Method for isolating spider silk proteins via high-shear solubilization

Abstract

The present disclosure relates to methods for isolating and purifying synthetic block copolymer proteins using solvent and high shear microfluidization techniques.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 901,053, filed September 16, 2019, the entire contents of which are incorporated herein by reference.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created on September 16, 2020, is entitled BTT-033WO_SL.txt and is 50,960 bytes in size. [Background technology]

[0003] background Spider silk polypeptides are large (>150 kDa, >1000 amino acids) polypeptides that can be divided into three domains: the N-terminal non-repetitive domain (NTD), the repetitive domain (REP), and the C-terminal non-repetitive domain (CTD). The NTD and CTD are relatively small (approximately 150 and 100 amino acids, respectively), well-characterized, and are thought to confer aqueous stability, pH sensitivity, and molecular alignment upon aggregation. The NTD also possesses a strong predicted secretion tag, which is often removed during heterologous expression. The native polypeptide is approximately 90% repetitive and folds into crystalline and amorphous regions that confer strength and flexibility, respectively, to silk fibers.

[0004] Silk polypeptides are derived from a variety of sources, including bees, moths, spiders, mites, and other arthropods. Some organisms produce multiple silk fibers with unique sequences, structural elements, and mechanical properties. For example, the orb-jawed spider (Argiope bruennichi) possesses six unique types of glands from which it produces various silk polypeptide sequences to polymerize fibers tailored to its environment or life cycle. These fibers are named for the glands from which they originate, and polypeptides are labeled with abbreviations for the gland (e.g., "Ma") and "Sp" for spidroin (an abbreviation for spider fibroin). In the orb-jawed spider, these types include Major Ampullate (MaSp, also known as dragline), Minor Ampullate (MiSp), Flagelliform (Flag), Aciniform (AcSp), Tubuliform (TuSp), and Pyriform (PySp). Given the variation among fiber types, domains, and different genera and species of organisms, the combination of these polypeptide sequences offers a vast number of potential traits that can be exploited for commercial production of recombinant fibers.To date, the majority of research using recombinant silk has focused on Major Ampullate Spidroin (MaSp).

[0005] Currently, recombinant silk fibers are not commercially available and, with a few exceptions, are not produced by microorganisms other than Escherichia coli and other Gram-negative prokaryotes. Recombinant silk produced to date has mainly consisted of either polymerized short silk sequence motifs or fragments of native repeat domains, sometimes in combination with NTDs and / or CTs.

[0006] However, in some cases, recombinant spider silk polypeptides form undesirable, insoluble aggregates during production and purification. Their ability to aggregate and form β-sheet structures makes solubilization of proteins based on silk sequences difficult. Solubilization of these proteins often requires chemical conditions unsuitable for biological molecules, such as high molar concentrations of chaotropes. Methods for resolubilizing peptides during purification often result in protein degradation, resulting in low yields and the production of fibers with poor tenacity and unpleasant texture. Therefore, improved methods for purifying these polypeptides to increase their solubility and improve silk protein recovery are needed. Summary of the Invention

[0007] Provided herein are methods for solubilizing silk proteins under conditions of poor chemical solvation, including various chaotrope solutions, by applying high physical energy, such as shear, impact, and cavitation generated by high-energy fluid processing devices.

[0008] summary In one aspect, provided herein is a method for isolating a recombinant spider silk protein from a host cell, the method comprising the steps of: providing an insoluble material comprising a recombinant spider silk protein; adding the insoluble material to an aqueous solution comprising a solvent; and applying shear force to the aqueous solution containing the insoluble material, thereby solubilizing the recombinant spider silk protein in the aqueous solution.

[0009] In some embodiments, the shear force is applied via microfluidization.

[0010] In some embodiments, the microfluidization results in about 6×10 6 s -1 ~10×10 6 s -1In some embodiments, the microfluidization produces a shear rate of at least about 6×10 6 s -1 In some embodiments, the microfluidization produces a shear rate of at least about 10×10 6 s -1 A shear rate of

[0011] In some embodiments, the microfluidization is carried out at 20,000 psi to 30,000 psi. In some embodiments, the microfluidization is carried out at 30,000 psi. In some embodiments, the microfluidization is carried out at 23,000 psi.

[0012] In some embodiments, a Microfluidizer M-110P or LM10 Microfluidizer.

[0013] In some embodiments, the microfluidizer comprises a G10Z interaction chamber, hi some embodiments, the microfluidizer comprises an F12Y interaction chamber.

[0014] In some embodiments, the shear force is applied at least two times. In some embodiments, the shear force is applied three times. In some embodiments, the shear force is the same in the at least two applications. In some embodiments, the shear force is different in the at least two applications.

[0015] In some embodiments, the insoluble material is derived from a cell culture comprising host cells, wherein the host cells express recombinant spider silk proteins.

[0016] In some embodiments, the method further comprises recovering insoluble material from the cell culture, wherein the insoluble material comprises recombinant spider silk protein.

[0017] In some embodiments, the solvent is a chaotropic agent, hi some embodiments, the chaotropic agent is urea, guanidine thiocyanate (GdnSCN), or guanidine chloride (GdnHCL).

[0018] In some embodiments, the insoluble material is added to the aqueous solution at about 5%, 10%, 15%, 20%, 25%, or 30% insoluble material by solvent volume.

[0019] In some embodiments, the chaotropic agent is present in the aqueous solution at a concentration of 0.1-10 M. In some embodiments, the aqueous solution comprises about 10 M urea, about 4 M-8 M GdnHCl, or about 3 M-6 M GdnSCN. In some embodiments, the aqueous solution comprises a chaotropic activity that is weaker than the chaotropic activity of an aqueous solution containing 10 M urea, 8 M GdnHCl, or 6 M GdnSCN.

[0020] In some embodiments, chaotropic activity is quantified using an agar-gelling assay.

[0021] In some embodiments, the aqueous solution contains about 15% insoluble material for 85% volume of 3M GdnSCN, about 15% insoluble material for 85% volume of 4M GdnHCl, or about 15% insoluble material for 85% volume of 10M urea.

[0022] In some embodiments, the insoluble material is incubated at 20°-30° C. In some embodiments, the insoluble material is incubated at room temperature. In some embodiments, the insoluble material is incubated at 30° C. or below. In some embodiments, the insoluble portion is incubated in the aqueous solution containing the solvent for 60-120 minutes.

[0023] In some embodiments, the insoluble material comprises a cell pellet.

[0024] In some embodiments, the step of recovering the insoluble material from the cell pellet comprises lysing the host cells.

[0025] In some embodiments, the lysing comprises heat treatment, chemical treatment, shear disruption, physical homogenization, sonication, or chemical homogenization.

[0026] In some embodiments, the step of recovering insoluble material of the cell culture further comprises centrifuging the lysed cells to obtain a first cell pellet.

[0027] In some embodiments, the step of recovering the insoluble material further comprises: incubating the cell pellet with a solution containing 4 M urea at a urea volume:pellet mass ratio of 10:1; and centrifuging the solution containing 4 M urea to obtain a second cell pellet, and then incubating the second cell pellet in the aqueous solution containing a solvent.

[0028] In some embodiments, the method further comprises isolating the recombinant spider silk protein from the aqueous solution, thereby producing an isolated recombinant spider silk protein.

[0029] In some embodiments, the recombinant spider silk protein is a highly crystalline silk protein, a silk protein with a high beta-sheet content, or a low solubility silk protein.

[0030] In some embodiments, the recombinant spider silk protein comprises the Uloborus diversus MiSP protein set forth in SEQ ID NO:23.

[0031] In some embodiments, the recombinant spider silk protein has a solubility threshold in a non-chaotropic solvent of less than 90%, 80%, 70%, 60%, or 50%.

[0032] In some embodiments, the cell culture comprises fungal cells, bacterial cells, or yeast cells. In some embodiments, the bacterial cells are Escherichia coli cells.

[0033] In some embodiments, the amount of isolated recombinant spider silk protein is measured using ELISA. In some embodiments, the amount of isolated recombinant spider silk protein is measured using size exclusion chromatography.

[0034] In some embodiments, the isolated recombinant spider silk protein is a full-length recombinant spider silk protein.

[0035] In some embodiments, the isolated recombinant spider silk protein comprises 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.

[0036] In some embodiments, the amount of full-length recombinant spider silk protein is measured using ELISA. In some embodiments, the amount of full-length recombinant spider silk protein is measured using size exclusion chromatography.

[0037] 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%.

[0038] In another aspect, provided herein is a method for isolating recombinant spider silk protein from a host cell, the method comprising the steps of: providing an insoluble material comprising a recombinant spider silk protein; adding the insoluble material to an aqueous solution comprising a solvent, wherein the aqueous solution comprises 15% (w / v) insoluble portion at a final 10 M urea concentration; applying shear force to the aqueous solution comprising the insoluble material via microfluidization, 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.

[0039] In another aspect, provided herein is a method for isolating recombinant spider silk protein from a host cell, the method comprising the steps of: providing an insoluble material comprising the recombinant spider silk protein; adding the insoluble material to an aqueous solution, the aqueous solution comprising about 15% (w / v) insoluble portion at a final 10 M urea concentration; applying a shear force via microfluidization, the shear force being about 10×10 to the aqueous solution. 6 s -1 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.

[0040] In another aspect, provided herein are compositions comprising recombinant spider silk proteins produced by the methods described herein.

[0041] In some embodiments, the composition comprises recombinant spider silk protein powder, 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.

[0042] In another aspect, provided herein are silk fibers comprising recombinant spider silk proteins produced by the methods disclosed herein. [The present invention 1001] 1. A method for isolating recombinant spider silk proteins from a host cell, the method comprising the steps of: a. providing an insoluble material comprising a recombinant spider silk protein; b. adding the insoluble material to an aqueous solution containing a solvent; c. applying shear force to the aqueous solution containing the insoluble material, thereby solubilizing the recombinant spider silk protein in the aqueous solution. The method comprising: [The present invention 1002] 1001. The method of claim 1001, wherein said shear force is applied via microfluidization. [The present invention 1003] The microfluidization resulted in approximately 6×10 6 s -1 ~10×10 6 s -1 The method of the present invention 1002, wherein a shear rate of [The present invention 1004] The microfluidization results in at least about 6×10 6 s -1 The method of the present invention 1002, wherein a shear rate of [The present invention 1005] The microfluidization results in at least about 10×10 6 s -1 The method of the present invention 1002, wherein a shear rate of [The present invention 1006] 1006. The method of any one of claims 1002 to 1005, wherein said microfluidization is carried out at 20,000 psi to 30,000 psi. [The present invention 1007] 1006. The method of claim 1006, wherein said microfluidization is carried out at 30,000 psi. [The present invention 1008] 1006. The method of claim 6, wherein said microfluidization is carried out at 23,000 psi. [The present invention 1009] Microfluidizer M-110P or LM10 Microfluidizer, any method of the preceding invention. [The present invention 1010] 1009. The method of claim 10, wherein said microfluidizer comprises a G10Z interaction chamber. [The present invention 1011] 1009. The method of claim 10, wherein said microfluidizer comprises an F12Y interaction chamber. [The present invention 1012] Any of the preceding methods of the present invention, wherein the shear force is applied at least twice. [The present invention 1013] 1013. The method of claim 1012, wherein the shear force is applied at least three times. [The present invention 1014] 1013. The method of claim 1012, wherein said shear force is the same during said at least two applications. [The present invention 1015] 1013. The method of claim 1012, wherein said shear force is different during said at least two applications. [The present invention 1016] the insoluble material is derived from a cell culture comprising host cells; the host cell expresses the recombinant spider silk protein; Any method of the preceding invention. [The present invention 1017] further comprising the step of recovering the insoluble material from the cell culture; the insoluble material comprises the recombinant spider silk protein; The method of the present invention 1016. [The present invention 1018] The method according to any one of claims 1001 to 1017, wherein the solvent is a chaotropic agent. [The present invention 1019] 1018. The method of claim 1018, wherein said chaotropic agent is urea, guanidine thiocyanate (GdnSCN), or guanidine chloride (GdnHCL). [The present invention 1020] 1020. The method of any of claims 1001 to 1019, wherein the insoluble material is added to the aqueous solution at about 5%, 10%, 15%, 20%, 25%, or 30% insoluble material by solvent volume. [The present invention 1021] 1020. The method of any one of claims 1019 to 1020, wherein said chaotropic agent is present in said aqueous solution at a concentration of 0.1 to 10 M. [The present invention 1022] 1020. The method of claim 1019, wherein the aqueous solution comprises about 10 M urea, about 4 M to 8 M GdnHCl, or about 3 M to 6 M GdnSCN. [The present invention 1023] 1021. The method of claim 1019 or 1020, wherein the aqueous solution comprises a chaotropic activity that is weaker than the chaotropic activity of an aqueous solution comprising 10 M urea, an aqueous solution comprising 8 M GdnHCl, or an aqueous solution comprising 6 M GdnSCN. [The present invention 1024] The method of claim 1023, wherein the chaotropic activity is quantified using an agar-gelling assay. [The present invention 1025] 1020. The method of claim 1020, wherein the aqueous solution contains about 15% insoluble material for 85% volume of 3M GdnSCN. [The present invention 1026] 1020. The process of claim 1020, wherein the aqueous solution contains about 15% insoluble material for 85% volume of 4M GdnHCl. [The present invention 1027] 1020. The method of claim 1020, wherein the aqueous solution contains about 15% insoluble material for 85% volume of 10M urea. [The present invention 1028] The method of any one of claims 1001 to 1027, wherein the insoluble material is incubated at 20°C to 30°C. [The present invention 1029] 1029. The method of claim 1028, wherein the insoluble material is incubated at room temperature. [The present invention 1030] 1028. The method of claim 1028, wherein the insoluble material is incubated at 30°C or below. [The present invention 1031] The method according to any one of claims 1001 to 1030, wherein the insoluble portion is incubated in the aqueous solution containing the solvent for 60 to 120 minutes. [The present invention 1032] The method of any one of claims 1001 to 1031, wherein the insoluble material comprises a cell pellet. [The present invention 1033] The method of any of claims 1001 to 1032, wherein the step of recovering said insoluble material from said cell pellet comprises lysing said host cells. [The present invention 1034] The method of claim 1033, wherein said dissolving comprises heat treatment, chemical treatment, shear disruption, physical homogenization, ultrasonic treatment, or chemical homogenization. [This invention 1035] The method of any one of claims 1033 to 1034, wherein the step of recovering said insoluble material of said cell culture further comprises centrifuging said lysed cells to obtain a first cell pellet. [The present invention 1036] The step of recovering the insoluble material comprises the steps of: a. incubating the cell pellet with a solution containing 4M urea at a 10:1 urea volume:pellet mass ratio; and b. centrifuging the solution containing 4M urea to obtain a second cell pellet, and then incubating the second cell pellet in the aqueous solution containing a solvent; Any of the methods of claims 1001 to 1035, further comprising: [This invention 1037] 1037. The method of any of claims 1001 to 1036, further comprising the step of isolating said recombinant spider silk protein from said aqueous solution, thereby producing an isolated recombinant spider silk protein. [The present invention 1038] 8. The method of any one of claims 1001 to 1037, wherein said recombinant spider silk protein is a highly crystalline silk protein, a silk protein with a high beta sheet content, or a low solubility silk protein. [This invention 1039] 1038. The method of claim 1038, wherein the recombinant spider silk protein comprises the Uloborus diversus MiSP protein set forth in SEQ ID NO: 23. [The present invention 1040] 1038. The method of claim 1038, wherein said recombinant spider silk protein has a solubility threshold in a non-chaotropic solvent of less than 90%, 80%, 70%, 60%, or 50%. [This invention 1041] The method of any of claims 1001 to 1040, wherein said cell culture comprises fungal cells, bacterial cells, or yeast cells. [The present invention 1042] The method according to any one of claims 1001 to 1041, wherein said bacterial cells are Escherichia coli cells. [This invention 1043] Any of the preceding methods of the invention, wherein the amount of isolated recombinant spider silk protein is measured using ELISA. [This invention 1044] 104. The method of any of claims 1001 to 1043, wherein the amount of isolated recombinant spider silk protein is measured using size exclusion chromatography. [This invention 1045] The method of any of claims 1001 to 1044, wherein said isolated recombinant spider silk protein is a full-length recombinant spider silk protein. [The present invention 1046] 1045. The method of claim 1045, wherein said isolated recombinant spider silk protein comprises 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 full-length recombinant spider silk protein. [This invention 1047] 1045. The method of claim 1045, wherein the amount of full-length recombinant spider silk protein is measured using ELISA. [This invention 1048] 104. The method of claim 1045, wherein the amount of full-length recombinant spider silk protein is measured using size exclusion chromatography. [This invention 1049] Any of the methods of claims 1001 to 1048, 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%, 95-95%, or 95-100%. [The present invention 1050] 1. A method for isolating recombinant spider silk proteins from a host cell, the method comprising the steps of: a. providing an insoluble material comprising a recombinant spider silk protein; b. adding the insoluble material to an aqueous solution containing a solvent, the aqueous solution containing 15% (w / v) of the insoluble portion at a final 10 M urea concentration; c. applying shear force to the aqueous solution containing the insoluble material via microfluidization, thereby solubilizing the recombinant spider silk protein in the aqueous solution; and d. isolating said recombinant spider silk protein from said aqueous solution, thereby producing an isolated recombinant spider silk protein. The method comprising: [This invention 1051] 1. A method for isolating recombinant spider silk proteins from a host cell, the method comprising the steps of: a. providing an insoluble material comprising a recombinant spider silk protein; b. adding the insoluble material to an aqueous solution, the aqueous solution containing about 15% (w / v) of the insoluble portion at a final 10 M urea concentration; c. applying a shear force via microfluidization, wherein the shear force is about 10×10 to the aqueous solution. 6 s -1 thereby solubilizing the recombinant spider silk protein in the aqueous solution; and d. isolating said recombinant spider silk protein from said aqueous solution, thereby producing an isolated recombinant spider silk protein. The method comprising: [This invention 1052] A composition comprising a recombinant spider silk protein produced by any of the methods of inventions 1001 to 1051. [This invention 1053] 1052. The composition of claim 1052, wherein said 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. [This invention 1054] A silk fiber comprising a recombinant spider silk protein produced by any of the methods of inventions 1001 to 1053. [Brief explanation of the drawings]

[0043] A brief description of some of the figures in the drawing These and other features, aspects, and advantages of the present methods and compositions described herein will become better understood with reference to the following description and accompanying drawings.

[0044] [Figure 1A] A shows the calculated shear rate as a function of pressure in various types of interaction chambers. [Figure 1B] B shows the calculated flow rate as a function of pressure in various types of single-slot interaction chambers. [Figure 2] SEC plot of the Uloborus dibruss silk protein Misp (SEQ ID NO: 23) after extraction with 10 M urea and microfluidization. The arrow points to the MiSp protein peak. DETAILED DESCRIPTION OF THE INVENTION

[0045] Detailed Description definition Terms used in the claims and specification are defined as set forth below unless otherwise specified.

[0046] Unless defined herein, scientific and technical terms used in connection with the methods and compositions described herein shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless the context clearly dictates otherwise, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature and techniques used in connection with biochemistry, enzymology, molecular and cell biology, microbiology, genetics, and polypeptide and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art.

[0047] The methods and techniques of the present invention are generally performed, unless otherwise indicated, according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (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, NY(1990);Taylor and Drickamer,Introduction to Glycobiology,Oxford Univ.Press(2003);Worthington Enzyme Manual,Worthington Biochemical Corp.,Freehold,NJ;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 See Press (1999).

[0048] All publications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0049] The following terms, unless otherwise specified, shall be understood to have the following meanings:

[0050] The term "in vitro" refers to processes that occur away from an organism, for example, in living cells grown in tissue culture.

[0051] The term "in vivo" refers to a process that occurs within a living organism.

[0052] As used herein, the term "clarifying" refers to a method of removing host cell biomass, such as whole cells, lysed cells, membranes, lipids, organelles, nuclei, non-spider silk proteins, or other unwanted cellular parts or products, or any other undesirable part of a cell culture. Purifying can also refer to the removal of impurities from a partially purified or separated spider silk composition. Impurities 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 other undesirable materials.

[0053] As used herein, the term "purity" refers to the fraction of all isolated components in a sample, e.g., an extracted sample, e.g., the amount of substantially full-length isolated recombinant spider silk protein as part of or as a fragment of the isolated recombinant spider silk protein, lipid, protein, membrane, or other molecule. In some embodiments, the full-length recombinant spider silk protein is at least 90-100% the length of the known full-length protein. In some embodiments, the full-length recombinant spider silk protein is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% the length of the known full-length protein.

[0054] As used herein, the term "yield" refers to the total amount of spider silk recovered, including spider silk protein fragments and substantially full-length spider silk, compared to the starting amount of spider silk.

[0055] The term "shear force" refers to a force acting parallel or tangential to a surface or planar cross-section of an object or material. The term "shear rate," in reference to fluids, refers to the rate of change of velocity at which one layer of fluid passes over an adjacent layer. For example, shear rate occurs when two parallel planes of fluid move at different speeds.

[0056] The term "soluble silk protein" refers to the protein remaining in the supernatant after thorough centrifugation, an example of thorough centrifugation is a 50 mL aliquot sample of silk protein found after centrifugation at 15,000 x g for 20 minutes at room temperature in a 50 mL conical centrifuge tube.

[0057] The term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides having a length of at least 10 bases. Such terms include DNA molecules (e.g., cDNA or genomic DNA or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-natural internucleoside linkages, or both. Nucleic acids can be in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or padlocked conformation.

[0058] Unless otherwise specified, for any sequence described herein in the general format of "SEQ ID NO:," for example, a "nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid having, at least in part, (i) the sequence set forth in SEQ ID NO: 1, or (ii) a sequence complementary to SEQ ID NO: 1. The alternative is dictated by the context. For example, if the nucleic acid is being used as a probe, the alternative is dictated by the requirement that the probe be complementary to the desired target.

[0059] "Isolated" RNA, DNA, or mixed polymers are those that are substantially separated from other cellular components that naturally accompany the natural polynucleotide in the natural host cell to which they belong, such as naturally associated ribosomes, polymerases, and genomic sequences.

[0060] The term "recombinant" refers to a biological molecule, e.g., a gene or polypeptide, that (1) has been removed from its natural environment, (2) is not associated in nature with all or part of a polynucleotide with which the gene is found, (3) is operably linked to a polynucleotide with which it is not linked in nature, or (4) is not naturally occurring. The term "recombinant" can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized in heterologous systems, as well as the polypeptides and / or mRNAs encoded by such nucleic acids.

[0061] As used herein, an endogenous nucleic acid sequence is considered "recombinant" when a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence in the genome of an organism to alter the expression of the endogenous nucleic acid sequence (or the polypeptide product encoded by that sequence). 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 itself endogenous (from the same host cell or its progeny) or exogenous (from a different host cell or its progeny). As an example, a promoter sequence replaces the native promoter of a gene present in the genome of a host cell (e.g., by homologous recombination) to alter the expression pattern of the gene. The gene is considered "recombinant" because it is separated from at least some of the sequences that naturally flank it. In certain embodiments, the heterologous nucleic acid molecule is not endogenous to the organism. In further embodiments, the heterologous nucleic acid molecule is a plasmid or molecule that has been integrated into a host chromosome by homologous or random integration.

[0062] A nucleic acid is also considered "recombinant" if it contains any alteration that does not occur naturally relative 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 introduced artificially, such as by human intervention. "Recombinant nucleic acid" also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0063] The term "percent sequence identity" in the context of nucleic acid sequences refers to the residues that are the same between two sequences when aligned for maximum correspondence. The length of sequence identity comparison can be over a stretch of at least about 9 nucleotides, typically at least about 20 nucleotides, more typically at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 nucleotides or more. There are many 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, programs in the Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignment and percent sequence identity of the regions of optimal overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (the entire contents of which are incorporated herein by reference). For example, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (word size of 6 and NOPAM factor for the scoring matrix) or using Gap with default parameters as provided in GCG Version 6.1, which is incorporated herein by reference.Alternatively, sequences can be compared using the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), in particular blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0064] As used herein, the term "substantial homology" or "substantial similarity," when referring to a nucleic acid or fragment thereof, indicates that when optimally aligned with another nucleic acid (or its complementary strand) using appropriate nucleotide insertions or deletions, there is nucleotide sequence identity, as measured by well-known sequence identity algorithms such as FASTA, BLAST, or Gap, as described above, of at least about 76%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98%, or 99% of the nucleotide bases.

[0065] Nucleic acids (also known as polynucleotides) can include both sense and antisense strands of RNA, cDNA, and genomic DNA, as well as synthetic forms and mixed polymers thereof. They can be chemically or biochemically modified or contain non-natural or derivatized nucleotide bases, as will be readily recognized by those skilled in the art. Such modifications include, for example, labels, methylation, substitution of one or more analogs of naturally occurring nucleotides, uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendant moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralens, etc.), chelators, alkylators, and internucleotide modifications such as modified linkages (e.g., alpha-anomeric nucleic acids, etc.). Synthetic molecules that mimic polynucleotides in their ability to bind to designated sequences using hydrogen bonding and other chemical interactions are also included. Such molecules are known in the art and include, for example, those in which peptide linkages replace phosphate linkages in the backbone of the molecule. Other modifications may include, for example, analogs in which the ribose ring contains bridging moieties or other structures, such as modifications found in "locked" nucleic acids.

[0066] The term "mutated," when used with reference to a nucleic acid sequence, means that nucleotides within a nucleic acid sequence have been inserted, deleted, or changed compared to a reference nucleic acid sequence. A single change can be made at one locus (point mutation), or multiple nucleotides can be inserted, deleted, or changed at a single locus. Furthermore, one or more changes can be made at any number of loci within a nucleic acid sequence. Nucleic acid sequences may be mutated by any method known in the art, including, but not limited to, mutagenesis techniques such as "error-prone PCR" (a method in which PCR is performed under conditions of low DNA polymerase copying fidelity, resulting in a high proportion of point mutations throughout the 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 in which site-specific mutations are generated in a cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).

[0067] 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 obtained by polymerase chain reaction (PCR) amplification or by treating a circular plasmid with a restriction enzyme. 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 can be ligated into the viral genome (discussed in more detail below). Some vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., vectors having an origin of replication that functions in the host cell). Other vectors, upon introduction into a host cell, integrate into the genome of the host cell, thereby replicating along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of genes operably linked to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors").

[0068] As used herein, the term "expression system" includes vehicles or vectors for the expression of a gene in a host cell as well as vehicles or vectors that result in the stable integration of a gene into a host chromosome.

[0069] "Operatively linked" or "operably linked" expression control sequences refer to a linkage that controls a gene of interest by placing the expression control sequence in contiguous with the gene of interest, as well as expression control sequences that function in trans or at some distance with respect to the gene of interest.

[0070] As used herein, the term "expression control sequence" refers to a polynucleotide sequence necessary to affect the expression of a coding sequence operably linked thereto. Expression control sequences are sequences that control the transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; signals for efficient RNA processing, such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that increase translation efficiency (e.g., ribosome binding sites); sequences that increase polypeptide stability; and, optionally, sequences that enhance polypeptide secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include, at a minimum, all elements whose presence is essential for expression, and may also include additional elements whose presence is advantageous, such as leader sequences and fusion partner sequences.

[0071] As used herein, the term "promoter" refers to a DNA region located 5' to the mRNA transcription start site to which RNA polymerase binds to initiate gene transcription.

[0072] 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. Such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications, either due to mutation or environmental influences, may occur in subsequent generations, such progeny may not, in fact, be identical to the parent cell, but are still within the scope of the term "host cell" as used herein. A recombinant host cell can be an isolated cell or cell line grown in culture, or it can be a cell that is present in a living tissue or organism.

[0073] As used herein, the term "polypeptide" includes both naturally occurring and non-naturally occurring proteins, as well as fragments, mutants, derivatives, and analogs thereof. Polypeptides can be monomeric or polymeric. Furthermore, polypeptides can contain a number of different domains, each with one or more distinct activities.

[0074] 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.

[0075] As used herein, the term "block" or "repeating unit" refers to a subsequence of more than about 12 amino acids in a natural silk polypeptide that is found repeatedly, possibly with moderate variation, in the natural silk polypeptide sequence and serves as the basic repeating unit in the silk polypeptide sequence. A block may, but does not necessarily, include a very short "motif." A "motif" refers to a sequence of about 2-10 amino acids that occurs in multiple blocks. For example, a motif may consist of the amino acid sequence GGA, GPG, or AAAAA (SEQ ID NO: 38). An arrangement of multiple blocks is a "block copolymer."

[0076] As used herein, the term "repeat domain" refers to a sequence selected from a set of contiguous (uninterrupted by substantially non-repetitive domains, excluding known silk spacer elements) repeat segments in a silk polypeptide. A natural silk sequence generally contains one repeat domain. In some embodiments, there is one repeat domain per silk molecule. A "macrorepeat" is a naturally occurring repetitive amino acid sequence that contains multiple blocks. In some embodiments, a macrorepeat has at least two repeats in the repeat domain. In further embodiments, the two repeats are not complete. As used herein, a "quasi-repeat" is an amino acid sequence that contains multiple blocks, where the blocks are similar but not identical in amino acid sequence.

[0077] As used herein, the term "repeat sequence" or "R" refers to a repeated amino acid sequence. In one embodiment, the repeat sequence comprises a macrorepeat, or a fragment of a macrorepeat. In another embodiment, the repeat sequence comprises a block. In a further embodiment, the single block is divided into two repeat sequences.

[0078] The term "about" refers to and includes the indicated value, as well as a range above and below that value. In certain embodiments, the term "about" refers to ±10%, ±5%, or ±1% of the given value. In certain embodiments, where applicable, the term "about" refers to the given value(s) ± one standard deviation around that value(s).

[0079] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural unless the context clearly dictates otherwise.

[0080] Ranges set forth herein should be understood to be inclusive and to be shorthand for all values ​​within the range. For example, the range 1 to 50 should be understood to include any number, combination of numbers, or subranges from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50. Additionally, the range of 2-5% includes 2% and 5% and every number or fraction of a number therebetween, for example: 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, and 4.75%.

[0081] Methods for solubilizing and purifying recombinant proteins Recombinant spider silk proteins expressed in cell culture must be purified from cellular components. In some cases, silk proteins are sequestered in insoluble cellular debris or form insoluble silk protein aggregates. Insoluble silk proteins are difficult to purify and also result in low recombinant silk protein recovery. In addition, some isolated spider silk protein solids can be insoluble. For example, MBI 18B silk powder has low solubility, and some model spider silks, such as U. diversus MiSp, are known to be poorly soluble. In such cases, various methods can be applied to the insoluble material, aggregates, or silk solids to release and solubilize the silk proteins for purification, thereby increasing the recombinant silk protein recovery. Additionally, even under conditions where silk dissolves (i.e., where silk protein dissolution is thermodynamically favorable), the rate of solubilization can be slow. This slow rate may be due to the large activation energy required for the solubilization reaction or the rate-limiting mass transfer of silk from cell debris particles. In both cases, where silk aggregates are insoluble or where silk is soluble but solubilization is slow, applying a large physical energy process, such as strong shear force through homogenization or microfluidization, can promote the solubilization of silk proteins. Strong shear force either imparts energy to the solution to increase the reaction rate, thereby overcoming the large activation energy, or breaks down cell particles and silk protein aggregates, suppressing mass transport effects. Therefore, applying physical energy in the form of shear force can increase the solubility, and therefore the recovery, of recombinant spider silk proteins.

[0082] Described herein are methods for solubilizing, isolating, and purifying recombinant spider silk proteins by applying high physical energy processes, such as shear force, shear rate, impact, and cavitation. In some embodiments, the high physical energy process is applied via homogenization or microfluidization. Exposing recombinant proteins to high physical energy processes can increase the solubilization of the protein in aqueous solutions, thereby improving the purification and recovery of the recombinant protein.

[0083] In some embodiments, the insoluble material is a cell pellet. In some embodiments, the insoluble material is a cell lysate. In some embodiments, the insoluble material is an isolated spider silk solid, bulk powder, or extrudate. The silk solid or recombinant silk solid is an isolated recombinant spider silk composition, such as a fiber, extrudate, powder, or pellet. The extrudate is an extruded recombinant spider silk composition, which has been extruded through a spinneret.

[0084] Physical Energy and Shear Force Physical energy can be applied to insoluble recombinant spider silk proteins to increase the solubility of the proteins. Physical energy refers to dynamic or mechanical energy exerted on insoluble recombinant spider silk by applying mechanical force, such as compression or contraction, pressure, flow rate, impact, cavitation, shear force, shear rate, shear stress, stretching, or any combination thereof, or other suitable mechanical force known in the art. The application of this mechanical energy increases the solubility of the recombinant protein in solution. Generally, any method that provides kinetic or mechanical energy that induces controlled damage to the recombinant protein can be used to solubilize the recombinant protein. The solubility of insoluble recombinant proteins can be increased, for example, by mechanical strain or shear force-induced pressure, where the recombinant protein or cell lysate is subjected to deformation, contraction, rapid stretching, rapid compression, or high shear rate pulses.

[0085] In some embodiments, shear forces include, but are not limited to, shear rate and other physical energy processes such as impaction, cavitation, and turbulent mixing produced by high energy fluid processors.

[0086] In some embodiments, the applied physical energy is shear rate, shear force, cavitation, impact, pressure, sonication, emulsion, or any other suitable application method known in the art. In some aspects, the physical energy is applied using homogenization, microfluidized microemulsion, or a French press. In some embodiments, the physical energy is shear force. In some embodiments, shear force generates shear rate. In some embodiments, the physical energy is pressure. In some embodiments, the physical energy is applied via homogenization or microfluidization. In some embodiments, the sonication is ultra-sonication.

[0087] A variety of devices capable of imparting physical energy are available, including high-energy fluid processors, microfluidizers, French presses, high-pressure homogenizers, bead mills, rotary blenders, and rotor / stator devices. In some embodiments described herein, recombinant proteins may be solubilized using a microfluidizer. Commercially available microfluidizers include the M110EH, M815, M700, LV1, LM10, LM20, M110Y, or M110P microfluidizers manufactured by Microfluidics Corp (Westwood, MA).

[0088] A microfluidizer consists of an exchangeable, fixed-geometry interaction chamber (e.g., G10Z, H10Z, H30Z, H210Z, L30Z, F20Y, or F12Y interaction chamber) and is pumped to initiate flow. The Y-shaped interaction chamber splits the incoming stream into two or more streams, which then converge at high speed, resulting in steep velocity and pressure gradients, shear, cavitation, and heating. The fluid in the interaction chamber is subjected to high velocity flow and uniform shear forces, resulting in shear rates. The intensity of homogenization can be varied by changing the interaction chamber geometry, temperature, pressure, or by processing the same material multiple times using the device. There is also a complex interplay between the concentration of the input material, the composition of the buffer, and the physicochemical properties of the solution, emulsion, or suspension. Several parameters, such as pressure, capillary diameter, temperature, number of homogenization passes, and buffer conditions, can affect the solubilization of recombinant proteins. Additionally, the interaction chamber can have one channel (single-slot interaction chamber) or three or more channels (multi-slot interaction chamber). Using a multi-slot interaction chamber, the volumetric flow rate through the interaction chamber can be increased, allowing for a larger sample volume to be processed. The flow rate of fluid in the microfluidizer chamber can reach 500 m / s using channels as small as 50 μm. Varying the pressure alters the shear rate as the fluid moves through the interaction chamber.

[0089] Any suitable homogenizer or microfluidizer known in the art can be used. Microfluidizers and high-pressure homogenizers are commercially available from various vendors, such as Microfluidics (Westwood, MA), Thomas Scientific (Swedesboro, NJ), CAT Scientific (Paso Robles, CA), and Thermo Fisher Scientific. In some embodiments, the microfluidizer comprises a Z-shaped interaction chamber. In some embodiments, the microfluidizer comprises a Y-shaped interaction chamber. In some embodiments, the microfluidizer is an M-110P or LM10 microfluidizer.

[0090] The shear rate generated by a particular combination of Microfluidizer interaction chamber and pressure used for sample processing can be determined using information provided by the chamber manufacturer. Examples of calculated shear rates as a function of pressure for various types of interaction chambers for a single-slot Microfluidizer brand are shown in Figure 1A. Figure 1B shows flow rates as a function of pressure for various types of interaction chambers for a single-slot Microfluidizer brand. Figures 1A and 1B are adapted from the 2014 Microfluidics Processor User Guide, created by Microfluidics™. As shown in Figure 1A, the shear rate for the same volume of fluid in two different chambers (e.g., an F12Y chamber and an L30Z chamber at 30,000 psi) is 10 × 10, respectively. 6 s -1 and the shear rate generated in these chambers is 2 × 10 6 s -1 A difference of nearly an order of magnitude was observed compared to the previous study. Thus, varying the pressure on the fluid passing through the different interaction chambers results in different amounts of shear force and shear rate. Also, the shear rate value can be varied and optimized by changing the fluid pressure in the selected interaction chamber.

[0091] Applying shear force to a fluid or solution generates a fluid shear rate. In some embodiments, the shear force is applied using a microfluidizer. In some embodiments, the shear rate is generated by a microfluidizer. In some embodiments, the shear rate is greater than 1×10 3 s -1 ~1×10 9 s -1 The shear rate can be between about 1×10 3 s -1 , 1.5×10 3 s -1 , 2 × 10 3 s -1 , 2.5×10 3 s -1 , 3×10 3 s -1 , 3.5×10 3 s -1 , 4×10 3 s -1 , 4.5×10 3 s -1 , 5×10 3 s -1 , 5.5×10 3 s -1 , 6×10 3 s -1 , 6.5×10 3 s -1 , 7×10 3 s -1 , 7.5×10 3 s -1 , 8×10 3 s -1 , 8.5×10 3 s -1 , 9×10 3 s -1 , 9.5×10 3 s -1 , 1×10 4 s -1 , 1.5×10 4 s -1 , 2 × 10 4 s -1 , 2.5×10 4 s -1 , 3×10 4 s -1 , 3.5×104 s -1 、4×10 4 s -1 、4.5×10 4 s -1 、5×10 4 s -1 、5.5×10 4 s -1 、6×10 4 s -1 、6.5×10 4 s -1 、7×10 4 s -1 、7.5×10 4 s -1 、8×10 4 s -1 、8.5×10 4 s -1 、9×10 4 s -1 、9.5×10 4 s -1 、1×10 5 s -1 、1.5×10 5 s -1 、2×10 5 s -1 、2.5×10 5 s -1 、3×10 5 s -1 、3.5×10 5 s -1 、4×10 5 s -1 、4.5×10 5 s -1 、5×10 5 s -1 、5.5×10 5 s -1 、6×10 5 s -1 、6.5×10 5 s -1 、7×10 5 s -1 、7.5×10 5 s -1 、8×10 5 s -1 、8.5×10 5 s -1 、9×10 5 s -1 、9.5×10 5s -1 、1×10 6 s -1 、1.5×10 6 s -1 、2×10 6 s -1 、2.5×10 6 s -1 、3×10 6 s -1 、3.5×10 6 s -1 、4×10 6 s -1 、4.5×10 6 s -1 、5×10 6 s -1 、5.5×10 6 s -1 、6×10 6 s -1 、6.5×10 6 s -1 、7×10 6 s -1 、7.5×10 6 s -1 、8×10 6 s -1 、8.5×10 6 s -1 、9×10 6 s -1 、9.5×10 6 s -1 、1×10 7 s -1 、1.5×10 7 s -1 、2×10 7 s -1 、2.5×10 7 s -1 、3×10 7 s -1 、3.5×10 7 s -1 、4×10 7 s -1 、4.5×10 7 s -1 、5×10 7 s -1 、5.5×10 7 s -1 、6×10 7 s -1 、6.5×10 7 s-1 , 7×10 7 s -1 , 7.5×10 7 s -1 , 8×10 7 s -1 , 8.5×10 7 s -1 , 9×10 7 s -1 , 9.5×10 7 s -1 , 1×10 8 s -1 , 1.5×10 8 s -1 , 2 × 10 8 s -1 , 2.5×10 8 s -1 , 3×10 8 s -1 , 3.5×10 8 s -1 , 4×10 8 s -1 , 4.5×10 8 s -1 , 5×10 8 s -1 , 5.5×10 8 s -1 , 6×10 8 s -1 , 6.5×10 8 s -1 , 7×10 8 s -1 , 7.5×10 8 s -1 , 8×10 8 s -1 , 8.5×10 8 s -1 , 9×10 8 s -1 , 9.5×10 8 s -1 , 1×10 9 s -1 In some embodiments, the shear rate is about 6.5×10 6 s -1 In some embodiments, the shear rate is about 9.5×10 6 s -1 is.

[0092] The pressure can be between about 500 and 50,000 psi, and can be at least about 500 psi, 750 psi, 1,000 psi, 2,000 psi, 3,000 psi, 4,000 psi, 5,000 psi, 10,000 psi, 15,000 psi, 20,000 psi, 25,000 psi, 20,000 psi, 25,000 psi, 40,000 psi, 45,000 psi, or 50,000 psi. This pressure can be increased to approximately 500-50,000 psi, 500-1,000 psi, 1,000-5,000 psi, 5,000-10,000 psi, 7,500-12,000 psi, 10,000-15,000 psi, 15,000-20,000 psi, 15,000-22,000 psi, 18,000-25,000 psi, 18,000-22,000 psi, The pressure can be between 20,000 and 25,000 psi, 25,000 and 30,000 psi, 27,500 and 30,000 psi, 27,500 and 32,000 psi, 30,000 and 32,000 psi, 30,000 and 35,000 psi, 35,000 and 40,000 psi, 40,000 and 45,000 psi, or 45,000 and 50,000 psi. In some embodiments, the pressure is about 10,000 psi, 20,000 psi, 23,000 psi, or 30,000 psi. In some embodiments, the pressure is about 23,000 psi. In some embodiments, the pressure is about 30,000 psi. In one embodiment, the pressure is between 10,000 and 30,000 psi.

[0093] The spider silk protein can be treated with physical energy at least once, i.e., pressure, shear force, and / or shear rate from a microfluidizer or homogenizer is applied to the spider silk protein once. In some embodiments, pressure, shear force, and / or shear rate is applied once. The spider silk protein can also be treated with physical energy multiple times, i.e., pressure, shear force, and / or shear rate is applied to the spider silk protein two, three, four, or more times. In some embodiments, pressure, shear force, and / or shear rate is applied three times. In some embodiments, pressure, shear force, and / or shear rate is applied two times.

[0094] The physical energy, i.e., applied pressure, shear force, and / or shear rate, can be the same for each iterative pass or run. For example, the sample can be treated at 30,000 psi for the first pass, second pass, and third pass. In other embodiments, the pressure can be varied for each pass or run. For example, the sample can be treated at 30,000 psi for the first pass, 23,000 psi for the second pass, and 10,000 psi for the third pass. In another example, the sample can be treated at 6.5×10 psi for the first pass, 23,000 psi for the second pass, and 10,000 psi for the third pass. 6 s -1 , and 9.5×10 for the second pass. 6 s -1 , and 5.5×10 for the third pass. 6 s -1 In some embodiments, the pressure is in absolute pressure per square inch (psia). In some embodiments, the pressure is in gauge pressure per square inch (psig).

[0095] In some aspects, the physical energy is acoustic energy applied using sonication. In such instances, the application of sound waves to a solution generates cavitation within the solution, leading to the nucleation, growth, and collapse of gas bubbles in the solution, resulting in mechanical and physical deformation of the recombinant protein in the solution, thus increasing its solubility.

[0096] Solvent and buffer conditions Buffer conditions for solutions of recombinant proteins and insoluble cell fractions, pellets, or lysates can also be varied to optimize recombinant protein homogenization or microfluidization and solubilization. Recombinant silk polypeptides aggregate and form β-sheet structures, resulting in undesirable insoluble aggregates during production and purification. Harsh chemical conditions, such as high molar concentrations of chaotropes, are required to solubilize these biological molecules. Furthermore, the conditions required to resolubilize peptides during purification often lead to protein degradation, resulting in low yields and poor fiber tenacity and texture. However, the combination of low concentrations of chaotropes and the application of high physical energy, such as microfluidization or homogenization, can increase solubility and suppress recombinant protein degradation.

[0097] In some embodiments, a solvent can be added to the insoluble cell fraction, pellet, or lysate to solubilize the recombinant spider silk protein. Any suitable solvent known in the art can be used, including, but not limited to, chaotropes and organic solvents. In some embodiments, the solvent is a chaotrope. Any suitable chaotrope known in the art can be used, including, but not limited to, guanidine chloride (GdnHCl), guanidine thiocyanate (GdnSCN), guanidine isothiocyanate, n-butanol, ethanol, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. In some embodiments, the solvent is guanidine chloride (GdnHCl). In some embodiments, the solvent is guanidine thiocyanate (GdnSCN). In some embodiments, the solvent is urea.

[0098] In some embodiments, the solvent is formulated in a water buffer. In some embodiments, the solvent is formulated in a 50 mM Tris, pH 7.5 buffer. The solvent can be formulated using any suitable buffer known in the art, such as, but not limited to, phosphate buffered saline (PBS) or Good's buffer, such as Tris, Tricine, MES, PIPES, ACES, MOPS, MOPSO, TES, HEPES, TAPS, Bicine, TES, bis-trispropane, bis-trismethane, ADA, HEPBS, CHES, AMP, CAPS, CAPSO, glycinamide, glycylglycine, or other suitable buffers.

[0099] The solvent (e.g., achaotrope) can be added directly to the insoluble cell fraction, pellet, or lysate, or can be added as a component of an aqueous buffer. The concentration of the solvent in the aqueous buffer can vary, as determined by one of skill in the art. In some embodiments, the concentration of the solvent in the aqueous buffer can be between 0.01-10 M, 0.01-0.1 M, 0.1-0.5 M, 0.5-1 M, 1-2 M, 2-3 M, 3-4 M, 4-5 M, 5-6 M, 6-7 M, 7-8 M, 8-9 M, 9-10 M, or 10 M or higher. In some embodiments, the concentration of the solvent in the aqueous buffer can be at least about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, 5M, 5.5M, 6M, 6.5M, 7M, 7.5M, 8M, 8.5M, 9M, 9.5M, or 10M or more.

[0100] In some embodiments, a solvent is added to the insoluble cell fraction, pellet, or lysate at a specific mass-to-volume ratio. In such embodiments, the total mass of the insoluble fraction, pellet, or lysate is determined, and a specific volume of a solution containing a specific concentration of solvent or chaotrope is added. For example, a cell pellet is weighed and resuspended in a solution containing a chaotropic agent so that the final volume of the cell population is 15% of the total volume of the sample (e.g., a 0.75 mg cell pellet is resuspended in 4.25 ml of a buffer solution containing 0.01-10 M chaotrope solution). In another example, a cell pellet is weighed and resuspended in an equal volume of a solution containing solvent to achieve a 50% cell population to solvent volume ratio.

[0101] In some embodiments, the ratio of cell population to solvent volume can be between 1-100%, 1-5%, 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% cell population. In some embodiments, the ratio of cell population to solvent volume can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% cell population.

[0102] In some embodiments, the ratio of cell population to solvent volume can be between 1-100%, 1-5%, 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% solvent volume. In some embodiments, the ratio of cell population to solvent volume can be at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% solvent volume.

[0103] In some embodiments, the final concentration of the aqueous buffer and solvent in the solution containing the insoluble cell portion, pellet, or lysate can be between 0.01-10 M, 0.01-0.1 M, 0.1-0.5 M, 0.5-1 M, 1-2 M, 2-3 M, 3-4 M, 4-5 M, 5-6 M, 6-7 M, 7-8 M, 8-9 M, or 9-10 M. In some embodiments, the final concentration of the aqueous buffer and solvent in the solution containing the cell lysate or pellet can be at least about 0.1M, 0.15M, 0.2M, 0.25M, 0.3M, 0.35M, 0.4M, 0.45M, 0.5M, 0.55M, 0.6M, 0.65M, 0.7M, 0.75M, 0.8M, 0.85M, 0.9M, 0.95M, 1M, 1.5M, 2M, 2.5M, 3M, 3.5M, 4M, 4.5M, 5M, 5.5M, 6M, 6.5M, 7M, 7.5M, 8M, 8.5M, 9M, 9.5M, or 10M.

[0104] Additional buffer modifiers that affect the structural properties of the vesicles may also be used, such as shear protectants, viscosity modifiers, and / or solutes. Excipients that improve the efficiency of homogenization or microfluidization, such as membrane softeners and molecular crowding agents, may also be added. Other modifications to the buffer may include specific pH ranges and / or concentrations of salts, organic solvents, small molecules, surfactants, zwitterions, amino acids, polymers, and / or any combination of the concentrations of the above.

[0105] In some embodiments, the insoluble cell fraction, pellet, or lysate is incubated with an aqueous solution containing a solvent for a defined period of time. The incubation time for the cell pellet or lysate with the solution can be varied to enhance solubilization of the spider silk protein or reduce potential protein degradation. The incubation time can be between 1 minute and 3 hours (180 minutes), 1 minute and 60 minutes, 3 minutes and 90 minutes, 60 minutes and 120 minutes, 90 minutes and 150 minutes, or 120 minutes and 180 minutes. The incubation time can be at least 1 minute, 5 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes, 105 minutes, 120 minutes, 135 minutes, 150 minutes, 165 minutes, 180 minutes, or longer. In some embodiments, the incubation time is 60 minutes. In some embodiments, the incubation time is 75 minutes. In some embodiments, the incubation time is 90 minutes. In some embodiments, the incubation time is 105 minutes. In some embodiments, the incubation time is 120 minutes.

[0106] The insoluble cell portion, pellet, or lysate can be incubated with the aqueous solution at 5-70° C. In some embodiments, the insoluble cell portion, pellet, or lysate is incubated with the aqueous solution at 5-10° C., 10-20° C., 10-15° C., 15-20° C., 20-30° C., 20-22° C., 20-25° C., 22-27° C., 25-27° C., 25-30° C., 27-30° C., 30-40° C., 40-50° C., 40-45° C., 45-50° C., 50-60° C., 50-55° C., 55-60° C., 60-70° C., 60-65° C., or 65-70° C. In some embodiments, the insoluble cell portion, pellet, or lysate is incubated with the aqueous solution at 20-30° C. In some embodiments, the insoluble cell portion, pellet, or lysate is incubated with the aqueous solution at 25° C. In some embodiments, the insoluble cell portion, pellet, or lysate is incubated with the aqueous solution at room temperature. In some embodiments, the insoluble cell portion, pellet, or lysate is incubated with the aqueous solution at 30° C. or below.

[0107] In some embodiments, the recombinant spider silk protein is expressed in the cytoplasm of the host cell. To isolate the protein, the host cell must be lysed to release the recombinant spider silk protein. Any suitable method can be used to lyse the host cells, including, but not limited to, heat treatment, chemical treatment, shear disruption, physical homogenization, microfluidization, sonication, or chemical homogenization. Chemical treatment includes incubating the cells with chemicals or enzymes known to disrupt the plasma membrane of prokaryotic and eukaryotic cells, such as detergents such as Triton X-100, Nonidet P-40, CHAPS, sodium dodecyl sulfate (SDS), or other suitable detergents.

[0108] After lysing the cells, the insoluble portion containing the recombinant spider silk protein can be recovered by centrifuging the cell lysate, resulting in a cell pellet of insoluble material containing the recombinant spider silk protein. The centrifugation speed required to pellet the insoluble recombinant protein can be determined by one of skill in the art. In some embodiments, the centrifugation speed is between 100 and 10,000 x g. In some embodiments, the centrifugation speed is 100 x g, 200 x g, 300 x g, 400 x g, 500 x g, 600 x g, 700 x g, 800 x g, 900 x g, 1000 x g, 2000 x g, 3000 x g, 4000 x g, 5000 x g, 6000 x g, 7000 x g, 8000 x g, 9000 x g, or 10,000 x g.

[0109] In some cases, the insoluble cell fraction or pellet can be resuspended or washed with a chaotrope, such as a urea solution, followed by another centrifugation to produce a second cell pellet. This second cell pellet is then incubated with an aqueous solvent, and physical force is applied to solubilize the recombinant spider silk protein. The molar concentration of the chaotrope in this wash solution can be between 0.1 and 10 M. In some embodiments, the chaotrope is urea. In some embodiments, the chaotrope is 4 M urea.

[0110] In some embodiments, biological or chemical impurities of non-spider silk proteins can be removed from cell lysates or cell pellets. Removal of impurities from cell lysates or cell pellets can be achieved by filtration, absorption (e.g., charcoal or solid absorption), dialysis, and coacervation, or by inducing phase separation using various chemicals. In other embodiments, phase separation can be chemically induced by adding kosmotropes and / or compounds used to precipitate proteins from solution.

[0111] In some embodiments, filtration, microfiltration, diafiltration, and / or ultrafiltration (e.g., against deionized water) are used to remove impurities. Suitable membranes for microfiltration may include those from 0.1 uM to 1 uM. Examples of membranes suitable for ultrafiltration include, but are not limited to, hydrophobic membranes (e.g., PES, PS, cellulose acetate) with molecular weight cutoffs between 50 kDa and 800 kDa, 100 kDa and 800 kDa, 200 kDa and 800 kDa, 300 kDa and 800 kDa, 400 kDa and 800 kDa, 500 kDa and 800 kDa, 600 kDa and 800 kDa, 700 kDa and 800 kDa, 100 kDa and 700 kDa, 200 kDa and 700 kDa, 300 kDa and 700 kDa, 400 kDa and 700 kDa, 500 kDa and 700 kDa, 600 kDa and 700 kDa, or 500 kDa and 600 kDa. In some embodiments, ultrafiltration separates the recombinant protein slurry into a water-containing retentate and a permeate containing impurities. Suitable conditions for ultrafiltration (e.g., membrane, temperature, volume displacement) can be determined using methods known in the art with the goal of maximizing filtrate density. In some embodiments, ultrafiltration provides a retentate having a density between 1 g / mL and 30 g / mL. In some embodiments, ultrafiltration involves a concentration step resulting in a concentrated retentate, followed by a diafiltration step that removes impurities and results in a protein slurry suspended in water. In some such embodiments, the concentrated retentate has a concentration factor of 2-fold to 12-fold volume reduction relative to the starting volume. In some embodiments, diafiltration provides a constant volume displacement between 3-fold and 10-fold.

[0112] Depending on the embodiment and type of impurity being removed, the method for removing the impurity may vary. Removal of lipid impurities from the isolated recombinant protein can be achieved by methods known in the art. Examples of such methods include, but are not limited to, absorption onto charcoal or other absorption media that specifically bind lipids. Removal of polysaccharide impurities from the isolated recombinant protein can be achieved by methods known in the art. Examples of such methods include, but are not limited to, treatment with enzymes that hydrolyze polysaccharides, followed by removal of the resulting small sugars by ultrafiltration. Examples of such enzymes include, but are not limited to, glucanases, lyticases, mannases, and chitinases.

[0113] quantitative Isolated recombinant spider silk proteins can be measured or quantified to assess the recovery (yield) and purity of the isolated proteins. Any suitable method can be used to measure or quantitate the amount of isolated full-length recombinant proteins and recombinant protein fragments, including, but not limited to, size-exclusion chromatography (SEC), enzyme-linked immunosorbent assay (ELISA), SDS-PAGE, Western blot (immunoblot), high-performance liquid chromatography (HPLC), SEC-HPLC, liquid chromatography-mass spectrometry (LC-MS), or fast protein liquid chromatography (FPLC), or other suitable methods known in the art, or combinations thereof. In one embodiment, the amount of full-length recombinant spider silk proteins and recombinant protein fragments is measured using Western blot. In another embodiment, the amount of full-length recombinant spider silk proteins and recombinant protein fragments is measured using enzyme-linked immunosorbent assay (ELISA). In another embodiment, the amount of full-length recombinant spider silk proteins and recombinant protein fragments is measured using size-exclusion chromatography (SEC).

[0114] 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 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 100% of the full-length recombinant spider silk protein, as determined by a suitable method.

[0115] In some embodiments, the isolated recombinant spider silk protein has a purity of 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 isolated recombinant spider silk protein has a purity of at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, or at least 60%. at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 100%.

[0116] In some embodiments, the purity of the isolated recombinant spider silk protein is increased relative to the starting insoluble material. For example, in cases where the starting insoluble material is an insoluble or poorly soluble isolated recombinant spider silk protein or silk powder, the purity of the isolated recombinant spider silk protein can be increased using the solubilization and isolation methods described herein. In some embodiments, the purity of the isolated recombinant spider silk protein is increased by 1-5%, 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% relative to the purity of the starting material. In some embodiments, the purity of the isolated recombinant spider silk protein is increased by at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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% compared to the purity of the starting material. In some embodiments, the purity of the insoluble material is 0-99%, 1-5%, 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 95-99.9%. In some embodiments, the purity of the insoluble material is at least 1%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, 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%, at least 99%, at least 99.5%, or at least 99.9%.

[0117] In some embodiments, the isolated recombinant spider silk protein comprises 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.

[0118] Recombinant spider silk compositions U.S. Patent No. 9,963,554, entitled "Methods and Compositions for Synthesizing Improved Silk Fibers," which is incorporated herein by reference, discloses compositions for synthetic block copolymers, recombinant microorganisms for producing them, and synthetic fibers containing these proteins. U.S. Patent Publication No. 2019 / 0100740, issued April 4, 2019, and entitled "Modified Strains for the Production of Recombinant Silk," which is incorporated herein by reference in its entirety, discloses Pichia pastoris cells selected or engineered to suppress degradation of recombinant proteins expressed by the yeast cells, and methods for culturing the yeast cells for the production of useful compounds. One skilled in the art can also produce synthetic block copolymers and fibers containing these proteins in Escherichia coli.

[0119] Several species of natural spider silk have been identified so far, and the mechanical properties of each species of naturally spun silk are thought to be closely related to their molecular composition. See, for example, Garb, JE, et al., Untangling spider silk evolution with spidroin terminal domains, BMC Evol. Biol., 10:243 (2010); Bittencourt, D., et al., Protein families, natural history and biotechnological aspects of spider silk, Genet. Mol. Res., 11:3 (2012); Rising, A., et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell. Mol. Life Sci., 68:2, pp. 169-184 (2011); and Humenik, M., et al., Spider silk: understanding the structure-function relationship of a natural fiber, Prog. Mol. Biol. Transl. Sci., 103, pp. 131-85 (2011). For example:

[0120] Tufted (AcSp) silks tend to be tough, combining moderately high strength with moderately high stretchability. AcSp silks are characterized by large block ("repeat aggregate") sizes, often incorporating polyserine and GPX motifs. Tubular (TuSp, or cylindrical) silks tend to be large in diameter, moderate in strength, and highly stretchable. TuSp silks are characterized by their polyserine and polythreonine content and short polyalanine sequences. Large ampullate (MaSp) silks tend to be high in strength and moderately stretchable. MaSp silks are either of two subtypes, MaSp1 or MaSp2. MaSp1 silks are generally less stretchable than MaSp2 silks and are characterized by polyalanine, GX, and GGX motifs. MaSp2 silks are characterized by polyalanine, GGX, and GPX motifs. MiSp silks tend to have moderate strength and moderate stretchability. MiSp silks are characterized by GGX, GA, and polyA motifs and often contain a spacer element of approximately 100 amino acids. Flagelliform (Flag) silks tend to have very high stretchability and moderate strength. Flag silks are usually characterized by GPG, GGX, and a short spacer motif.

[0121] The properties of each silk species may vary from species to species, and spiders with different lifestyles (e.g., stationary web-weaving spiders versus wandering, feeding spiders) or evolutionarily older spiders may produce silks that differ from the above description (for a description of spider diversity and classification, see Hormiga, G., and Griswold, C.E., Systematics, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, pp. 487-512 (2014); and Blackedge, T.A. et al., Reconstructing web evolution and spider diversification in the molecular era, Proc. Natl. Acad. Sci. USA, 106:13, pp. 5229-5234 (2009)). However, synthetic block copolymer polypeptides with sequence similarity and / or amino acid composition similarity to the repeat domains of natural silk proteins can be used to produce consistent silk-like fibers on a commercial scale that recapitulate the properties of the corresponding natural silk fibers.

[0122] In some embodiments, the recombinant spider silk is a highly crystalline silk protein, a silk protein with a high beta sheet content, or a low solubility silk protein. In some embodiments, the recombinant spider silk protein has a solubility threshold in a non-chaotropic solvent of less than 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 10%, or 5%. In some embodiments, the solubility threshold is the amount of protein that dissolves in the non-chaotropic solvent after centrifugation.

[0123] Silk nucleotide and peptide sequences In some embodiments, a list of putative silk sequences can be compiled by searching GenBank for related terms, such as "spidroin," "fibroin," and "MaSp," and these sequences can be pooled with additional sequences obtained from independent sequencing. These sequences are then translated into amino acids, duplicate entries are filtered, and manually divided into their respective domains (NTD, REP, CTD). In some embodiments, the candidate amino acid sequences are reverse-translated into DNA sequences optimized for expression in Pichia (Komagataella) pastoris. The DNA sequences are then cloned into expression vectors, and Pichia (Komagataella) pastoris is transformed with them. In some embodiments, the various silk domains that are successfully expressed and secreted are then combinatorially assembled to construct fiber-forming silk molecules.

[0124] Silk polypeptides characteristically consist of repeat domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). The repeat domains exhibit a hierarchical structure. The repeat domains contain a series of blocks (also known as repeat units). These blocks are sometimes perfectly and sometimes imperfectly repeated (forming quasi-repeat domains) throughout the repeat domain of silk. The length and composition of the blocks vary between different silk species and between different species. Table 1 lists block sequences for selected species and silk species; further examples are described in Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships, and biomedical applications, Cell Mol. Life Sci., 68:2, pp. 169-184 (2011); and Gatesy, J. et al., Extreme diversity, conservation, and convergence of spider silk fibroin sequences, Science, 291:5513, pp. 2603-2605 (2001). In some cases, blocks are arranged in a regular pattern and may form larger macrorepeats that occur multiple times (usually 2-8 times) in the repeat domain of the silk sequence. Blocks repeated within the repeat domain or macrorepeat may be separated by spacing elements from macrorepeats repeated within the repeat domain. Block sequences may include a glycine-rich region followed by a poly(A) region. Short (approximately 1-10) amino acid motifs can occur multiple times within a block. A subset of commonly accepted motifs is shown in Figure 1. Blocks from different natural silk polypeptides can be selected without regard to circular permutation (i.e., identified blocks that are otherwise similar between silk polypeptides may not align due to circular permutation).Thus, for example, a "block" of SGAGG (SEQ ID NO: 39) is, for purposes of the methods and compositions described herein, identical to GSGAG (SEQ ID NO: 40), which is also identical to GGSGA (SEQ ID NO: 41); they are all merely circular permutations of each other. The particular permutation chosen for a given silk sequence can be determined, among other things, by convenience (usually starting with G). Silk sequences obtained from the NCBI database can be divided into blocks and non-repetitive regions.

[0125] (Table 1) Example of block arrangement TIFF0007814051000001.tif96167TIFF0007814051000002.tif246167TIFF0007814051000003.tif106167

[0126] According to certain embodiments of the present invention, fiber-forming block copolymer polypeptides derived from block and / or macro-repeat domains are described in International Publication No. WO / 2015 / 042164, which is incorporated herein by reference. Natural silk sequences obtained from protein databases such as GenBank or by de novo sequencing are domain-disrupted (N-terminal, repeat, and C-terminal domains). The N-terminal and C-terminal domain sequences selected for post-synthetic assembly into fibers contain native amino acid sequence information and other modifications described herein. The repeat domains are broken down into repeat sequences, which typically contain one to eight representative blocks that capture key amino acid information, depending on the silk species, while reducing the size of the DNA encoding the amino acids to easily synthesizable fragments. In some embodiments, a properly formed block copolymer polypeptide comprises at least one repeat domain containing at least one repeat sequence, optionally flanked by an N-terminal domain and / or a C-terminal domain.

[0127] In some embodiments, the repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence is 150-300 amino acid residues. In some embodiments, the repeat sequence comprises multiple blocks. In some embodiments, the repeat sequence comprises multiple macrorepeats. In some embodiments, the blocks or macrorepeats are divided into multiple entire repeat sequences.

[0128] In some embodiments, repeat sequences must begin with glycine and cannot end with phenylalanine (F), tyrosine (Y), tryptophan (W), cysteine ​​(C), histidine (H), asparagine (N), methionine (M), or aspartic acid (D) to meet DNA assembly requirements. In some embodiments, some repeat sequences can be altered relative to the native sequence. In some embodiments, repeat sequences can be altered, such as by adding serine to the C-terminus of the polypeptide (to avoid termination at F, Y, W, C, H, N, M, or D). In some embodiments, repeat sequences can be modified by filling in incomplete blocks with homologous sequences from another block. In some embodiments, repeat sequences can be modified by rearranging the order of blocks or macrorepeats.

[0129] In some embodiments, unique N-terminal and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain can be created by removing a leading signal sequence, for example, as identified by SignalP (Peterson, TN, et. Al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pg. 785-786 (2011)).

[0130] In some embodiments, the N-terminal domain sequence, the repeat sequence, or the C-terminal domain sequence is selected from 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 clavipes, Nephila madagascariensis, Nephila pilipes, Nephilengys cruentata, Parawixia bistriata, Peucetia viridans, Plectreurys It can be derived from T. tristis, Poecilotheria regalis, Tetragnatha kauaiensis, or Uloborus diversus.

[0131] In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to an alpha mating factor nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence can be operably linked to a 3X FLAG nucleotide coding sequence. In some embodiments, the silk polypeptide nucleotide coding sequence is operably linked to another affinity tag, such as a nucleotide coding sequence of 6-8 His residues (SEQ ID NO: 42).

[0132] secretion signal The amount of protein secreted by cells varies greatly among proteins and depends, in part, on the secretion signal operably linked to the nascent protein. Several secretion signals are known in the art, some of which are commonly used for the production of secreted recombinant proteins. Among these, the secretion signal of Saccharomyces cerevisiae α-mating factor (αMF) stands out, consisting of an N-terminal 19-amino acid signal peptide (also referred to herein as pre-αMF(sc)) followed by a 70-amino acid leader peptide (also referred to herein as pro-αMF(sc)). Incorporation of pro-αMF(sc) into the Saccharomyces cerevisiae αMF secretion signal (also referred to herein as pre-αMF(sc) / pro-αMF(sc)) has been shown to be important for increasing protein secretion yields. Studies have been conducted to achieve secretion of recombinant proteins by adding pro-αMF(sc) or its functional variants to signal peptides other than pre-αMF(sc). These have shown varying degrees of effectiveness, enhancing secretion of certain recombinant proteins in certain recombinant host cells, but showing no effect or reduced secretion of other recombinant proteins.

[0133] As described in U.S. Application No. 15 / 724,196, the use of multiple distinct secretion signals can improve the secretion yield of recombinant proteins. Compared to recombinant host cells containing multiple polynucleotide sequences encoding recombinant proteins operably linked to a single secretion signal (e.g., pre-αMF(sc) / pro-αMF(sc)), recombinant host cells containing the same number of polynucleotide sequences encoding recombinant proteins operably linked to at least two distinct secretion signals exhibit increased secretion yields of recombinant proteins. Without wishing to be bound by theory, the use of at least two distinct secretion signals may allow the recombinant host cell to engage distinct cellular secretion pathways to enable efficient secretion of recombinant proteins, thus preventing oversaturation of any one secretion pathway.

[0134] At least one of the distinct secretion signals comprises a signal peptide that may be selected from Table 2 or 3, or is a functional variant having 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 comprising 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 translocation of the nascent recombinant protein to the ER post-translationally (i.e., protein synthesis precedes translocation so that the nascent recombinant protein is in the cytosol before being translocated to the ER). In other embodiments, the signal peptide mediates translocation of the nascent recombinant protein to the ER co-translationally (i.e., protein synthesis and translocation to the ER occur simultaneously). The advantage of using a signal peptide to mediate cotranslational translocation to the ER is that it prevents recombinant proteins prone to rapid folding from adopting a conformation that prevents translocation to the ER and, therefore, secretion.

[0135] (Table 2) Secretion signals TIFF0007814051000004.tif43163

[0136] Table 3. Recombinant secretion signals TIFF0007814051000005.tif60163

[0137] Expression vector The expression vectors described herein can be produced according to the teachings of the present specification in light of techniques known in the art. Sequences, such as vector sequences or transgene-encoding sequences, are commercially available from companies such as Integrated DNA Technologies, Coralville, IA, or DNA 2.0, Menlo Park, CA. Exemplary herein are expression vectors that direct high-level expression of chimeric silk polypeptides.

[0138] Another standard source of the polynucleotides described herein is polynucleotides isolated from organisms (e.g., bacteria), cells, or selected tissues. Nucleic acids from a selected source can be isolated using standard procedures, which generally involve sequential phenol and phenol / chloroform extractions followed by ethanol precipitation. After precipitation, the polynucleotides can be treated with a restriction endonuclease to cleave the nucleic acid molecule into fragments. Fragments of a selected size can be separated using several techniques, such as 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 provide starting material of the appropriate size for cloning.

[0139] Another method for obtaining the nucleotide components of an expression vector or construct is PCR. The general procedure for PCR is taught in MacPherson et al., PCR: A PRACTICAL APPROACH, (IRL Press at Oxford University Press, (1991)). PCR conditions for each reaction used can be determined empirically. Numerous parameters affect the success of the reaction. These parameters include annealing temperature and time, extension time, Mg2+ and ATP concentrations, pH, and the relative concentrations of primers, template, and deoxyribonucleotides. Exemplary primers are described in the Examples below. After amplification, the resulting fragments can be detected by agarose gel electrophoresis, followed by visualization with ethidium bromide staining and ultraviolet illumination.

[0140] Another method for obtaining polynucleotides is by enzymatic digestion. For example, a nucleotide sequence can be generated by digesting a suitable vector with the appropriate recognition restriction enzyme. Using standard techniques, the restriction fragments can be blunt-ended by treatment with the large fragment of E. coli DNA polymerase I (Klenow) in the presence of four deoxynucleotide triphosphates (dNTPs).

[0141] Polynucleotides are inserted into a suitable backbone, such as a plasmid, using methods well known in the art. For example, insert and vector DNA can be contacted with a restriction enzyme under appropriate conditions to create complementary or blunt ends on each molecule, which then pair with each other and are joined with a ligase. Alternatively, synthetic nucleic acid linkers can be attached to the ends of the polynucleotide. These synthetic linkers can contain nucleic acid sequences that correspond to specific restriction sites in the vector DNA. Other means are known and available in the art. A variety of sources are available for the component polynucleotides.

[0142] In some embodiments, an expression vector comprising an R, N, or C sequence is transformed into a host organism for expression and secretion. In some embodiments, the expression vector comprises a secretion signal. In some embodiments, the expression vector comprises a termination signal. In some embodiments, the expression vector is designed to integrate into the host cell genome and comprises the following: a region of homology to the target genome, a promoter, a secretion signal, a tag (e.g., a Flag tag), a termination / polyA signal, a Pichia selectable marker, an E. coli selectable marker, an E. coli origin of replication, and a restriction site for release of the fragment of interest.

[0143] The vector of the present invention can further comprise a targeting sequence that directs the integration of the spider silk protein coding sequence into a specific location in the genome of the host cell. Examples of such targeting sequences include, but are not limited to, nucleotide sequences that are identical to nucleotide sequences present in the genome of the host cell. In some embodiments, the targeting sequence is identical to a repetitive element in the genome of the host cell. In some embodiments, the targeting sequence is identical to a transposable element in the genome of the host cell.

[0144] In some embodiments, provided herein are recombinant host cells comprising the vectors described herein. In some embodiments, these vectors are stably integrated into the genome (e.g., chromosome) of the recombinant host cell, e.g., using homologous recombination or targeted integration. Examples of suitable sites for genomic integration include, but are not limited to, the Ty1 locus in the Saccharomyces cerevisiae genome, the rDNA and HSP82 loci in the Pichia pastoris genome, and transposable elements whose copies are scattered throughout the genome of the recombinant host cell. In other embodiments, these vectors are not stably integrated into the genome of the recombinant host cell, but rather are extrachromosomal.

[0145] Host cell transformants Host cells transformed with a nucleic acid molecule or vector that expresses a spider silk polypeptide, and their progeny, are provided. These cells can also carry the nucleic acid sequence on the vector, which can, but need not, replicate freely. In other embodiments, the nucleic acid is integrated into the genome of the host cell.

[0146] In some embodiments, microorganisms or host cells that enable large-scale production of block copolymer polypeptides comprise a combination of: 1) the ability to produce large (>50 kDa) polypeptides; 2) tolerance to large-scale contaminants (such as viral or bacterial contamination); and 3) existing know-how for growing and processing microorganisms on a large scale (1-2000 m). 3 ) a bioreactor.

[0147] In some embodiments, the host cell expresses the recombinant spider silk protein intracellularly and the protein remains within the host cell, hi some embodiments, the host cell expresses the recombinant spider silk protein intracellularly and the protein is secreted.

[0148] A variety of host organisms can be genetically engineered / transformed to contain the block copolymer polypeptide expression system. Preferred organisms for expression of recombinant silk polypeptides include plants, algae, yeast, fungi, gram-positive bacteria, and gram-negative bacteria. In some embodiments, the host organism is selected from the group consisting of Arxula adeninivorans, Aspergillus aculeatus, Aspergillus awamori, Aspergillus ficuum, Aspergillus fumigatus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Aspergillus sojae, Aspergillus tubigensis, Bacillus alcalophilus, and the like. 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 thuringiensis), Candida boisinii (Candidaboidinii, Chrysosporium lucknowense, Escherichia coli, Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis, Kluyveromyces marxianus, Myceliopthora thermophila, Neurospora crassa, Ogataea polymorpha, Penicillium camemberti, Penicillium canescens, Penicillium chrysogenum 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 miehei), Rhizomucor pusillus, Rhizopus arrhizus, Streptomyces lividans, Saccharomycescerevisiae, Schwanniomyces occidentalis, Trichoderma harzianum, Trichoderma reesei, or Yarrowia lipolytica.

[0149] Additional strains that can be used as recombinant host cells are known in the art. It should be understood that the term "recombinant host cell" is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations, due either to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "recombinant host cell" as used herein.

[0150] Genetically engineered host cell lines Another widely used cell line for recombinant protein production is the bacterium Escherichia coli. However, during cultivation of E. coli strains, recombinantly expressed proteins become insoluble, resulting in poor isolation and reduced recombinant protein yield. Another widely used microorganism is the methylotrophic yeast Pichia pastoris. P. pastoris can be grown to high cell densities, provides tightly controlled methanol-inducible transgene expression, and efficiently secretes heterologous proteins in defined media. However, during cultivation of P. pastoris strains, recombinantly expressed proteins are degraded before they can be recovered, resulting in a mixture of proteins containing fragments of the recombinantly expressed protein and reduced yield of the full-length recombinant protein.

[0151] In some embodiments, the engineered strains described herein with reduced protease activity recombinantly express silk-like polypeptide sequences. In some embodiments, the silk-like polypeptide sequences are 1) block copolymer polypeptide compositions, i.e., compositions produced by mixing and matching repeat domains derived from silk polypeptide sequences, and / or 2) block copolymer polypeptides produced via recombinant expression, i.e., polypeptides large enough (approximately 40 kDa) to form useful fibers when secreted by industrially scalable microorganisms. Genetically engineered large block copolymer polypeptides (approximately 40 kDa to approximately 100 kDa) can be expressed in the engineered microorganisms described herein from silk repeat domain fragments containing sequences derived from nearly the entire published amino acid sequence of a spider silk polypeptide. In some embodiments, the silk polypeptide sequences are adapted and designed to produce highly expressed and secreted polypeptides capable of fiber formation. In some embodiments, knocking out protease genes or reducing protease activity in the host engineered strain prevents degradation of the silk-like polypeptide.

[0152] In some embodiments, to attenuate protease activity in Pichia pastoris, the genes encoding these enzymes are inactivated or mutated to reduce or eliminate activity. This can be done by mutating the gene itself via modification of gene regulatory elements, or by inserting a gene into the gene itself. This can be achieved by standard yeast genetic techniques. An example of such a technique is gene replacement via double homologous recombination, in which homologous regions flanking the gene to be inactivated are cloned into a vector adjacent to a selectable marker gene (such as an antibiotic resistance gene or a gene that complements an auxotrophy of the yeast strain).

[0153] Alternatively, the homologous region can be PCR amplified and linked to the selectable marker gene via overlap PCR. Such DNA fragments are then transformed into Pichia pastoris using methods known in the art, such as electroporation. Transformants grown under selective conditions are then analyzed for gene disruption events using standard techniques, such as PCR on genomic DNA or Southern blot analysis. In another experiment, gene inactivation can be achieved by a single homologous recombination step, in which the 5' end of the gene's ORF is cloned into a promoterless vector that also contains a selectable marker gene. Such vectors are linearized by digestion with restriction enzymes, cleaving the vector to leave only the target gene homologous fragment, and then transformed into Pichia pastoris. Integration into the target gene site is confirmed by PCR on genomic DNA or Southern blot analysis. In this way, replication of the gene fragment cloned into the vector is achieved within the genome, resulting in two copies of the target locus: the first copy, whose ORF is incomplete and therefore expresses only a short, inactive protein (if any), and the second copy, which lacks a promoter to drive transcription.

[0154] Alternatively, transposon mutagenesis is used to inactivate the target gene, and a library of such mutants can be screened by PCR for insertion events in the target gene.

[0155] The functional phenotype (i.e., deficiency) of the engineered / knockout strain can be assessed using techniques known in the art. For example, the lack of protease activity in the engineered strain can be confirmed using any of a variety of methods known in the art, such as assays for the hydrolytic activity of a chromogenic protease substrate, band shifts of the substrate protein of the selected protease, etc.

[0156] The attenuation of protease activity described herein can be achieved using mechanisms other than knockout mutations. For example, the desired protease can be attenuated by altering its amino acid sequence, by modifying the nucleic acid sequence, placing the gene under the control of a weakly active promoter, downregulating it, expressing interfering RNA, ribozymes, or antisense sequences targeting the gene of interest, or using other techniques known in the art. In preferred strains, the protease activity of the proteases encoded by PAS_chr4_0584 (YPS1-1) and PAS_chr3_1157 (YPS1-2) is attenuated by any of the methods described above. In some aspects, methylotrophic yeast strains, particularly Pichia pastoris strains, are described in which the YPS1-1 and YPS1-2 genes are inactivated. In some embodiments, genes encoding additional proteases can also be knocked out according to the methods provided herein to further reduce the protease activity of the desired protein product expressed by the strain.

[0157] In some embodiments, the P. pastoris strains disclosed herein are engineered to express silk-like polypeptides. Methods for producing preferred embodiments of silk-like polypeptides are provided in WO 2015 / 042164, particularly paragraphs 114-134, which are incorporated herein by reference. Disclosed therein are synthetic proteinaceous copolymers based on recombinant spider silk protein fragment sequences from MaSp2, such as from the Argiope bruennichi species. Silk-like polypeptides are described that contain 2 to 20 repeat units, each of which has a molecular weight greater than about 20 kDa. Each repeat unit in the copolymer has greater than about 60 amino acid residues organized into several "quasi-repeat units." In some embodiments, the repeat units of the polypeptides described herein have at least 95% sequence identity to the MaSp2 dragline silk protein sequence. [Example]

[0158] Below are examples of specific embodiments for carrying out the methods described herein. These examples are provided for illustrative purposes only and are not intended to limit the scope of the disclosure in any way. Care has been taken to ensure accuracy with respect to numbers used (amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0159] The practice of the methods described herein will employ, unless otherwise indicated, conventional methods in protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, within the skill of one of ordinary skill in the art. Such techniques are fully explained in the literature, see, e.g., 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 3 rd Ed. (Plenum Press) Vols A and B (1992).

[0160] Example 1: Microfluidization enhances the solubility of recombinant silk proteins Large physical energies were used to solubilize model silk proteins from aggregates of insoluble cellular material in a variety of aqueous solvent conditions.

[0161] The model silk UD MiSp 64kDa is a recombinantly expressed 64kD protein derived from the Uloborus diversus ampullate gland spidroin gene sequence (GenBank: DQ399332.1, SEQ ID NO: 23) linked to an N-terminal histidine hexamer (SEQ ID NO: 43). This protein was expressed using Escherichia coli C41(DE3) (Lucigen) transformed with a T7 expression vector encoding the MiSp protein. Cells were grown in minimal medium, MiSp gene expression was induced with isopropyl β-D-1-thiogalactopyranoside (IPTG), and lysed by homogenization. The insoluble cell lysate was pelleted by centrifugation. A 4M urea solution was mixed with the insoluble material in a 10:1 (by mass) ratio for 1 hour, and the insoluble fraction was collected by centrifugation. The homogenizer used yielded approximately 0.7 x 10 6 s -1 However, this UD MiSp 64kD model silk is known to be poorly soluble, and the shear rate generated by the homogenizer was insufficient to solubilize the UD MiSp 64kDa silk.

[0162] The cell biomass and pellet containing insoluble silk proteins were resuspended in aqueous buffer (50 mM Tris, pH 7.5) and then resuspended in a solution containing the selected chaotrope at a 15% w / v ratio (cell pellet mass:solution volume). The final chaotrope concentrations were 10 M urea, 4 M GdnHCl, 8 M GdnHCl, 3 M GdnSCN, or 6 M GdnSCN, assuming a pellet density of 1 g / L. The solution was stirred at room temperature for at least 1 hour to mix and break down macroscopic clumps of material. 100 mL aliquots were processed by passing them three times through an F12Y interaction chamber operating at 30,000 psi (gauge pressure, M-110P, Microfluidics Inc.). A water bath was used to limit heating of the sample during processing steps. For the control, another set of 100 mL aliquots was stirred at room temperature for 3 hours instead of being subjected to microfluidization.

[0163] Silk protein solubilization was assayed by centrifuging 50 mL aliquots at 15,000 × g for 20 minutes at room temperature, and the supernatant and cell pellet were separated. Soluble silk protein was determined as the silk protein remaining in the supernatant after centrifugation. Insoluble silk remaining in the cell pellet was assayed by extracting the pellet with 50 mL of water containing 5 M GdnSCN. The silk concentration of the two fractions was assessed by ELISA using an anti-His6 antibody ("His6" disclosed as SEQ ID NO: 43), and the results are shown in Table 1.

[0164] Table 1. Increased solubility of model silks after high-energy treatment in aqueous buffers and selected chaotrope solutions. The concentrations of silk in the two fractions were determined by ELISA. TIFF0007814051000006.tif113144

[0165] For all conditions evaluated, microfluidization increased the amount of silk in the soluble fraction. A notable example was the 10 M urea and 4 M GdnHCl condition, where very little silk (6% in 4 M GdnHCl or 0% in 10 M urea) was dissolved in the control, but more than 75% was dissolved after microfluidization. In addition, both the 3 M and 6 M GdnSCN buffers achieved 100% solubilized silk protein after microfluidization, whereas the control treatments at the same chaotrope concentrations failed to completely solubilize the silk. Thus, the use of microfluidization reduced the concentration of chaotrope required to solubilize silk protein.

[0166] The presence of silk proteins in the soluble fraction was also confirmed by size-exclusion chromatography (SEC). Using SEC HPLC, the soluble fractions of protein samples extracted with 10 M urea and microfluidization (high-energy line) were compared with those extracted with 10 M urea alone (control line) (Figure 2). The elution time of the model silk protein (MiSp peak) was identified with purified protein standards (data not shown). The column was run with a denaturing mobile phase (5 M GdnSCN), and the eluate was detected with a refractive index detector. The application of microfluidization shear force significantly improved the solubilization and recovery of silk proteins compared to the use of urea alone. The purity of the solubilized silk proteins was assessed by calculating the % refractive index curve (RU) area of ​​the SEC peak for some samples. The purity of selected samples is shown in Table 2.

[0167] (Table 2) TIFF0007814051000007.tif111128

[0168] Example 2: Optimization of shear rate pressure Next, we evaluated silk solubilization by varying the size of the interaction chamber and the microfluidization pressure.

[0169] Insoluble cell biomass containing silk proteins was prepared as described in Example 1 and suspended in urea to a final concentration of 10 M urea. Samples were processed at two distinct shear rates, controlled by the type of interaction chamber and operating pressure, according to the manufacturer's instructions (Microfluidics Processor User Guide, Microfluidics, Inc.). Specifically, 6.5 x 10 6 s -1 (G10Z Interaction Chamber, 23,000 psi, Microfluidics Inc. LM10), or 9.5 × 10 6 s -1The predicted shear rate was targeted (F12Y interaction chamber, 30,000 psi, Microfluidics Inc. M-110P). Silk protein solubilization was assayed using the centrifugation protocol described in Example 1. Silk concentration in the soluble or insoluble fractions was assessed by SEC HPLC as previously described. Silk protein was measured by the area of ​​the SEC refractive index peak using a bovine serum albumin protein standard to derive silk concentration.

[0170] Table 3 shows the solubilization of silk after two treatments. The shear rate was 9.5 x 10 6 s -1 If we increase it to 6.5×10 6 s -1 The yield of solubilized silk protein increased by 40% compared to samples treated at lower shear rates (47% yield at 23,000 psi compared to 66% yield obtained using 30,000 psi). Thus, increasing the shear rate could improve the solubilization and recovery of silk proteins.

[0171] Table 3. Effect of changing shear rate on silk protein solubilization with 10 M urea. Silk concentration was estimated from SEC HPLC peak area. Yield is the amount of recovered silk protein normalized to the total amount of silk protein in the starting material. The total amount of silk protein in the starting material was extracted by incubation with 5 M GdnSCN. TIFF0007814051000008.tif43160

[0172] While the present invention has been particularly shown and described with reference to preferred 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 or scope of the invention.

[0173] All references, issued patents, and patent applications cited within the body of this specification are hereby incorporated by reference in their entirety for all purposes.

Claims

1. 1. A method for isolating recombinant spider silk proteins from a host cell, the method comprising the steps of: a. recovering insoluble material from a cell culture comprising host cells, wherein recovering the insoluble material comprises lysing the host cells, wherein the host cells express a recombinant spider silk protein, and further wherein the insoluble material comprises the recombinant spider silk protein; b. adding the insoluble material to an aqueous solution containing a chaotropic agent; c) applying shear force via microfluidization to the aqueous solution containing the insoluble material, thereby solubilizing the recombinant spider silk protein in the aqueous solution.

2. The microfluidization resulted in 6×10 ６ s －１ ~10 x 10 ６ s －１ 10. The method of claim 1, wherein a shear rate of

3. 3. The method of claim 1 or 2, wherein the microfluidization is carried out at 20,000 psi to 30,000 psi (about 138 MPa to 207 MPa).

4. 3. The method of claim 1 or 2, wherein the microfluidization is carried out using a microfluidizer, wherein the microfluidizer comprises a Z-shaped interaction chamber or a Y-shaped interaction chamber.

5. The shear force is applied at least twice; or The shear force is applied at least twice and the shear force is the same during the at least two applications; or the shear force is applied at least two times, and the shear force is different during the at least two applications; The method of claim 1.

6. 2. The method of claim 1, wherein the chaotropic agent is urea, guanidine thiocyanate (GdnSCN), or guanidine chloride (GdnHCL).

7. 10. The method of claim 1, wherein the insoluble material is added to the aqueous solution at 5%, 10%, 15%, 20%, 25%, or 30% (w / v) insoluble material relative to the solution volume.

8. 7. The method of claim 6, wherein the chaotropic agent is present in the aqueous solution at a concentration of 0.1 to 10 M.

9. 7. The method of claim 6, wherein the aqueous solution comprises 10 M urea, 4 M to 8 M GdnHCl, or 3 M to 6 M GdnSCN.

10. The aqueous solution contains 15% (w / v) insoluble material relative to the solution volume, and the chaotropic agent is 3 M GdnSCN, 4 M GdnHCl, or 10 M urea. The method of claim 7.

11. 10. The method of claim 1, wherein the insoluble material is incubated at 20°C to 30°C.

12. 2. The method of claim 1, wherein the insoluble material is incubated in the aqueous solution containing the chaotropic agent for 60 to 120 minutes.

13. The method of claim 1 , wherein the insoluble material comprises a cell pellet.

14. 10. The method of claim 1, wherein the lysing comprises heat treatment, chemical treatment, shear disruption, physical homogenization, sonication, or chemical homogenization.

15. 15. The method of claim 14, wherein recovering the insoluble material from the cell culture further comprises centrifuging the lysed cells to obtain a first cell pellet.

16. The step of recovering the insoluble material comprises the steps of: a. Incubating the cell pellet with a solution containing 4M urea at a 10:1 urea volume:pellet mass ratio; and b. centrifuging the solution containing 4M urea to obtain a second cell pellet, and then incubating the second cell pellet in the aqueous solution containing a solvent; The method of claim 1 further comprising:

17. 10. The method of claim 1, further comprising isolating said recombinant spider silk protein from said aqueous solution, thereby producing an isolated recombinant spider silk protein.

18. 2. The method of claim 1, wherein the recombinant spider silk protein is a highly crystalline silk protein, a silk protein with a high beta sheet content, or a low solubility silk protein.

19. 19. The method of claim 18, wherein the recombinant spider silk protein comprises the Uloborus diversus MiSP protein set forth in SEQ ID NO:

23.

20. 19. The method of claim 18, wherein the recombinant spider silk protein has a solubility threshold in a non-chaotropic solvent of less than 90%, 80%, 70%, 60%, or 50%.

21. 10. The method of claim 1, wherein the cell culture comprises fungal cells, bacterial cells, or yeast cells.

22. 22. The method of claim 21, wherein the bacterial cell is Escherichia coli.

23. 2. The method of claim 1, wherein the isolated recombinant spider silk protein is a full-length recombinant spider silk protein.

24. 2. The method of claim 1, wherein the isolated recombinant spider silk protein comprises at least 60% of full-length recombinant spider silk protein, based on the total amount of isolated recombinant spider silk protein.

25. 10. The method of claim 1, wherein the isolated recombinant spider silk protein is at least 30% pure.

26. 1. A method for isolating recombinant spider silk proteins from a host cell, the method comprising the steps of: a. providing insoluble material from a cell culture comprising host cells, wherein the insoluble material comprises recombinant spider silk protein; b. adding the insoluble material to an aqueous urea solution, the aqueous solution containing 15% (w / v) insoluble material at a final 10 M urea concentration; c. applying shear force to the aqueous solution containing the insoluble material via microfluidization, thereby solubilizing the recombinant spider silk protein in the aqueous solution; and d. Isolating said recombinant spider silk protein from said aqueous solution, thereby producing an isolated recombinant spider silk protein.

27. 1. A method for isolating recombinant spider silk proteins from a host cell, the method comprising the steps of: a. providing insoluble material from a cell culture comprising host cells, wherein the insoluble material comprises recombinant spider silk protein; b. adding the insoluble material to an aqueous urea solution, the aqueous solution containing 15% (w / v) insoluble material at a final 10 M urea concentration; c. applying shear force via microfluidization, wherein the shear force is 10×10 to the aqueous solution. ６ s －１ thereby solubilizing the recombinant spider silk protein in the aqueous solution; and d. Isolating said recombinant spider silk protein from said aqueous solution, thereby producing an isolated recombinant spider silk protein.

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