some embodiments, the P. pastoris strains disclosed herein are modified to express silk-like polypeptides. The method for producing silk-like polypeptides in preferred embodiments is presented in WO2015 / 042164, which is incorporated herein by reference, particularly in paragraphs 114 to 134. WO2015 / 042164 discloses synthetic proteinaceous copolymers based on recombinant spider silk protein fragment sequences such as those derived from MaSp2 from the genus Nephila. Silk-like polypeptides containing 2 to 20 repeating units are described, and in the above polypeptides, the molecular weight of each repeating unit is greater than about 20 kDa. Within each repeating unit of the copolymer, there are more than about 60 amino acid residues organized into a number of "quasi-repeating units". In some embodiments, the sequence identity of the repeating units of the polypeptides described in this disclosure to the sequence of the MaSp2 dragline silk protein is at least 95%.

[0103] Methods for production and purification of recombinant proteins The method provided herein involves fermenting an inoculum of the recombinant host cells provided herein in a suitable fermentation broth and a suitable fermentation vessel under suitable fermentation conditions to produce a recombinant protein having a desired cumulative yield and / or cumulative titer and / or cumulative productivity.

[0104] In some embodiments, the recombinant host cell secretes a recombinant protein. In various embodiments, the recombinant host cell can be a prokaryote that does not secrete a recombinant protein. In certain embodiments, the recombinant host cell is Escherichia coli.

[0105] In various embodiments, the recombinant host cell can be a eukaryote that secretes a recombinant protein, or a prokaryote such as a gram-negative or gram-positive bacterium that secretes a recombinant protein. In some embodiments, the recombinant host cell is Pichia pastoris. In certain embodiments, the recombinant host cell is a Pichia pastoris strain having the activity of one or more proteases inactivated (e.g., by functional knockout). Further, the specific embodiments discussed below are applicable to the production of recombinant hydrophobic or partially hydrophobic proteins such as silk proteins.

[0106] U.S. Patent No. 9,963,554, "Methods and Compositions for Synthesizing Improved Silk Fibers", which is incorporated herein by reference, discloses compositions for synthetic block copolymers, recombinant microorganisms for their production, and synthetic fibers containing proteins. U.S. Patent Application No. 15 / 724,196, "Modified Strains for the Production of Recombinant Silk", which is incorporated herein by reference, discloses modified Pichia pastoris selected or genetically engineered to reduce the degradation of recombinant proteins expressed by yeast cells, and methods for culturing yeast cells for the production of useful compounds. Other suitable microbial strains, including Escherichia coli, can be cultured and used in the production of useful compounds.

[0107] Fermentation In some embodiments, the inoculum of the recombinant host cell can be derived from a seed stock (i.e., a series of fermentations that produce an increasing value of a sufficient number of recombinant host cells). Depending on the embodiment, the number of seeds can range from 2 to 7, 3 to 7, 3 to 6, or 3 to 5 types of seeds.

[0108] In some embodiments, the inoculum of the recombinant host cell has a dry cell weight % (DCW) of at least 0.2 g / L, at least 0.5 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 1 g / L, at least 2 g / L, at least 3 g / L, at least 4 g / L, or at least 5 g / L; 0.2 g / L to 3 g / L, 0.2 g / L to 2 g / L, or 0.2 / L to 1 g / L; 0.5 g / L to 3 g / L, 0.5 g / L to 2 g / L, or 0.5 / L to 1 g / L; 1 g / L to 3 g / L, 1 g / L to 2 g / L, or 0.5 / L to 1 g / L; or, 3 g / L to 1 g / L per liter of medium. The DCW can be measured using a biophotometer (e.g., Eppendorf Bio Photometer D30).

[0109] In most embodiments, the size of the inoculum will depend on the size of the fermentation vessel. In embodiments where the size of the fermentation vessel is less than 150 L, the DCW can range from 0.1 g / L to 0.5 g / L. In embodiments where the size of the fermentation vessel exceeds 150 L, the DCW can range from 2 to 4 g / L.

[0110] Depending on the specific embodiment, a suitable fermentation broth is any fermentation broth in which the recombinant host cell can survive (i.e., maintain growth and / or viability). Non-limiting examples of suitable fermentation broths include aqueous media containing nutrients necessary for the growth and / or viability of the recombinant host cell. Non-limiting examples of such nutrients include carbon sources, nitrogen sources, phosphate sources, salts, minerals, bases, acids, vitamins (e.g., biotin), amino acids, and metals (e.g., iron, zinc, calcium, copper, sodium, potassium, cobalt, magnesium, manganese).

[0111] In some embodiments, any of the above nutrients may be restricted to inhibit cell growth and improve the productivity, yield, or titer of the recombinant protein. The carbon source can be any carbon source fermentable by the recombinant host cell. Non-limiting examples of suitable carbon sources include monosaccharides, disaccharides, polysaccharides, acetate, ethanol, methanol, methane, and combinations thereof. Non-limiting examples of monosaccharides include dextrose (glucose), fructose, galactose, xylose, arabinose, and combinations thereof. Non-limiting examples of disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of polysaccharides include starch, glycogen, cellulose, and combinations thereof.

[0112] The nitrogen source can be any nitrogen source assimilable (i.e., metabolizable) by the recombinant host cell. Non-limiting examples of suitable nitrogen sources include anhydrous ammonia, ammonium sulfate, ammonium nitrate, diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, sodium nitrate, urea, peptone, protein hydrolysate, yeast extract, and any of the above enriched with air or oxygen.

[0113] In some embodiments, prior to addition to the fermentation broth, any or all of the nutrients can be sterilized using heat or ozone treatment to reduce or remove microbial contaminants. For example, the carbon source can be caramelized or sterilized using heat prior to addition to the fermentation broth. Similarly, the carbon source can be ozone-treated prior to addition to the fermentation broth. Suitable methods of ozone treatment are discussed in Dziugan et al., Ozonation as an effective way to stabilize new kinds of fermentation media used in biotechnological production of liquid fuel additives, Biotechnology for Biofuels, 9:150 (2016).

[0114] The fermentation broth can contain an acid or a base to adjust and / or maintain the pH. In some such embodiments, the pH is 4.0 - 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, or 4.5; 4.5 - 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, or 5.0; 5.0 - 8.0, 7.5, 7.0, 6.5, 6.0, or 5.5; 5.5 - 8.0, 7.5, 7.0, 6.5, or 6.0; 6.0 - 8.0, 7.5, 7.0, or 6.5; 6.5 - 8.0, 7.5, or 7.0; 7.0 - 8.0, or 7.5; or 7.5 - 8.0.

[0115] Non-limiting examples of suitable acids include aspartic acid, acetic acid, hydrochloric acid, and sulfuric acid. Non-limiting examples of suitable bases include sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, calcium carbonate, ammonia, and diammonium phosphate. In some embodiments, strong acids or strong bases are used to limit dilution of the fermentation broth.

[0116] In some embodiments, the fermentation broth contains such nutrients, or such an amount of such nutrients, such that a desired oxygen uptake rate (OUR) is achieved and / or maintained. In some such embodiments, the desired OUR is at least 40 mmol O2 / L / hour, at least 80 mmol O2 / L / hour, at least 100 mmol O2 / L / hour, at least 105 mmol O2 / L / hour, at least 110 mmol O2 / L / hour, at least 115 mmol O2 / L / hour, at least 120 mmol O2 / L / hour, or at least 140 mmol O2 / L / hour, at least 160 mmol O2 / L / hour, at least 180 mmol O2 / L / hour, at least 200 mmol O2 / L / hour, or at least 220 mmol O2 / L / hour; from 40 mmol O2 / L / hour to 220 mmol O2 / L / hour, from 60 mmol O2 / L / hour to 220 mmol O2 / L / hour, from 80 mmol O2 / L / hour to 220 mmol O2 / L / hour, or from 100 mmol O2 / L / hour to 220 mmol O2 / L / hour; from 100 mmol O2 / L / hour to 140 mmol O2 / L / hour, from 100 mmol O2 / L / hour to 135 mmol O2 / L / hour, from 100 mmol O2 / L / hour to 130 mmol O2 / L / hour, or from 100 mmol O2 / L / hour to 125 mmol O2 / L / hour; from 110 mmol O2 / L / hour to 125 mmol O2 / L / hour, or from 110 mmol O2 / L / hour to 120 mmol O2 / L / hour; or from 115 mmol O2 / L / hour to 120 mmol O2 / L / hour. The OUR can be calculated by one skilled in the art using the direct method described in Bioreaction Engineering Principles 3rd Edition, 2011, Spring Science + Business Media, p. 449.

[0117] In some embodiments, the fermentation broth contains such nutrients, or such an amount of such nutrients, such that the production of the recombinant protein by the recombinant host cell increases in relation to the production of by-products. Non-limiting examples of such by-products include ethanol. In some embodiments, in a 72-hour fermentation, the recombinant host cell produces ethanol at a cumulative yield of less than 0.1 g / L, less than 1 g / L, less than 5 g / L, less than 10 g / L, or less than 15 g / L; from 0.1 g / L to 15 g / L, from 1 g / L to 15 g / L, from 5 g / L to 15 g / L, from 10 g / L to 15 g / L, or from 0.5 g / L to 15 g / L; or from 0.1 g / L to 1.5 g / L, from 0.2 g / L to 1.5 g / L, from 0.5 g / L to 1.5 g / L, from 0.7 g / L to 1.5 g / L, or from 1.0 g / L to 1.5 g / L.

[0118] In some embodiments, the fermentation broth contains such nutrients, or such an amount of such nutrients, such that the desired dissolved oxygen (DO) content is reached and / or maintained. In some such embodiments, the desired DO content is at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 100%; or from 2% to 40%, from 2% to 5%, from 5% to 40%, from 2% to 20%, from 5% to 20%, from 2 to 15%, or from 5% to 15%.

[0119] In some embodiments, the fermentation broth contains such nutrients, or such an amount of such nutrients, such that the desired respiratory quotient (RQ; i.e., the ratio of carbon dioxide produced to oxygen consumed) is achieved and / or maintained. In some such embodiments, the desired RQ is less than 2, less than 1.75, less than 1.5, or less than 1.25; or from 1 to 1.1, from 1 to 1.2, from 1 to 1.3, from 1 to 1.4, or from 1 to 1.5.

[0120] In some embodiments, the fermentation broth contains such nutrients, or such an amount of such nutrients, such that the desired doubling time of the recombinant host cell is reached and / or maintained. In some such embodiments, the desired doubling time is at least 4 hours, 8 hours, 12 hours, 16 hours, 18 hours, 22 hours, 26 hours, 30 hours, 34 hours, or 36 hours; or 4 hours to 12 hours, 4 hours to 10 hours, 4 hours to 8 hours, 6 hours to 12 hours, 6 hours to 10 hours, or 6 hours to 8 hours.

[0121] In some embodiments, the fermentation broth contains one or more additional proteins. Adding such additional proteins can serve to disrupt protease activity from the recombinant protein produced by the recombinant host cell in embodiments where the recombinant host cell secretes the recombinant protein. Non-limiting examples of additional proteins include bovine serum albumin (BSA) and casamino acids. Other additional proteins are well known in the art.

[0122] The nutrients can be added to the fermentation broth either in a single bolus, incrementally, or continuously. In embodiments where the nutrients are added continuously, the nutrients can be added at a rapid, slow, or exponential rate.

[0123] In embodiments where the nutrients are added continuously to the fermentation broth, the nutrients can be added by continuous addition of a medium containing the nutrients. In these embodiments, an equal volume of aqueous medium in the fermentation broth can be removed from the fermentation to maintain the total volume of the fermentation broth the same. In some embodiments, the recombinant host cell can be removed from the fermentation broth and re-added to the medium containing the nutrients prior to addition to the fermentation broth.

[0124] A suitable fermentation vessel is any fermentation vessel in which the recombinant host cell can survive (grow and / or maintain viability). Non-limiting examples of suitable fermentation vessels include culture plates, vials, flasks, or fermenters. Non-limiting examples of suitable fermenters include stirred tank fermenters, air-lift fermenters, bubble column reactors, fixed bed bioreactors, and any combination thereof.

[0125] Suitable fermentation conditions are any conditions under which the recombinant host cell can survive (grow and / or maintain viability). Non-limiting examples of such fermentation conditions include a suitable volume of fermentation broth, a suitable pH of the fermentation broth, a suitable DO in the fermentation broth, a suitable temperature, a suitable oxygen addition, a suitable agitation of the recombinant host cell, and a suitable duration of fermentation.

[0126] In various embodiments, the suitable temperature can be any temperature suitable for the growth and / or viability of the recombinant host cell and / or for the production of the recombinant protein. In some embodiments, the temperature is at least 15°C, 20°C, 25°C, 30°C, 35°C; 15°C to 35°C, 15°C to 25°C, 15°C to 20°C, 20°C to 35°C, 20°C to 30°C, 20°C to 25°C, 25°C to 35°C, or 25°C to 30°C.

[0127] Suitable oxygen addition can be any oxygen addition suitable for the growth and / or viability of the recombinant host cell and / or for the production of the recombinant host cell. Such oxygen addition can be achieved by providing suitable aeration and / or suitable agitation of the fermentation vessel and / or the fermentation broth. In some embodiments, the suitable aeration is at least 1.5 vvm, at least 1.6 vvm, at least 1.7 vvm, at least 1.8 vvm, at least 1.9 vvm, or at least 2 vvm; 1.5 vvm to 2 vvm, 1.5 vvm to 1.9 vvm, 1.5 vvm to 1.8 vvm, 1.5 vvm to 1.7 vvm, 1.5 vvm to 1.6 vvm, 1.6 vm to 2 vvm, 1.7 vvm to 2 vvm, 1.8 vvm to 2 vvm, or 1.7 vvm to 1.9 vvm.

[0128] Depending on the embodiment and the type of fermentation, suitable agitation of the recombinant host cells in the fermentation broth can vary.

[0129] Depending on the embodiment, a bubble column may be used for aeration. The bubble column can vary in complexity based on a particular embodiment (e.g., it may be single-phase or multi-layered) and can impart various gas velocities. Non-limiting examples of suitable gas velocities include, but are not limited to, 0.003 - 0.08 m / s. Non-limiting examples of bubble reactors are included in Kantarci et al., Bubble Column Reactors, Process Biochemistry 40:2263-2283 (2005).

[0130] In some embodiments, the fermentation broth contains an agent (an "antifoaming agent") that reduces foaming during fermentation. As defined herein, foam is the dispersion of gas in a continuous liquid phase located within the fermentation vessel or near its upper part. Depending on the embodiment, the antifoaming agent may be selected and optimized to reduce its interaction with any recombinant protein product. Non-limiting examples of antifoaming agents include silicon-based oils, emulsions, and polymers; polypropylene glycol; polyethylene glycol-based antifoaming agents; polyalkylene glycol-based antifoaming agents; bifunctional ethylene / propylene oxide (EO / PO) block copolymers; fatty acid-based antifoaming agents; polyester-based antifoaming agents; oil-based antifoaming agents, and any combination of the foregoing. Suitable antifoaming agents are discussed in Junker, Foam and its Mitigation in Fermentation Systems, Biotechnol.Prog., 23:767-784 (2007). In embodiments where the recombinant protein is a hydrophobic protein such as silk protein, the antifoaming agent may be selected such that it solubilizes or does not solubilize the hydrophobic protein.

[0131] The desired cumulative yield of the recombinant protein can be any cumulative yield that contributes to low production costs. As used herein, the cumulative yield is calculated as the ratio of the mass of the produced recombinant protein to the mass of the carbon source catabolized by the recombinant host cell during the fermentation process (i.e., the mass of the provided carbon source - the mass of the carbon source remaining in the fermentation broth; for example, if 100 grams of glucose is provided to the recombinant host cell, and at the end of fermentation, 25 grams of recombinant protein is produced and 10 grams of glucose remains, the cumulative yield of the recombinant protein is 27.7%). Assuming that all other reference values are equal, a higher cumulative yield results in a lower production cost than a lower cumulative yield. In some embodiments, the cumulative yield of the recombinant silk protein in the carbon source after 72 hours of fermentation is at least 1%, at least 5%, at least 30%, or at least 100%; 1% - 5%, 5% - 10%, 10% - 35%, 35% - 50%, or 50% - 100%.

[0132] The desired cumulative titer of the recombinant protein can be any cumulative titer that contributes to low production costs. As used herein, the cumulative titer is calculated as the grams of the produced recombinant protein per titer of the fermentation broth (i.e., g / L) during the fermentation process. Assuming that all other reference values are equal, a higher cumulative titer results in a lower production cost than a lower cumulative titer. In some embodiments, the cumulative titer of the recombinant protein after 72 hours of fermentation is at least 2 g / L, at least 5 g / L, at least 15 g / L, or at least 30 g / L; 1 g / L - 100 g / L, 5 g / L, 15 g / L, or 30 g / L; 10 g / L - 100 g / L, 80 g / L, or 75 g / L; or 5 g / L - 30 g / L.

[0133] The desired cumulative productivity of the recombinant protein can be any cumulative productivity that contributes to low production costs. As used herein, cumulative productivity is calculated as grams of recombinant protein produced per liter of fermentation broth per hour (i.e., g / L / hour) during the fermentation process. Assuming that all other reference values are equal, higher cumulative productivity results in lower production costs than lower cumulative productivity. In some embodiments, the cumulative productivity of the recombinant protein is at least 0.001 g / L / hour, at least 0.025 g / L / hour, at least 0.05 g / L / hour, at least 0.1 g / L / hour, or at least 0.2 g / L / hour; from 0.001 g / L / hour to 0.5 g / L / hour.

[0134] The methods provided herein can be implemented at any fermentation scale and / or according to any fermentation procedure known in the art. The fermentation procedure can be fed-batch, batch, continuous, or any combination thereof. In some embodiments, the method begins with one or more batch fermentations followed by one or more continuous fermentations, and the inoculum of the recombinant host cell, suitable fermentation broth, suitable fermentation vessel, and / or suitable fermentation conditions can vary between one or more batch fermentations and / or one or more continuous fermentations. In some embodiments, the temperature of the batch fermentation is higher than the temperature of the continuous fermentation. In some such embodiments, the temperature of the batch fermentation is higher than 27°C and the temperature of the continuous fermentation is less than 27°C.

[0135] In some embodiments, the fermentation proceeds stepwise. Such steps can include a growth step, a production step, and / or a recovery step. In some embodiments, the steps differ from each other in the inoculum of the recombinant host cell, suitable fermentation broth, suitable fermentation vessel, and / or one or more suitable fermentation conditions.

[0136] Method for isolating recombinant protein Depending on the embodiment, various methods can be used to isolate and recover the target recombinant protein. As described above, some but not all of these methods are specific to recombinant host cells that secrete the target recombinant protein. Further, some of these methods are specific to the target protein that is hydrophobic.

[0137] Figure 1 shows a process flow for isolating a recombinant protein according to an embodiment of the present invention. Those skilled in the art will understand that some of the steps shown in Figure 1 can be performed in a different order and / or repeatedly. The disclosed embodiments are not intended to limit the scope of the methods provided herein, and the skilled person will recognize that the method can vary based on the recombinant host cell used, the desired cumulative yield, cumulative titer, and / or cumulative productivity, or other factors.

[0138] In any step A02, the biomass (i.e., intact or disrupted recombinant host cells and cell debris) is removed from the fermentation broth containing the recombinant host cells. In various embodiments, removing the biomass can also include removing insoluble fermentation impurities (e.g., antifoaming agents and other components of the fermentation broth that can precipitate during protein solubilization).

[0139] In various embodiments, biomass can be removed according to size, weight, density, or combinations thereof. Removing biomass based on size can be achieved, for example, by filtration using a filter press, a candle stick filter, or other filtration systems used in the industry with a molecular weight cut-off smaller than the size of the recombinant host cells. Removing biomass based on weight or density can be achieved, for example, by gravity sedimentation or centrifugation using a settler, a low g-force decanter centrifuge, a disk stack separator, a two-phase nozzle centrifuge, a solid discharge centrifuge, or a liquid cyclone. By removing biomass as disclosed herein, a centrifugate (i.e., the light phase or clear cell broth) containing protein and a solid (heavy phase) containing biomass and insoluble fermentation impurities are obtained. Suitable conditions for removing biomass (e.g., g-force, sedimentation time, centrifugation time, the proportion of solids at the centrifuge input, the feed rate of the centrifuge) can be determined using methods known in the art that are interlinked to minimize biomass and insoluble fermentation impurities in the clear cell broth. In some embodiments, removing biomass results in a clear cell broth having a wet packed solid volume of less than 5%, less than 1%, less than 0.5%, or less than 0.1%. In some embodiments, removing biomass results in a clear cell broth containing protein at a concentration of 1 g / L to 50 g / L. In some embodiments, the clear cell broth is subjected to polishing centrifugation to remove the remaining solids. In some embodiments, the solid obtained by removing biomass is subjected to at least one more round of protein solubilization and biomass removal, and all centrifugates are ultimately combined for further processing according to the methods provided herein.

[0140] Depending on the embodiment, step A02 may be carried out before and / or after step A04. Step A02 may be carried out several times. For example, several rounds of centrifugation and / or filtration may be carried out to remove the biomass before and / or after step A04.

[0141] After step A04, the recombinant protein is solubilized. In some embodiments where step A02 is not carried out, the recombinant host cell and the recombinant protein associated with the recombinant host cell may be centrifuged into a pellet of biomass (hereinafter "cell pellet"), and the supernatant discarded, to isolate the recombinant protein together with the recombinant host cell prior to solubilization. This step may be beneficial where the recombinant protein is insoluble and / or where the recombinant protein aggregates by itself and / or with the recombinant host cell and / or adheres to the surface of the recombinant host cell. In other embodiments, the recombinant protein is solubilized in the whole cell broth. In some embodiments, the recombinant protein is solubilized in the clear cell broth produced by carrying out step A02.

[0142] In some embodiments, solubilization of the recombinant protein can be achieved by adding a solubilizing agent to the whole cell broth, the clarified cell broth, or the cell pellet. Non-limiting examples of suitable solubilizing agents include surfactants, hydrotropes, SDS, urea, cysteine, guanidine thiocyanate, enzymes that hydrolyze polysaccharides (e.g., glucanase, lichenase, mannanase, chitinase), high pH water (H2O with a pH of 11 - 12), or other known chaotropes. Different solubilizing agents may be selected for different types of recombinant proteins. Suitable conditions for solubilizing the protein (e.g., type and amount of extractant, temperature, incubation time, agitation, pH) can be determined using methods known in the art that are coordinated to maximize the yield of the recombinant protein and minimize lysis of the recombinant host cell and solubilization of impurities. As described above, in certain embodiments where the recombinant protein is insoluble and / or the recombinant protein aggregates with itself and / or within or near the recombinant host cell, the recombinant host cell can be centrifuged and the supernatant can be discarded before adding the solubilizing agent to the pellet.

[0143] In some embodiments, various techniques may be used to perforate or permeabilize the membrane of the recombinant host cell in order to remove excess protein from the membrane before solubilization and / or precipitation. Such methods include chemical disruption, mechanical disruption, or sonication. Mechanical disruption of the cell membrane includes homogenization, shear force, freeze / thaw, heating, pressure, sonication, and filtration. Chemical disruption includes detergents such as Triton, sodium dodecyl sulfate; or chaotropic agents such as urea and guanidine. Other methods are well known in the art.

[0144] In certain embodiments, urea is used as a solubilizing agent to solubilize the recombinant protein and prevent disruption of the recombinant host cell. The concentration of urea can be varied to prevent disruption of the recombinant host cell. Depending on the embodiment, the amount of urea concentration can vary from 4M to 10M. In various embodiments, the recombinant host cell and the recombinant protein can be incubated with urea for 1 to 2 hours, 1 to 3 hours, or 1 to 4 hours. Depending on the embodiment, other known chaotropes such as guanidine thiocyanate are used to solubilize the recombinant protein.

[0145] In certain embodiments, high pH H2O or an aqueous buffer is used to solubilize the recombinant protein and prevent disruption of the recombinant host cell. The pH of the high pH H2O or aqueous buffer can be varied to prevent disruption of the recombinant host cell. Depending on the embodiment, the pH of the high pH H2O can range from pH 10 to pH 12.5, pH 10.5 to pH 12.5, pH 11 to pH 12.5, pH 11.5 to pH 12.5, pH 12 to pH 12.5, pH 10 to pH 12, pH 10.5 to pH 11.0, pH 10.5 to pH 11.5, pH 10.5 to pH 12, pH 10.5 to pH 12.5, pH 11 to pH 11.5, pH 11 to pH 12, pH 11.5 to pH 12.5, or pH 12 to pH 12.5. In various embodiments, the recombinant host cell and the recombinant protein can be incubated with high pH H2O for at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 115 minutes, or at least 120 minutes.

[0146] In certain embodiments, homogenization is used to lyse host cells. The homogenization pressure (psi) can be 5,000 to 100,000 psi, 5,000 to 10,000 psi, 10,000 to 20,000 psi, 20,000 to 30,000 psi, 30,000 to 40,000 psi, 40,000 to 50,000 psi, 50,000 to 60,000 psi, 60,000 to 70,000 psi, 70,000 to 80,000 psi, 80,000 to 90,000 psi, 90,000 to 100,000 psi. The homogenization can be a single process or multiple processes. In some embodiments, the homogenization is a single process, a two-process, a three-process, a four-process, or a five-process.

[0147] After step A06, impurities are removed from the fermentation broth. Step A06 may be performed before and / or after step A04 and / or step A08. Step A06 may be repeated any number of times. Removal of impurities from the fermentation broth can be achieved by filtration, absorption (e.g., charcoal or solid state absorption), dialysis, and phase separation induced by coacervation or by using various chemicals. In embodiments where the phase separation is induced by coacervation, the coacervation can be induced by cooling the fermentation broth to a temperature sufficient to induce phase separation. In other embodiments, the phase separation can be chemically induced by adding cosmotrops and / or compounds used to precipitate proteins from solution. Detailed embodiments of impurity removal using phase separation are described below with respect to Figure C. In some embodiments where the recombinant protein is thermostable, the fermentation broth can be subjected to high temperature to denature other proteins and then centrifuged to separate the denatured proteins from the proteins in solution, thereby removing the other proteins.

[0148] In some embodiments, impurities are removed using filtration (e.g., for deionized water), microfiltration, diafiltration, and / or ultrafiltration. Membranes suitable for microfiltration may include from 0.1 uM to 1 uM. Non-limiting examples of membranes suitable for ultrafiltration include hydrophobic membranes (e.g., PES, PS, cellulose acetate) having a molecular weight cut-off of 50 kDa to 800 kDa, 100 kDa to 800 kDa, 200 kDa to 800 kDa, 300 kDa to 800 kDa, 400 kDa to 800 kDa, 500 kDa to 800 kDa, 600 kDa to 800 kDa, 700 kDa to 800 kDa, 100 kDa to 700 kDa, 200 kDa to 700 kDa, 300 kDa to 700 kDa, 400 kDa to 700 kDa, 500 kDa to 700 kDa, 600 kDa to 700 kDa, or 500 kDa to 600 kDa. In some embodiments, ultrafiltration provides a retentate containing a recombinant protein slurry in water and a permeate containing impurities as concentrated water. Suitable conditions for ultrafiltration (e.g., membrane, temperature, volume exchange) can be determined using methods known in the art that are coordinated to maximize the osmotic density. In some embodiments, ultrafiltration provides a retentate having a density of from 1 g / mL to 30 g / mL. In some embodiments, ultrafiltration includes a concentration step to obtain a concentrated retentate and a subsequent diafiltration step to remove impurities and obtain a protein slurry suspended in water. In some such embodiments, the concentrated retentate has a concentration factor of a 2-fold to 12-fold volume reduction relative to the starting volume. In some embodiments, diafiltration provides a constant volume exchange of from 3-fold to 10-fold.

[0149] Depending on the embodiment and the type of impurities to be removed, the method for removing impurities can vary. Removing lipid impurities from an isolated recombinant protein can be achieved by methods known in the art. Non-limiting examples of such methods include absorption onto charcoal or other absorption media that specifically bind to lipids. Removing polysaccharide impurities from an isolated recombinant protein can be achieved by methods known in the art. Non-limiting examples of such methods include removing small saccharides produced by ultrafiltration after treatment with an enzyme that hydrolyzes polysaccharides. Non-limiting examples of such enzymes include glucanase, licase, mannanase, and chitinase.

[0150] In step A08, the solubilized recombinant protein is isolated. The solubilized recombinant protein can be isolated in a number of different ways, including using extraction buffer, size exclusion chromatography, gel filtration, ultrasonic protein extraction, and ion exchange chromatography. In some embodiments where the biomass is not removed in any of the steps A02, the recombinant protein can be isolated together with the recombinant host cell.

[0151] In some embodiments, the recombinant protein is precipitated as a single isolation step or in addition to other isolation steps. Precipitating the solubilized recombinant protein can be achieved by adding a precipitating agent to the fermentation broth. Non-limiting examples of such precipitating agents include sulfate ions (e.g., ammonium sulfate, sodium sulfate, sulfuric acid), or citrate ions (e.g., sodium citrate). In some embodiments, the precipitating agent is an acid. In some embodiments, the precipitating agent is a salt. In one embodiment, the precipitating agent is H2SO4.

[0152] Any suitable acid can be used to adjust or change the pH of a solution containing solubilized recombinant protein. Suitable acids include mineral acids such as hydrochloric acid (HCl), sulfuric acid (H2SO4), nitric acid (HNo3), boric acid (H3BO3), phosphoric acid (H3PO4), hydrofluoric acid (HF), hydrobromic acid (HBr), perchloric acid (HClO4), hydroiodic acid (HI); citric acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, oxalic acid, lactic acid, malic acid, benzoic acid, carbonic acid, uric acid, taurine, p-toluenesulfonic acid, trifluoromethanesulfonic acid, aminomethylphosphonic acid, and trichloroacetic acid (TCA); or any combination thereof, or other suitable acids known in the art. Acidic salts of any of the acids disclosed above can also be used.

[0153] In some embodiments, the recombinant protein precipitates at pH 4 to 10. In some embodiments, the precipitation occurs at pH 4, 5, 6, 7, 8, 9, or 10. In some embodiments, the precipitation occurs at at least pH 4, at least pH 4.5, at least pH 5, at least pH 5.5, at least pH 6, at least pH 6.5, at least pH 7, at least pH 7.5, at least pH 8, at least pH 8.5, at least pH 9, at least pH 9.5, at least pH 10. In one embodiment, the precipitation occurs at pH 7. In some embodiments, the precipitation is at pH 4 to 5, pH 5 to 6, pH 6 to 7, pH 7 to 8, pH 8 to 9, or pH 9 to 10.

[0154] The precipitation can be repeated once, twice, or as many times as necessary. In some embodiments, more than two precipitation steps are performed and the pH of each precipitation is the same. In other embodiments, more than two precipitation steps are performed and the pH of each precipitation is different. For example, the first precipitation can be performed at pH 4 and the second precipitation can be performed at pH 7.

[0155] Isolating the precipitated recombinant protein can be achieved based on size, weight, density, or combinations thereof, as disclosed herein. In some embodiments, such isolation results in a concentrated solution and a permeate containing the suspended recombinant protein slurry and waste. Suitable conditions for precipitating the recombinant protein (e.g., dilution prior to addition of the divalent anion, type and amount of the divalent anion, incubation temperature, incubation time), and suitable conditions for isolating the precipitated recombinant protein can be determined using methods known in the art that are coordinated to maximize the yield of the recombinant protein in the suspended recombinant protein slurry. In some embodiments, the yield of the precipitated recombinant protein in the suspended silk protein slurry is 20% - 99%. In some embodiments, the suspended silk protein slurry has a wet fill solid content of 30% - 65%. In some embodiments, the suspended silk protein slurry contains silk protein at a concentration of 10 g / L - 50 g / L. In some embodiments, the steps of precipitating the silk protein and isolating the precipitated silk protein are repeated at least once (using the same or different processing conditions) to further wash away aqueous soluble impurities.

[0156] In any step A10, the isolated recombinant protein is concentrated. Concentrating the isolated recombinant protein can be achieved by evaporation at high temperature and / or reduced pressure (e.g., partial vacuum). Suitable conditions for concentrating the isolated recombinant protein (e.g., temperature, pressure, duration) can be determined using methods known in the art that are coordinated to obtain the isolated recombinant protein with an increased dry solid content. In some embodiments, concentrating results in a 20% - 70% reduction in volume from the original volume. In some embodiments, concentrating results in a concentrated, isolated recombinant protein containing 3% - 20% dry solids.

[0157] In any step A12, the isolated recombinant protein is dry. Drying the suspended silk protein slurry to obtain silk protein powder can be achieved by spray drying, drum drying, freeze drying, or fluid bed drying. In some embodiments, the powder has a water content of less than 10%, less than 9%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0158] Figure 2 shows a process flow for isolating a recombinant protein according to an embodiment of the present invention. Those skilled in the art will understand that some of the steps shown in Figure 2 can be performed in a different order and / or repeatedly. The disclosed embodiments are not intended to limit the scope of the methods provided herein, and those skilled in the art will recognize that the methods can vary based on the recombinant host cell used, the desired cumulative yield, cumulative titer, and / or cumulative productivity, or other factors.

[0159] In step B05, the recombinant host cell is lysed and / or otherwise disrupted, releasing the contents of the recombinant host cell for use in the fermentation broth. Depending on the embodiment, the recombinant host cell can be disrupted using a variety of different methods. Suitable methods for lysing and / or disrupting host cells include using heat such as the high temperature short time (HTST) method, high shear cell disruption, physical homogenization, and chemical homogenization.

[0160] In any step B04, the recombinant protein is solubilized as described above with respect to step A04. Step B04 can be performed before or after step B05. In some embodiments, step B04 can be performed before and after step B05.

[0161] In any step B02, the biomass is removed as described above with respect to step A02. Additionally, other methods of removing biomass from the lysed and / or disrupted cells can include centrifugation and filtration when the recombinant protein is solubilized.

[0162] In any step B06, impurities are removed as described above with respect to step A06. Steps B02 and B06 can be carried out before or after other steps and can be carried out repeatedly. In some embodiments, step B06 may be carried out before and after step B08.

[0163] In step B08, the recombinant protein is isolated. Suitable methods for isolating the recombinant protein are as described above with respect to step A08. Further, methods for isolating the recombinant protein can also include removing phospholipids using additional membranes in filtration and / or degumming.

[0164] In any step B10, the recombinant protein is concentrated as described above with respect to step A10. In any step B12, the recombinant protein is dried as described above with respect to step B10.

[0165] Figure 3 shows a process flow for recombinant protein purification according to an embodiment of the present invention. Those skilled in the art will understand that some of the steps shown in Figure 3 can be carried out in a different order and / or repeatedly. The disclosed embodiments are not intended to limit the scope of the methods provided herein, and those skilled in the art will recognize that the methods can vary based on various factors.

[0166] In step C02, a strong chaotrope is used to create an aqueous two-phase solution and denature the recombinant protein. Suitable chaotropes include, but are not limited to, guanidine thiocyanate (GD-SCN), guanidine hydrochloride (GD-HCl), guanidine iodide, urea, lithium perchlorate, lithium acetate, magnesium chloride, sodium dodecyl sulfate (SDS), potassium iodide (KI), or any combination thereof. Depending on the embodiment, the chaotrope and the protein may be heated to promote protein denaturation.

[0167] In some embodiments, a cosmotrope (also referred to herein as a “precipitant”) is added to the solution to promote phase separation. Suitable cosmotropes include the precipitants referenced above. In other embodiments, a chaotrope at a high starting concentration is used to denature the recombinant protein, and then the concentration of the chaotrope is slowly diluted to obtain phase separation.

[0168] In step C04, a viscous layer of phase separation is obtained. Depending on the type of phase separation, various methods can be used to obtain the viscous layer, such as decanting / extracting the non-viscous layer, or the viscous layer can be extracted using a Hamilton syringe or pipette. Other methods are known to those skilled in the art.

[0169] As step C06, the viscous layer of phase separation is further processed to remove impurities. Suitable dialyzates include double-diluted H2O at a low concentration, or GD-SCN. Depending on the embodiment, various methods of dialysis that can be implemented include cassette dialysis, or other suitable methods known in the art. In some embodiments, tangential flow filtration (TFF) is used to dialyze the viscous layer.

[0170] In some embodiments, the isolated recombinant spider silk protein is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% full-length recombinant spider silk protein.

[0171] In some embodiments, the purity of the isolated recombinant spider silk protein is 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%. In some embodiments, the purity of the isolated recombinant spider silk protein is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0172] In some embodiments, the full-length recombinant spider silk protein is measured or quantified. Size exclusion chromatography (SEC), SDS-PAGE, immunoblot (western blot), high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), or fast protein liquid chromatography (FPLC), or any other suitable method known in the art, or any combination thereof, can be used to measure or quantify the amount of the full-length recombinant protein. In one embodiment, the amount of the full-length recombinant spider silk protein is measured using western blot. In another embodiment, the amount of the full-length recombinant spider silk protein is measured using size exclusion chromatography (SEC).

Example

[0173] The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and do not limit the scope of the present invention in any way. Although efforts have been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), any experimental errors and deviations are of course to be tolerated.

[0174] In the practice of the present invention, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the skill of the art are used. Such techniques are well described in the literature. See, for example, T.E. Creighton, Proteins: Structures and Molecular Properties (W.H. Freeman and Company, 1993), A.L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.), Remington’s Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rd Ed. (Plenum Press) Vols A and B (1992).

[0175] Example 1: Purification of 18B Using One-Step Alkaline Conditions A high pH solution was used to solubilize the recombinant protein without disrupting the host cells secreting the recombinant protein. The pH buffer solution concentration and incubation time were tested to measure the solubility of the recombinant spider silk protein from the Nephila clavipes MaSp2 block (“18B”, SEQ ID NO: 38) having a C-terminal 3×FLAG tag (SEQ ID NO: 40) expressed in P. pastoris. The FLAG tag is linked to a glycine residue (G) linker at the C-terminus of the 18B peptide sequence.

[0176] Specifically, Pichia pastoris expressing the 18B recombinant protein was inoculated into a cell culture fermentation broth and incubated to express the 18B protein. The culture broth was centrifuged to collect the cells, and the cell pellet was resuspended in distilled water at a ratio of 1:1 (equal amounts of cell pellet and water), or 1:3 (one part cell pellet and two parts water). The pH of the cell pellet suspension was adjusted to a final pH of 11.8 - 11.9 with 2 - 10 M NaOH. The cell pellet suspension was incubated at room temperature for 15 - 30 minutes with stirring. The pH was adjusted with NaOH and maintained at pH 11.8 - 11.9 during the incubation. The cell pellet suspension was centrifuged, and the supernatant containing the recombinant protein was collected. The supernatant was lyophilized to concentrate the 18B protein, and the amount of the collected 18B protein was evaluated by size exclusion chromatography (SEC) (Figures 4A and 4B) as described below.

[0177] Size exclusion chromatography (SEC) was used to analyze the relative amounts of high, low, and medium molecular weight impurities, monomeric 18B, and aggregated 18B. The 18B powder was dissolved in 5 M guanidine thiocyanate (GdSCN), injected into a Yarra SEC - 3000 SEC - HPLC column, and the components were separated based on molecular weight. Refractive index was used as the detection modality. 18B aggregates, 18B monomers, low molecular weight (1 - 8 kDa) impurities, medium molecular weight impurities (8 - 50 kDa), and high molecular weight impurities (110 - 150 kDa) were quantified. The relevant compositions were reported as mass % and area %. Bovine serum albumin (BSA) was used as a general protein standard under the assumption that more than 90% of all proteins showed dn / dc values (refractive index response coefficients) within about 7% of each other. Poly(ethylene oxide) was used as the retention time standard, and a BSA calibration substance was used as a check standard to ensure consistent performance of the method. As a control, samples in which the 18B protein was solubilized with urea were also evaluated.

[0178] Alkaline extraction of 18B from P. pastoris cell pellets at pH 11.9 yielded full-length 18B protein normalized to the amount of 18B protein isolated using 5 M GdSCN with an extraction yield of 70 - 75%. The purity of the samples was calculated using the SEC area percentage (%) of the extracted 18B protein. The purity of the 18B monomer in the alkaline extract was ˜35% monomer area, 35% medium molecular weight impurity area, and 28% low molecular weight impurity area percentage (%) (Figures 4A and 4B). In comparison, solubilization of the 18B protein with 10 M urea yielded a low yield of 18B protein with ˜26% monomer area, 27% medium molecular weight impurity area, and 45% low molecular weight impurity area. These data indicate that the alkaline solubilization and extraction method yielded a higher 18B yield and higher purity of the isolated 18B protein.

[0179] Example 2: Further Purification of the Isolated Silk Polypeptide To further purify the 18B spider protein, the 18B sample isolated from the above alkaline extraction was subjected to ultrafiltration and tangential flow filtration using a 750 k MW filter and 8 diavolumes of water. Samples containing unfiltered protein, unultrafiltered protein, and protein after 1, 3, 6, and 8 diavolumes of water were evaluated by SEC as described above. The SEC area percentage (%) for 18B monomer, medium molecular weight impurities, low molecular weight impurities, and high molecular weight impurities in each sample is shown in Figure 5. The sample of unfiltered protein is shown in the leftmost bar (“unconditioned feed”), the sample of unultrafiltered protein is shown in the second bar from the left (“unconditioned UFR”), and the samples of 1, 3, 6, or 8 diavolumes are shown in the left middle, right middle, second from the right, and rightmost bars, respectively (Figure 5). Increasing the diavolume of the wash resulted in an increase in the area percentage (%) of the 18B monomer and a decrease in the area percentage (%) of the low molecular weight impurities.

[0180] Example 3: Purification of 18B Using Two-Step Alkaline Extraction To increase the recovery rate of 18B protein from cells, a two-step extraction process was also implemented. The pH of the whole-cell broth of P. pastoris cells expressing 18B was adjusted to pH 11.8 with 2M NaOH as the first alkaline extraction step and incubated for 30 - 60 minutes. A control sample of the whole-cell broth of P. pastoris cells expressing 18B was incubated with 5M GdSCN for about 15 minutes to solubilize and extract the 18B protein. The cells were pelleted and the supernatant was collected. The remaining pellet from the first alkaline extraction step was re-extracted as the second extraction step by adding water at pH 11.8 at a pellet:water ratio of 1:1, 1:2, or 1:3. The supernatants from the first and second alkaline extracts containing the recombinant 18B protein were collected. The supernatants were lyophilized to concentrate the 18B protein and the samples were evaluated by SEC as described above in Example 1. Two individual experimental runs are shown for each extraction condition and GdSCN subject (Figure 6A). Increasing the amount of alkaline water (1:2 and 1:3 ratios) increased the amount of 18B protein recovered. However, the purity of the double-extracted 18B monomer protein was highest in a single extraction. Increasing the more alkaline water compared to the pellet used in the second extraction also increased the purity of the 18B monomer (Figure 6B).

[0181] Next, samples from the extraction were purified by ultrafiltration and tangential flow filtration using the 750k MW filter described above and water with a maximum dia volume of 8. The purity of the resulting silk polypeptide composition was evaluated by SEC (Figures 7A and 7B). Figure 7A shows the area percentages (%) of the 18B monomer, medium MW impurities, and low molecular weight impurities. Increasing the dia volume during tangential flow filtration resulted in an increase in the 18B monomer peak area. Figure 7B shows the SEC peaks for each sample, the starting material ("SM"), the unfiltered ultrafiltration concentrate ("UF R"), and the tangential flow filtration dia volume samples 1, 2, 3, 4, 6, and 8 (DF 1, 2, 3, 4, 6, 8).

[0182] Example 4: Further isolation of silk polypeptides from alkaline extracts by varying pH By adjusting the pH of the extract, the 18B recombinant protein was precipitated from the alkaline extract. In this experiment, alkaline extraction from the cell culture broth was first carried out by adjusting the pH of the whole cell culture broth to a final pH of 11.8 - 11.9 by adding NaOH, thereby preparing an alkaline cell suspension. The cell suspension was incubated at room temperature for 15 - 30 minutes with stirring. After incubation, the cell suspension was centrifuged and the alkaline supernatant containing the solubilized 18B protein was collected to produce an 18B alkaline extract.

[0183] Next, samples of the 18B alkaline extract were treated under different pH conditions to precipitate the 18B protein. H2SO4 was added to the samples of the alkaline extract to a final pH of 4, 5, 6, 7, 8, 9, or 10. Next, the precipitate containing the 18B recombinant protein was isolated from the alkaline extract. The precipitate samples were evaluated by SEC as described above. Figure 8 shows the percentage (%) of SEC area purity of the high molecular weight (HMW) peak, 18B monomer and aggregate peaks, medium MW (IMW), and low MW (LMW) peaks for each pH condition. Figure 9 shows the percentage (%) of the yield of 18B protein with respect to the pH of each precipitant tested. Among all conditions, for the one-step precipitation method followed by the initial alkaline extraction of the 18B protein, it was found that pH 7 was the most effective for the precipitation step, having an area percentage (%) of about 70% showing a purity of about 70%. Figure 10 shows the SEC profile for the 18B precipitate at pH 6.

[0184] In addition to diafiltration centrifugation, tangential flow filtration (TFF) was also carried out to isolate the alkaline extract. However, diafiltration centrifugation was more effective than TFF in removing impurities, generally resulting in a protein recovery of 60 - 70% with an 18B protein purity exceeding 70%.

[0185] The 18B protein precipitate obtained at pH 6 was lyophilized, wet-spun into fibers, and subjected to tensile strength measurement. When the lyophilized 18B protein was dissolved in formic acid, the final protein amount was 36% by weight. The dissolved protein was extruded into a 100% ethanol coagulation bath at 40 μL / min to produce fibers. The 18B fibers produced by this method had a tensile strength of 19.4 cN / tex.

[0186] Example 5: Recovery of P0 Using Alkaline Conditions vs. Salt Precipitation The concentrations of the pH buffer solution and the incubation time were tested to determine their use in the solubilization of recombinant silk protein P0 (SEQ ID NO: 39) in Escherichia coli cell lysates for extraction from cell culture broth.

[0187] Escherichia coli expressing the P0 recombinant protein with a C-terminal 6×His tag (SEQ ID NO: 46) was seeded into cell culture fermentation broth and incubated to express the P0 protein. The culture broth was centrifuged at 15,000 rcf to pellet the cells. The supernatant was removed, and the cell pellet was resuspended in H2O at a ratio of 1:4 (cell pellet: buffer) or 1:9 (cell pellet: buffer) and incubated for 15 - 60 minutes. The pH of the resuspended cell pellet was adjusted with NaOH to a final pH of 9, 10, 10.5, or 11. As a control, the resuspended cell pellet sample was also incubated with 5 M guanidine thiocyanate (GdSCN) and sonicated for 1.5 minutes. The samples were vortexed and homogenized using a rotary mixer. The lysate was clarified by centrifugation at 15,000 rcf for 5 minutes, and the clarified supernatant containing the P0 protein was retained. The supernatant was filtered using 0.25 μm and analyzed by BCA, ELISA, and immunoblotting.

[0188] Samples were normalized to a protein concentration of 1 mg / mL, and the amount of solubilized P0 in each sample was evaluated by Western blot using an anti-His antibody (Figure 11). Lane H1 is a control sample lysed by sonication in 5 M GdSCN. Lanes B1–B4 are samples mixed at a 1:4 ratio of cell pellet:buffer at pH 9, pH 10, pH 10.5, and pH 11, and lanes B7–B10 are samples mixed at a 1:9 ratio of cell pellet:buffer at pH 9, pH 10, pH 10.5, and pH 11. Lanes C2–C4 are samples incubated with GdSCN for 15, 30, or 60 minutes.

[0189] In an exemplary method, Escherichia coli expressing the P0 recombinant protein was seeded into cell culture fermentation broth and incubated to express the P0 protein. The culture broth was centrifuged at 15,000 rcf to pellet the cells. The cell pellet was resuspended in H2O at a cell pellet:liquid ratio of 1:1 or 1:3, and the cell suspension was homogenized at 10,000–40,000 psi to lyse the E. coli cells. The lysate was clarified by centrifugation, retaining the cell pellet containing insoluble P0. The cell pellet was resuspended in H2O, and the pH of the cell pellet suspension was adjusted to a final pH of 11.5 with 2–10 M NaOH. The cell pellet suspension was incubated at room temperature for 15–60 minutes with stirring. The pH was adjusted with NaOH to maintain pH 11.5 during incubation. After incubation, the cell suspension was centrifuged, and the supernatant containing the recombinant P0 protein was collected.

[0190] As a further method, insoluble P0 can also be extracted from the cell pellet using an alkaline buffer containing 10 M urea. After resuspending the cell pellet in H2O, the pH of the cell pellet suspension was adjusted to a final pH of 11.5 with 2–10 M NaOH, and urea was added to a final concentration of 10 M. The cell pellet suspension was incubated at room temperature for 15–60 minutes with stirring.

[0191] In all methods, the isolated recombinant P0 protein can be further generated by additional clarification steps such as filtration, centrifugation, precipitation, or chromatography.

[0192] equivalent Although the invention has been specifically shown and described with reference to preferred embodiments and various alternative embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0193] All references, issued patents, and patent applications cited in the instant specification are hereby incorporated by reference in their entirety for all purposes.

[0194] Unofficial Sequence Listing TIFF2025108573000008.tif165170

[0195] Sequence Information SEQUENCE LISTING <110> BOLT THREADS, INC. <120> ALKALINE PURIFICATION OF SPIDER SILK PROTEINS <150> US 62 / 772,588 <151> 2018-11-28 <160> 46 <170> PatentIn version 3.5 <210> 1 <211> 181 <212> PRT <213> Aliatypus gulosus <400> 1 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> 2 <211> 126 <212> PRT <213> Plectreurys tristis <400> 2 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> 3 <211> 239 <212> PRT <213> Plectreurys tristis <400> 3 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> 4 <211> 182 <212> PRT <213> Araneus gemmoides <400> 4 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 Ser Ser Ala Ser Gln Ala Ser Ala Ser Ala Phe Ala Gln Gln Ser Ser 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> 5 <211> 180 <212> PRT <213> Argiope aurantia <400> 5 Gly Ser Leu Ala Ser Ser Phe Ala Ser Ala Leu Ser Ala Ser Ala Ala 1 5 10 15 Ser Val Ala Ser Ser Ala Ala Ala Gln Ala Ala Ser Gln Ser 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 Leu 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> 6 <211> 199 <212> PRT <213> Deinopis spinosa <400> 6 Gly Ala Ser Ala Ser Ala Tyr Ala Ser Ala Ile Ser Asn Ala Val Gly 1 5 10 15 Pro Tyr Leu Tyr Gly Leu Gly Leu Phe Asn Gln Ala Asn Ala Ala Ser 20 25 30 Phe Ala Ser Ser Phe Ala Ser Ala Val Ser Ser Ala Val Ala Ser Ala 35 40 45 Ser Ala Ser Ala Ala Ser Ser Ala Tyr Ala Gln Ser Ala Ala Ala Gln 50 55 60 Ala Gln Ala Ala Ser Ser Ala Phe Ser Gln Ala Ala Ala Gln Ser Ala 65 70 75 80 Ala Ala Ala Ser Ala Gly Ala Ser Ala Gly Ala Gly Ala Ser Ala Gly 85 90 95 Ala Gly Ala Val Ala Gly Ala Gly Ala Val Ala Gly Ala Gly Ala Val 100 105 110 Ala Gly Ala Ser Ala Ala Ala Ala Ser Gln Ala Ala Ala Ser Ser Ser 115 120 125 Ala Ser Ala Val Ala Ser Ala Phe Ala Gln Ser Ala Ser Tyr Ala Leu 130 135 140 Ala Ser Ser Ser Ala Phe Ala Asn Ala Phe Ala Ser Ala Thr Ser Ala 145 150 155 160 Gly Tyr Leu Gly Ser Leu Ala Tyr Gln Leu Gly Leu Thr Thr Ala Tyr 165 170 175 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> 7 <211> 171 <212> PRT <213> Nephila clavipes <400> 7 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 Leu 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 Gln Ala 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> 8 <211> 268 <212> PRT <213> Argiope trifasciata <400> 8 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> 9 <211> 420 <212> PRT <213> Nephila clavipes <400> 9 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> 10 <211> 376 <212> PRT <213> Latrodectus hesperus <400> 10 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> 11 <211> 200 <212> PRT <213> Argiope trifasciata <400> 11 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 Ser Ser Ser Ser Phe Leu Ser Thr Ser Ser Ser Ser Ala Ser Tyr Ser 180 185 190 Gln Ala Ser Ala Ser Ser Thr Ser 195 200 <210> 12 <211> 357 <212> PRT <213> Uloborus diversus <400> 12 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> 13 <211> 32 <212> PRT <213> Euprosthenops australis <400> 13 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 Ala 20 25 30 <210> 14 <211> 42 <212> PRT <213> Tetragnatha kauaiensis <400> 14 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> 15 <211> 42 <212> PRT <213> Argiope aurantia <400> 15 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> 16 <211> 46 <212> PRT <213> Deinopis spinosa <400> 16 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> 17 <211> 42 <212> PRT <213> Nephila clavata <400> 17 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> 18 <211> 174 <212> PRT <213> Deinopis Spinosa <400> 18 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> 19 <211> 149 <212> PRT <213> Latrodectus hesperus <400> 19 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> 20 <211> 161 <212> PRT <213> Nephila clavipes <400> 20 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 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 Ala <210> 21 <211> 186 <212> PRT <213> Nephilengys cruentata <400> 21 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> 22 <211> 132 <212> PRT <213> Uloborus diversus <400> 22 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> 23 <211> 198 <212> PRT <213> Uloborus diversus <400> 23 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> 24 <211> 190 <212> PRT <213> Araneus ventricosus <400> 24 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> 25 <211> 166 <212> PRT <213> Dolomedes tenebrosus <400> 25 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> 26 <211> 177 <212> PRT <213> Nephilengys cruentata <400> 26 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 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> 27 <211> 174 <212> PRT <213> Nephilengys cruentata <400> 27 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> 28 <211> 22 <212> PRT <213> Saccharomyces cerevisiae <400> 28 Methionine, Phenylalanine, Serine, Leucine, Lysine, Alanine, Leucine, Leucine, Proline, Leucine, Alanine, Leucine, Leucine, Leucine, Valine, Serine 1 5 10 15 Alanine, Asparagine, Glutamine, Valine, Alanine, Alanine 20 <210> 29 <211> 23 <212> PRT <213> Pichia pastoris <400> 29 Methionine, Serine, Phenylalanine, Serine, Serine, Asparagine, Valine, Proline, Glutamine, Leucine, Phenylalanine, Leucine, Leucine, Leucine, Valine, Leucine 1 5 10 15 Leucine, Threonine, Asparagine, Isoleucine, Valine, Serine, Glycine 20 <210> 30 <211> 20 <212> PRT <213> Pichia pastoris <400> 30 Methionine, Lysine, Leucine, Serine, Threonine, Asparagine, Leucine, Isoleucine, Leucine, Alanine, Isoleucine, Alanine, Alanine, Alanine, Serine, Alanine 1 5 10 15 Valine, Valine, Serine, Alanine 20 <210> 31 <211> 18 <212> PRT <213> Gallus gallus <400> 31 Methionine, Arginine, Serine, Leucine, Leucine, Isoleucine, Leucine, Valine, Leucine, Cysteine, Phenylalanine, Leucine, Proline, Leucine, Alanine, Alanine 1 5 10 15 Leucine, Glycine <210> 32 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 32 Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu Asp Leu Glu Gly Asp Phe 35 40 45 Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55 60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val 65 70 75 80 Ser Leu Asp Lys Arg Glu Ala Glu Ala 85 <210> 33 <211> 89 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 33 Methionine, Arginine, Phenylalanine, Proline, Serine, Isoleucine, Phenylalanine, Threonine, Alanine, Valine, Leucine, Phenylalanine, Alanine, Alanine, Serine, Serine 1 5 10 15 Alanine, Leucine, Alanine, Alanine, Proline, Valine, Asparagine, Threonine, Threonine, Threonine, Glutamic acid, Aspartic acid, Glutamic acid, Threonine, Alanine, Glutamine 20 25 30 Isoleucine, Proline, Alanine, Glutamic acid, Alanine, Valine, Isoleucine, Glycine, Tyrosine, Serine, Aspartic acid, Leucine, Glutamic acid, Glycine, Aspartic acid, Phenylalanine 35 40 45 Aspartic acid, Valine, Alanine, Valine, Leucine, Proline, Phenylalanine, Serine, Asparagine, Serine, Threonine, Asparagine, Asparagine, Glycine, Leucine, Leucine 50 55 60 Phenylalanine, Isoleucine, Asparagine, Threonine, Threonine, Isoleucine, Alanine, Serine, Isoleucine, Alanine, Alanine, Lysine, Glutamic acid, Glutamic acid, Glycine, Valine 65 70 75 80 Serine, Leucine, Glutamic acid, Lysine, Arginine, Glutamic acid, Alanine, Glutamic acid, Alanine 85 <210> 34 <211> 92 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 34 Methionine, Phenylalanine, Serine, Leucine, Lysine, Alanine, Leucine, Leucine, Proline, Leucine, Alanine, Leucine, Leucine, Leucine, Valine, Serine 1 5 10 15 Alanine, Asparagine, Glutamine, Valine, Alanine, Alanine, Alanine, Proline, Valine, Asparagine, Threonine, Threonine, Threonine, Glutamic acid, Aspartic acid, Glutamic acid 20 25 30 Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu 35 40 45 Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn 50 55 60 Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu 65 70 75 80 Glu Gly Val Ser Leu Glu Lys Arg Glu Ala Glu Ala 85 90 <210> 35 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 35 Met Ser Phe Ser Ser Asn Val Pro Gln Leu Phe Leu Leu Leu Val Leu 1 5 10 15 Leu Thr Asn Ile Val Ser Gly Ala Pro Val Asn Thr Thr Thr Glu Asp 20 25 30 Glu Thr Ala Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu 35 40 45 Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn 50 55 60 Asn Gly Leu Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys 65 70 75 80 Glu Glu Gly Val Ser Leu Glu Lys Arg Glu Ala Glu Ala 85 90 <210> 36 <211> 90 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 36 Met Lys Leu Ser Thr Asn Leu Ile Leu Ala Ile Ala Ala Ala Ser Ala 1 5 10 15 Val Val Ser Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala 20 25 30 Gln Ile Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp 35 40 45 Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu 50 55 60 Leu Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly 65 70 75 80 Val Ser Leu Glu Lys Arg Glu Ala Glu Ala 85 90 <210> 37 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 37 Met Arg Ser Leu Leu Ile Leu Val Leu Cys Phe Leu Pro Leu Ala Ala 1 5 10 15 Leu Gly Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile 20 25 30 Pro Ala Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp 35 40 45 Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe 50 55 60 Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser 65 70 75 80 Leu Glu Lys Arg Glu Ala Glu Ala 85 <210> 38 <211> 943 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 38 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 Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Gly 385 390 395 400 Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 405 410 415 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 420 425 430 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly 435 440 445 Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr 450 455 460 Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser 465 470 475 480 Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala 485 490 495 Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln 500 505 510 Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro 515 520 525 Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala 530 535 540 Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln 545 550 555 560 Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr 565 570 575 Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro 580 585 590 Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly 595 600 605 Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala 610 615 620 Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly 625 630 635 640 Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 645 650 655 Ser Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly 660 665 670 Gln Gly Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala 675 680 685 Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly 690 695 700 Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly 705 710 715 720 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly 725 730 735 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala 740 745 750 Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln 755 760 765 Arg Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly 770 775 780 Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly 785 790 795 800 Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala 805 810 815 Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser 820 825 830 Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro 835 840 845 Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly 850 855 860 Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly 865 870 875 880 Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala 885 890 895 Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln 900 905 910 Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly 915 920 925 Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala 930 935 940 <210> 39 <211> 800 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 39 Met Ala Gly Ala Gly Ala Gly Tyr Arg Gly Gln Ala Gly Tyr Ile Gln 1 5 10 15 Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Val Gly 20 25 30 Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly 35 40 45 Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Arg Gln Ala Gly Tyr Gly 50 55 60 Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Ala 65 70 75 80 Gly Arg Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala 85 90 95 Ala Ala Ala Gly Ala Asp Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly 100 105 110 Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ser Gly Ala Gly Ala 115 120 125 Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala 130 135 140 Gly Ala Ala Ala Ala Gly Ala Gly Ala Gly Tyr Leu Gly Gln Ala Gly 145 150 155 160 Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Gly Ala Gly 165 170 175 Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Thr Gly Ala Ala 180 185 190 Ala Ser Ala Ala Ala Ser Ser Ala Gly Ala Gly Ala Gly Tyr Arg Gly 195 200 205 Gln Ala Gly Tyr Ile Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala 210 215 220 Ala Gly Ala Gly Val Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly 225 230 235 240 Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly 245 250 255 Arg Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala 260 265 270 Ala Ala Gly Ala Gly Ala Gly Arg Gln Ala Gly Tyr Gly Gln Gly Ala 275 280 285 Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Asp Ala Gly Tyr Gly 290 295 300 Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala 305 310 315 320 Ala Ser Gly Ala Gly Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln 325 330 335 Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Ala Gly 340 345 350 Tyr Leu Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly 355 360 365 Ala Ala Ala Gly Ala Gly Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly 370 375 380 Gln Gly Thr Gly Ala Ala Ala Ser Ala Ala Ala Ser Ser Ala Gly Ala 385 390 395 400 Gly Ala Gly Tyr Arg Gly Gln Ala Gly Tyr Ile Gln Gly Ala Gly Ala 405 410 415 Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Val Gly Tyr Gly Gly Gln 420 425 430 Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala 435 440 445 Ala Gly Ala Gly Ala Gly Arg Gln Ala Gly Tyr Gly Gln Gly Ala Gly 450 455 460 Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Ala Gly Arg Gln Ala 465 470 475 480 Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly 485 490 495 Ala Asp Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly 500 505 510 Ala Ser Ala Gly Ala Ala Ala Ser Gly Ala Gly Ala Gly Tyr Gly Gly 515 520 525 Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala 530 535 540 Ala Gly Ala Gly Ala Gly Tyr Leu Gly Gln Ala Gly Tyr Gly Gln Gly 545 550 555 560 Ala Gly Ala Ser Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Tyr Gly 565 570 575 Gly Gln Ala Gly Tyr Gly Gln Gly Thr Gly Ala Ala Ala Ser Ala Ala 580 585 590 Ala Ser Ser Ala Gly Ala Gly Ala Gly Tyr Arg Gly Gln Ala Gly Tyr 595 600 605 Ile Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly 610 615 620 Val Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser 625 630 635 640 Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Arg Gln Ala Gly 645 650 655 Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala 660 665 670 Gly Ala Gly Arg Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala 675 680 685 Gly Ala Ala Ala Ala Gly Ala Asp Ala Gly Tyr Gly Gly Gln Ala Gly 690 695 700 Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Ser Gly Ala 705 710 715 720 Gly Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala 725 730 735 Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Ala Gly Tyr Leu Gly Gln 740 745 750 Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Gly 755 760 765 Ala Gly Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Thr Gly 770 775 780 Ala Ala Ala Ser Ala Ala Ala Ser Ser Ala Gly Gly Gly Gly Gly Gly 785 790 795 800 <210> 40 <211> 24 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 40 Asp Tyr Lys Asp Asp Asp Asp Lys Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 10 15 Asp Tyr Lys Asp Asp Asp Asp Lys 20 <210> 41 <211> 5 <212> PRT <213> Unknown <220> <223> Description of Unknown: silk protein sequence <400> 41 Ala Ala Ala Ala Ala 1 5 <210> 42 <211> 5 <212> PRT <213> Unknown <220> <223> Description of Unknown: silk protein sequence <400> 42 Ser Gly Ala Gly Gly 1 5 <210> 43 <211> 5 <212> PRT <213> Unknown <220> <223> Description of Unknown: silk protein sequence <400> 43 Gly Ser Gly Ala Gly 1 5 <210> 44 <211> 5 <212> PRT <213> Unknown <220> <223> Description of Unknown: silk protein sequence <400> 44 Gly Gly Ser Gly Ala 1 5 <210> 45 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <220> <221> MISC_FEATURE <222> (1)..(8) <223> This sequence may encompass 6-8 residues <400> 45 His His His His His His His His 1 5 <210> 46 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 46 His His His His His His 1 5

Claims

1. A method for isolating a recombinant spider silk protein from a host cell culture solution, the method comprising the following steps: a. A step of obtaining a cell culture solution, wherein the cell culture solution contains a host cell and a growth medium, and the host cell expresses a recombinant spider silk protein; b. A step of collecting a part of the cell culture solution containing the recombinant spider silk protein; c. A step of incubating the part of the cell culture solution in an aqueous solution under alkaline conditions, thereby solubilizing the recombinant spider silk protein in the aqueous solution; d. A step of isolating the recombinant spider silk protein from the aqueous solution, thereby producing an isolated recombinant spider silk protein sample.

2. The method according to claim 1, wherein the alkaline conditions include an alkaline pH of 9 to 14.

3. The method according to claim 2, wherein the alkaline pH is 11 to 12.

4. The method according to any one of the preceding claims, wherein the isolated recombinant spider silk protein is a full-length recombinant spider silk protein.

5. The method according to claim 4, wherein the isolated recombinant spider silk protein sample contains at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the full-length recombinant spider silk protein compared to the isolated total recombinant spider silk protein.

6. The method according to claim 5, wherein the ratio of the full-length recombinant spider silk protein is measured using Western blot.

7. The method according to claim 5, wherein the ratio of the full-length recombinant spider silk protein is measured using size exclusion chromatography.

8. The method according to any one of the preceding claims, wherein the purity of the isolated recombinant spider silk protein is 5 to 10%, 10 to 15%, 15 to 20%, 20 to 25%, 25 to 30%, 30 to 35%, 35 to 40%, 45 to 50%, 50 to 55%, 55 to 60%, 60 to 65%, 65 to 70%, 70 to 75%, 75 to 80%, 80 to 85%, 85 to 90%, 09 to 95%, or 95 to 100%.

9. The yield of the isolated recombinant spider silk protein is at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100% compared to recombinant spider silk isolated by the urea or guanidine thiocyanate method, the method according to any one of the preceding claims.

10. The method according to any one of the preceding claims, wherein the step of isolating the recombinant spider silk protein comprises precipitating the recombinant spider silk protein by changing the alkaline conditions of the aqueous solution.

11. The method according to claim 10, wherein changing the alkaline conditions comprises adjusting the alkaline pH of the cell culture to a lowered pH of 4-10.

12. The method according to claim 11, wherein the lowered pH is pH 4, 5, 6, 7, 8, 9, or 10.

13. The method according to claim 11, wherein the lowered pH is pH 6-7.

14. The method according to any one of claims 10-13, wherein adjusting the alkaline pH comprises adding an acid to the aqueous solution.

15. wherein the acid is H ２ SO4, the method according to claim 14.

16. The method according to any one of the preceding claims, wherein the portion of the cell culture comprises a supernatant, whole cell broth, or cell pellet.

17. The method according to any one of the preceding claims, wherein the step of collecting the portion of the cell culture comprises removing the host cells from the growth medium and reconstituting the host cells in the aqueous solution.

18. The method according to any one of the preceding claims, wherein the step of collecting the portion of the cell culture comprises lysing the host cells.

19. The method according to claim 18, wherein lysing comprises heat treatment, shear disruption, physical homogenization, sonication, or chemical homogenization.

20. The method according to any one of the preceding claims, wherein the portion of the cell culture comprises the host cells and the growth medium derived from the cell culture.

21. The method according to any one of the preceding claims, wherein the aqueous solution comprises a diluted growth medium.

22. The method according to any one of the preceding claims, wherein the step of incubating the portion of the cell culture under alkaline conditions is carried out for 10-120 minutes.

23. The method according to claim 22, wherein the step of incubating the portion of the cell culture under alkaline conditions is carried out for at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 105 minutes, or at least 120 minutes.

24. The method according to claim 22, wherein the step of incubating the portion of the cell culture under alkaline conditions is carried out for 15 to 30 minutes.

25. The method according to any one of the preceding claims, wherein the step of incubating the portion of the cell culture under alkaline conditions further comprises stirring the portion of the cell culture.

26. The method according to any one of the preceding claims, further comprising removing insoluble biomass from the aqueous solution under alkaline conditions.

27. The method according to claim 26, wherein removing the insoluble biomass comprises filtration, centrifugation, gravitational sedimentation, adsorption, dialysis, or phase separation.

28. The method according to claim 27, wherein the filtration is ultrafiltration, microfiltration, or diafiltration.

29. The method according to any one of claims 26 to 28, wherein removing the insoluble biomass is repeated at least once.

30. The method according to any one of the preceding claims, further comprising removing impurities before or after isolating the recombinant spider silk protein.

31. The method according to claim 30, wherein removing the impurities comprises filtration, centrifugation, gravitational sedimentation, adsorption, dialysis, or phase separation.

32. The method according to claim 31, wherein the filtration is ultrafiltration, microfiltration, or diafiltration.

33. The method according to claim 31, wherein the centrifugation is ultracentrifugation or diafiltration centrifugation.

34. The method according to claim 31, wherein the adsorption is carbon adsorption.

35. The method according to any one of claims 31 to 34, wherein removing impurities is repeated at least once.

36. The method according to any one of the preceding claims, further comprising concentrating the isolated recombinant spider silk protein to produce a concentrated spider silk protein.

37. The method according to claim 36, wherein concentrating comprises precipitation, filtration, ultrafiltration, centrifugation, dialysis, evaporation, or lyophilization.

38. The method according to any of the preceding claims, further comprising drying the isolated recombinant spider silk protein.

39. The method according to any of the preceding claims, further comprising producing silk fibers from the isolated recombinant spider silk.

40. The method according to claim 39, wherein the silk fibers comprise a tensile strength of at least 19 cN / tex.

41. The method according to any of the preceding claims, wherein the recombinant spider silk protein is 18B or P0.

42. The method according to any of the preceding claims, wherein the cell culture comprises fungal cells, bacterial cells, or yeast cells.

43. The method according to any of the preceding claims, wherein the yeast cells are Pichia pastoris cells.

44. A method for isolating a recombinant spider silk protein, the method comprising the following steps: a. obtaining a cell culture comprising a host cell and a growth medium, wherein the host cell expresses a recombinant spider silk protein; b. collecting a portion of the cell culture comprising the recombinant spider silk protein; c. incubating the portion of the cell culture in an aqueous solution under alkaline conditions to solubilize the recombinant spider silk protein in the aqueous solution; d. adjusting the aqueous solution to a non-alkaline pH to precipitate the solubilized recombinant spider silk protein; e. isolating the recombinant spider silk protein from the portion of the cell culture to produce an isolated recombinant spider silk protein.

45. A composition comprising a recombinant spider silk protein produced by the method according to any one of the preceding claims.

46. The composition according to claim 45, wherein the recombinant spider silk comprises at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% full-length recombinant spider silk.

47. Silk fibers comprising a recombinant spider silk protein produced by the method according to any one of claims 1 to 44.

48. The silk fiber according to claim 47, wherein the silk fiber comprises a tensile strength of at least 19 cN / tex.

49. A composition comprising a cell culture solution containing a growth medium and a host cell containing a recombinant spider silk protein in an alkaline buffer solution.

50. The composition according to any one of claims 45 to 49, wherein the alkaline buffer solution has a pH of 9 to 14.

51. The composition according to claim 50, wherein the pH is 11 to 12.

52. The composition according to any one of claims 49 to 51, wherein the spider silk protein is 18B or P0.

53. The composition according to any one of claims 49 to 52, wherein the cell culture solution contains fungal cells, bacterial cells, or yeast cells.

54. The composition according to claim 53, wherein the bacterial cells are Escherichia coli cells.

55. The composition according to claim 53, wherein the yeast cells are Pichia pastoris cells.

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