Basic purification of spider silk proteins

Abstract

This disclosure relates to methods for producing and purifying synthetic block copolymer proteins, expression constructs for secreting said synthetic block copolymer proteins, recombinant microorganisms for producing said synthetic block copolymer proteins, and synthetic fibers containing these proteins that reproduce many properties of natural silk.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 772,588, filed November 28, 2018, the contents of which are incorporated herein by reference in their entirety.

[0003] sequence list

[0004] This application contains a sequence list, which has been submitted via EFS-Web and incorporated herein by reference in its entirety. The ASCII copy was created on December 10, 2019, named BTT-013WO_SL.txt, and has a file size of 66,352 bytes. Background Technology

[0005] Spider silk polypeptides are large (>150 kDa, >1000 amino acids) polypeptides that can be broken down into three domains: N-terminal non-repeating domain (NTD), repetitive domain (REP), and C-terminal non-repeating domain (CTD). The NTD and CTD are relatively small (approximately 150 and 100 amino acids, respectively), well-studied, and believed to confer aqueous stability, pH sensitivity, and post-aggregation molecular arrangement. The NTD also possesses a strong predictive secretion tag, which is often removed during heterologous expression. The repetitive region constitutes approximately 90% of the natural polypeptide and folds into crystalline and amorphous regions, which respectively confer silk fiber strength and flexibility.

[0006] Silk polypeptides originate from a variety of sources, including bees, moths, spiders, mites, and other arthropods. Some organisms produce a variety of silk fibers with specific sequences, structural elements, and mechanical properties. For example, the orb-weaving spider has six unique types of glands that produce different sequences of silk polypeptides that can polymerize into fibers adapted to their environment or life cycle microenvironment. The fiber is named after the gland from which it originates, and the polypeptide is labeled with the gland abbreviation (e.g., "Ma") and the abbreviation for spider silk protein (sp. "Sp"). In the orb-weaving spider, these types include the large ampulla (MaSp, also known as the drag silk), the small ampulla (MiSp), the flagellate (Flag), the variegated (AcSp), the tubular (TuSp), and the piriform (PySp). This combination of polypeptide sequences, varying across fiber types, domains, and genera and species, produces a wide range of potential properties that can be utilized through the commercial production of recombinant fibers. To date, most work on recombinant silk has focused on the large ampulla (MaSp) spider silk protein.

[0007] Currently, recombinant silk fibers are not commercially available (with a few exceptions) and cannot be produced in microorganisms other than *Escherichia coli* and other Gram-negative prokaryotes. Recombinant silks produced to date primarily consist of fragments of polymerized short filamentary sequence motifs or primitive repeating domains, sometimes combined with NTDs and / or CTDs. This leads to small-scale production of recombinant silk peptides using intracellular expression (mg on a laboratory scale, kg on a bioprocessing scale), followed by purification by chromatography or bulk precipitation. These methods fail to achieve viable commercial scalability that can compete with existing technologies and textile fibers in terms of price. Other production hosts already used for silk peptide manufacturing include transgenic goats, transgenic silkworms, and plants. Commercial-scale production of silk has not yet been achieved with these hosts, likely due to slow engineering cycles and poor scalability.

[0008] Furthermore, recombinant silk peptides form undesirable insoluble aggregates during production and purification. Methods for re-solubilizing peptides during purification often lead to protein degradation, resulting in low fiber yield, poor fiber toughness, and a poor hand feel. In addition, standard protein solubilization methods require the use of cholecystokinins, such as urea, guanidine hydrochloride, or guanidine thiocyanate, which must be collected and properly disposed of after protein separation. Therefore, improved methods are needed to purify these peptides in a sustainable and environmentally friendly process. Summary of the Invention

[0009] In one aspect, this document provides a method for isolating recombinant spider silk protein from a host cell culture, comprising: obtaining a cell culture containing host cells and a growth medium, wherein the host cells express recombinant spider silk protein; collecting a portion of the cell culture containing the recombinant spider silk protein; incubating the portion of the cell culture in an aqueous solution under alkaline conditions to solubilize the recombinant spider silk protein in the aqueous solution; and isolating the recombinant spider silk protein from the aqueous solution to produce an isolated recombinant spider silk protein sample.

[0010] In some embodiments, alkaline conditions include an alkaline pH value of 9 to 14. In one embodiment, the alkaline pH value is 11 to 12.

[0011] In some embodiments, the isolated recombinant spider silk protein is full-length recombinant spider silk protein. In one embodiment, the isolated recombinant spider silk protein sample comprises at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the total isolated recombinant spider silk protein as full-length recombinant spider silk protein. In one embodiment, the percentage of full-length recombinant spider silk protein is measured by Western blotting. In another embodiment, the percentage of full-length recombinant spider silk protein is measured by size exclusion chromatography.

[0012] In some embodiments, the purity of the isolated recombinant spider silk protein is 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%. In some embodiments, the yield of the isolated recombinant spider silk protein is at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100% relative to recombinant spider silk isolated by urea or guanidine thiocyanate methods.

[0013] In some embodiments, isolating recombinant spider silk protein includes precipitating the recombinant spider silk protein by altering the alkaline conditions of the aqueous solution. In one embodiment, altering the alkaline conditions includes adjusting the alkaline pH of the fraction of the cell culture to a decreased pH value from 4 to 10. In one embodiment, the decreased pH value is 4, 5, 6, 7, 8, 9, or 10. In one embodiment, the decreased pH value is from 6 to 7.

[0014] In some embodiments, adjusting the alkaline pH involves adding an acid to the aqueous solution. In one embodiment, the acid is H₂SO₄.

[0015] In some embodiments, the portion of the cell culture comprises a supernatant, whole-cell broth, or cell clumps. In some embodiments, collecting the portion of the cell culture includes removing the host cells from the growth medium and reconstituted the host cells in the aqueous solution.

[0016] In some embodiments, collecting the portion of the cell culture includes lysing the host cells. In many embodiments, lysis includes heat treatment, shearing, physical homogenization, sonication, or chemical homogenization.

[0017] In some embodiments, the portion of the cell culture comprises the host cells and the growth medium derived from the cell culture.

[0018] In various embodiments, the aqueous solution contains a diluted growth medium.

[0019] In some embodiments, the portion of the cell culture is incubated under alkaline conditions for 10 to 120 minutes. In some embodiments, the portion of the cell culture is incubated under alkaline conditions for at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 105 minutes, or at least 120 minutes. In some embodiments, the portion of the cell culture is incubated under alkaline conditions for 15 to 30 minutes.

[0020] In various embodiments, the portion of the cell culture incubated under alkaline conditions further includes stirring the portion of the cell culture.

[0021] In various embodiments, the method further includes removing unsolvated biomass from the aqueous solution under alkaline conditions. In some embodiments, the removal of unsolvated biomass includes filtration, centrifugation, gravity sedimentation, adsorption, dialysis, or phase separation. In some embodiments, filtration is ultrafiltration, microfiltration, or dialysis filtration. In some embodiments, the removal of unsolvated biomass is repeated at least once.

[0022] In various embodiments, the method further includes removing impurities before or after the separation of recombinant spider silk proteins. In some embodiments, impurity removal includes filtration, centrifugation, gravity sedimentation, adsorption, dialysis, or phase separation. In various embodiments, filtration is ultrafiltration, microfiltration, or dialysis filtration. In some embodiments, centrifugation is ultracentrifugation or dialysis. In one embodiment, adsorption is carbon adsorption. In some embodiments, impurity removal is repeated at least once.

[0023] In various embodiments, the method further includes concentrating the isolated recombinant spider silk protein to produce concentrated spider silk protein. In some embodiments, concentration includes precipitation, filtration, ultrafiltration, centrifugation, dialysis, evaporation, or lyophilization.

[0024] In various embodiments, the method further includes drying and separating the recombinant spider silk protein.

[0025] In various embodiments, the method further includes producing silk fibers from separated, reconstituted spider silk. In one embodiment, the silk fibers have a toughness of at least 19 cN / tex.

[0026] In some embodiments, the recombinant spider silk protein is 18B or PO.

[0027] In some implementations, the cell culture includes fungal cells, bacterial cells, or yeast cells.

[0028] In some implementations, the yeast cells are Pichia pastoris cells.

[0029] In another aspect, this document provides a method for isolating recombinant spider silk protein, the method comprising: obtaining a cell culture containing host cells and a growth medium, wherein the host cells express recombinant spider silk protein; collecting a portion of the cell culture containing the recombinant spider silk protein; incubating the portion of the cell culture in an aqueous solution under alkaline conditions to solubilize the recombinant spider silk protein in the aqueous solution; adjusting the aqueous solution to a non-alkaline pH to precipitate the solubilized recombinant spider silk protein; and isolating the recombinant spider silk protein from the portion of the cell culture to produce isolated recombinant spider silk protein.

[0030] In another aspect, this document provides compositions comprising recombinant spider silk protein produced by any of the disclosed methods.

[0031] In some implementations, the reconstituted 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% of full-length reconstituted spider silk.

[0032] In another respect, this article provides silk fibers comprising recombinant spider silk proteins produced by any of the disclosed methods.

[0033] In some implementations, the filament fibers have a toughness of at least 19 cN / tex.

[0034] In another aspect, this document provides compositions comprising cell cultures in an alkaline buffer, the cell cultures comprising growth medium and host cells, the host cells comprising recombinant spider silk proteins.

[0035] In one embodiment, the pH of the alkaline buffer solution is between 9 and 14. In another embodiment, the pH is between 11 and 12.

[0036] In some embodiments, the spider silk protein is 18B or PO. In some embodiments, the cell culture includes fungal cells, bacterial cells, or yeast cells. In one embodiment, the bacterial cells are Escherichia coli cells. In one embodiment, the yeast cells are Pasteurella multocida cells. Attached Figure Description

[0037] Figure 1 An exemplary process flow for isolating recombinant spider silk protein from cell supernatant is shown.

[0038] Figure 2 An exemplary process flow for isolating recombinant spider silk proteins from cell lysates is shown.

[0039] Figure 3 An exemplary process flow for separating recombinant spider silk proteins using a separation agent is shown.

[0040] Figure 4A Size exclusion chromatography (SEC) analysis of purified 18B spider silk protein isolated from cell clumps using an alkaline pH buffer is shown. The 18B monomer peak is indicated by an arrow. Figure 4B A comparison of the quantity and purity of 18B spider silk purified using urea extraction or alkaline extraction is shown.

[0041] Figure 5 The area of ​​purified 18B spider silk monomers and impurities after tangential flow filtration (TFF), as measured by SEC, is shown.

[0042] Figure 6A The total yield of 18B spider silk protein after two-step extraction is shown. Results from two different runs are also shown. Figure 6B The purity of 18B after two-step extraction is shown as measured by SEC area percentage.

[0043] Figure 7A The area percentages of 18B monomer, low molecular weight (LMW) impurities, and medium molecular weight (IMW) impurities after alkaline extraction of whole-cell broth are shown. The extracted protein was concentrated using tangential flow filtration. Figure 7B The SEC analysis of the recovered 18B spider silk protein is shown. The 18B monomer peaks of various tangential flow filtration fractions are indicated by arrows.

[0044] Figure 8 The area percentages of 18B monomer, high molecular weight (HMW) impurities, low molecular weight (LMW) impurities, and medium molecular weight (IMW) impurities after alkaline extraction and pH precipitation of whole-cell broth are shown. Proteins extracted using dialysis centrifugation were concentrated.

[0045] Figure 9The yield of 18B monomers after alkaline extraction and pH precipitation in whole-cell broth is shown. The extracted protein was concentrated using dialysis centrifugation.

[0046] Figure 10 SEC analysis of purified 18B spider silk protein after acid precipitation at pH 6 is shown. The 18B monomer peak is indicated by arrows. The extracted protein was concentrated using dialysis centrifugation.

[0047] Figure 11 Immunoblotting of soluble P0 protein after extraction from Escherichia coli lysate using various pH buffers or urea is shown. Detailed Implementation

[0048] definition

[0049] Unless otherwise defined herein, scientific and technical terms related to this invention shall have the meanings commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature used in conjunction with the following and the following techniques are those well-known and commonly used in the art: biochemistry, enzymology, molecular and cell biology, microbiology, genetics and peptide and nucleic acid chemistry, and hybridization as described herein.

[0050] Unless otherwise specified, the methods and techniques of the present invention are generally performed in accordance with conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989); Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, with a 2002 supplement); Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990); Taylor and Drickamer, Introduction to Glycobiology, Oxford University 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 Press (1999).

[0051] All publications, patents and other references mentioned in this article are incorporated herein by reference in their entirety.

[0052] Unless otherwise specified, the following terms shall be understood to have the following meanings:

[0053] As used herein, the terms “fermented” and “fermentation” describe the cultivation of host cells under conditions intended to produce a desired product, including but not limited to conditions for the growth of the host cells.

[0054] As used in this article, the term "fermented broth" refers to an aqueous culture medium used to culture host cells during the fermentation process.

[0055] As used in this article, “inoculum” refers to a certain amount of host cells added to the fermenting broth to initiate fermentation.

[0056] As used herein, the term "clarifying" refers to a method of removing host cell biomass, such as whole cells, lysed cells, cell membranes, lipids, organelles, cell nuclei, non-spider silk proteins, or any other unwanted cellular portion or product, or any other unwanted portion of a cell culture. Clarifying may also refer to the removal of impurities from a partially purified or separated spider silk composition. Impurities may include, but are not limited to, non-spider silk proteins, degraded spider silk proteins, large aggregates of proteins, chemicals used in the purification and separation process, or any other undesirable material.

[0057] As used herein, the term "purity" refers to the percentage of full-length isolated recombinant spider silk protein in all isolated components of a sample (such as an extracted sample), including partially or degraded isolated recombinant spider silk protein, lipids, proteins, cell membranes, or other molecules.

[0058] As used herein, the term "yield" refers to the percentage of full-length recombinant spider silk protein isolated from a cell culture relative to the amount of full-length or total silk protein in a control sample. This percentage can be referenced to the total amount of full-length spider silk protein in cell lysates, crude alkaline extracts, partially purified or filtered alkaline extracts, purified solutions subjected to alkaline extraction methods, or purified solutions subjected to control extraction methods (such as urea or GdSCN as described herein).

[0059] The term "polynucleotide" or "nucleic acid molecule" refers to a polymer of nucleotides with a length of at least 10 bases. This term includes DNA molecules (e.g., cDNA, genomic DNA, or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as DNA or RNA analogs containing non-natural nucleotide analogs, non-native nucleoside bonds, or both. Nucleic acids can exist in any topological conformation. For example, nucleic acids can be single-stranded, double-stranded, triple-stranded, quadruple-stranded, partially double-stranded, branched, hairpin-shaped, circular, or padlocked conformations.

[0060] Unless otherwise specified, and as an example of all sequences described herein in the general format “SEQ ID NO:”, “a nucleic acid containing SEQ ID NO:1” means a nucleic acid having at least a portion of the following sequence: (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is determined by the context. For example, if the nucleic acid is used as a probe, the choice between the two depends on the requirement that the probe is complementary to the desired target.

[0061] "Separated" RNA, DNA, or mixed polymers are RNA, DNA, or mixed polymers that are substantially separated from other cellular components naturally associated with their native host cells, such as ribosomes, polymerases, and genome sequences.

[0062] The term "recombinant" refers to a biomolecule (e.g., a gene or polypeptide) that: (1) has been removed from its naturally occurring environment; (2) is not associated with all or part of the polynucleotide of the gene found in nature; (3) is operatively linked to a polynucleotide not linked to it in nature; or (4) does not exist in nature. The term "recombinant" may also be used for cloned DNA isolates, chemically synthesized polynucleotide analogs or polynucleotide analogs biosynthesized from heterologous systems, and polypeptides and / or mRNA encoded by such nucleic acids.

[0063] As used herein, if a heterologous sequence is placed adjacent to an endogenous nucleic acid sequence, causing an alteration in the expression of that endogenous nucleic acid sequence, then that endogenous nucleic acid sequence (or the polypeptide product encoded by that sequence) in the organism's genome is considered a "recombinant" herein. In this context, a heterologous sequence is a sequence that is not naturally adjacent to an endogenous nucleic acid sequence, regardless of whether the heterologous sequence itself is endogenous (derived from the same host cell or its descendants) or exogenous (derived from different host cells or their descendants). For example, with respect to the original promoter of a gene in the host cell's genome, the promoter sequence can be replaced (e.g., through homologous recombination), resulting in an altered expression pattern of the gene. This gene will now become a "recombinant" because it is dissociated from at least some of the sequences naturally adjacent to it. In one embodiment, the heterologous nucleic acid molecule is not homologous to the organism. In a further embodiment, the heterologous nucleic acid molecule is integrated into the host chromosome as a plasmid or molecule through homologous or random integration.

[0064] A nucleic acid is also considered a "recombinant" if it contains any modifications that are not naturally present in the corresponding nucleic acid in the genome. For example, if an endogenous coding sequence contains artificially introduced modifications, such as insertions, deletions, or point mutations introduced through human intervention, then that endogenous coding sequence is considered a "recombinant." "Recombinant nucleic acids" also include nucleic acids integrated into the host cell chromosome at heterologous sites and nucleic acid constructs existing as episomes.

[0065] In the context of nucleic acid sequences, the term "sequence identity percentage" refers to a quantitative value of the alignment of residues between two sequences when a maximum correspondence alignment is performed. The length of a sequence identity comparison may be 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 or more nucleotides. Many different algorithms are known in the art for measuring nucleotide sequence identity. For example, polynucleotide sequences can be compared using FASTA, Gap, or Bestfit, which are programs in version 10.0 of the Wisconsin Package of Genetics Computer Group (GCG), Madison, Wis. FASTA provides the alignment and sequence identity percentage of the region with the best overlap between the query and search sequences. (Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated hereinously by reference).) For example, the percentage of sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (word length of 6 and a scoring matrix of NOPAM factors) or using Gap, as provided in GCG version 6.1 (incorporated hereby by reference), with its default parameters. 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)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res.25:3389-3402 (1997)).

[0066] When referring to nucleic acids or fragments thereof, the terms "substantial homology" or "substantial similarity" mean that, when best aligned with appropriate nucleotide insertions or deletions of another nucleic acid (or its complementary strand), nucleotide sequence identity exists at 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, as measured by any recognized sequence identity algorithm such as FASTA, BLAST, or Gap discussed above.

[0067] Nucleic acids (also known as polynucleotides) can include RNA, cDNA, genomic DNA, and synthetic forms described above, as well as sense and antisense strands of mixed polymers. As will be readily understood by those skilled in the art, they may be chemically or biochemically modified or may contain non-natural or derived nucleotide bases. Such modifications include, for example, labeling, methylation, substitution of one or more naturally occurring nucleotides with analogs, and internucleotide modifications such as uncharged linkages (e.g., methylphosphonates, phosphotriesters, aminophosphates, carbamates, etc.), charged linkages (e.g., thiophosphates, dithiophosphates, etc.), overhangs (e.g., peptides), intercalators (e.g., acridine, psoralen, etc.), chelating agents, alkylating agents, and modified linkages (e.g., α-anomeric nucleic acids, etc.). Synthetic molecules that mimic the ability of polynucleotides to bind to a specified sequence through 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 molecular backbone. Other modifications may include, for example, ribose rings containing bridging portions or analogues of other structures, such as those found in “locked” nucleic acids.

[0068] The term "mutated," when applied to nucleic acid sequences, refers to the possibility that nucleotides in the nucleic acid sequence may have been inserted, deleted, or altered compared to a reference nucleic acid sequence. A single alteration (point mutation) can be made at a locus, or multiple nucleotides can be inserted, deleted, or altered at a single locus. Furthermore, one or more alterations can be made at any number of loci within the nucleic acid sequence. Nucleic acid sequences can be mutated by any method known in the art, including but not limited to mutagenesis techniques such as "error-prone PCR" (a process of performing PCR under conditions of low replication fidelity of DNA polymerase to obtain a high point mutation rate along the entire length of the PCR product; see, for example, Leung et al., Technique, 1:11-15 (1989) and Caldwell & Joyce, PCR Methods Applic. 2:28-33 (1992)); and "oligonucleotide directed mutagenesis" (a process that enables site-specific mutations to be generated in any cloned DNA segment of interest; see, for example, Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).

[0069] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is ligated. One class of vectors is the "plasmid," which generally refers to a circular double-stranded DNA loop to which an additional DNA segment can be ligated, but also includes linear double-stranded molecules, such as those obtained by polymerase chain reaction (PCR) amplification or by treating circular plasmids with restriction enzymes. Other vectors include granules, bacterial artificial chromosomes (BACs), and yeast artificial chromosomes (YACs). Another class of vectors is the viral vector, in which an additional DNA segment can be ligated into a viral genome (discussed in more detail below). Some vectors are capable of autonomous replication in the host cell to which they are introduced (e.g., vectors with an origin of replication that functions in the host cell). Other vectors, upon introduction into the host cell, can be integrated into the host cell's genome and thus replicated along with the host genome. Furthermore, certain preferred vectors are capable of directing the expression of genes operatively ligated to them. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0070] As used herein, the term "expression system" includes mediators or vectors used to express genes in host cells and mediators or vectors used to stably integrate genes into the host chromosome.

[0071] "Operatively linked" or "operably linked" expression control sequences refer to those where the expression control sequence is adjacent to the gene of interest to control the gene of interest, or acts in a trans manner or at a certain distance to control the expression control sequence of the gene of interest.

[0072] As used herein, the term "expression control sequence" refers to a polynucleotide sequence essential to the expression of a coding sequence operatively linked to it. Expression control sequences are sequences that control transcription, post-transcriptional events, and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter, and enhancer sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences stabilizing cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., ribosome binding sites); sequences that enhance peptide stability; and sequences that enhance peptide secretion when needed. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences typically include promoters, ribosome binding sites, and transcription termination sequences. The term "control sequence" is intended to include at least all components whose presence is essential for expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion chaperone sequences.

[0073] As used in this article, the term "promoter" refers to the DNA region to which RNA polymerase binds to initiate gene transcription and to the location at the 5' position of the mRNA transcription start site.

[0074] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to refer to a cell in which a recombinant vector has been introduced. It should be understood that such terms are intended to refer not only to the specific subject cell but also to the progeny of such cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may actually differ from the parent cell but are still included within the scope of the term "host cell" as used herein. Recombinant host cells can be isolated cells or cell lines grown in a culture or cells residing in living tissue or an organism.

[0075] The term "peptide" encompasses naturally occurring and non-natural proteins, as well as their fragments, mutants, derivatives, and analogs. Peptides can be monomeric or polymeric. Furthermore, a peptide can contain multiple distinct domains, each possessing one or more different activities.

[0076] 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 may be natural or synthetic.

[0077] As used herein, the term "block" or "repetitive unit" refers to a subsequence of more than about 12 amino acids in a natural silk polypeptide sequence that is repeatedly found (possibly with modest variation) within the sequence and serves as a basic repeating unit in the silk polypeptide sequence. A block may, but does not necessarily, include a very short "motif." As used herein, a "motif" refers to a sequence of about 2-10 amino acids appearing in multiple blocks. For example, a motif may consist of the amino acid sequences GGA, GPG, or AAAAA (SEQ ID NO:41). The sequence of multiple blocks is a "block copolymer."

[0078] As used herein, the term "repetitive domain" refers to a sequence selected from a collection of continuous (not interrupted by substantially non-repetitive domains, excluding known filament spacer elements) repetitive segments in a filament polypeptide. A native filament sequence generally contains one repetitive domain. In some embodiments of the invention, each filament molecule contains one repetitive domain. As used herein, a "macrorepetitive" is a naturally occurring repetitive amino acid sequence comprising more than one block. In embodiments, the macrorepetitive is repeated at least twice in the repetitive domain. In further embodiments, the two repetitions are imperfect. As used herein, a "quasi-repetitive" is an amino acid sequence comprising more than one block, such that the blocks are similar in amino acid sequence but not identical.

[0079] As used herein, "repetitive sequence" or "R" refers to a repetitive amino acid sequence. In embodiments, the repetitive sequence includes a macrorepetitive or a fragment of a macrorepetitive. In another embodiment, the repetitive sequence includes a block. In a further embodiment, a single block is split into two repetitive sequences.

[0080] It must be noted that, unless the context clearly specifies otherwise, the singular forms “a,” “an,” and “the” as used in the specification and appended claims include plural references.

[0081] Any ranges disclosed herein include the extreme values ​​of the range. For example, a range of 2-5% includes 2% and 5%, and any numbers or fractions of numbers in between, such as: 2.25%, 2.5%, 2.75%, 3%, 3.25%, 3.5%, 3.75%, 4%, 4.25%, 4.5%, and 4.75%.

[0082] Recombinant spider silk composition

[0083] Several types of primitive spider silk have been identified. It is believed that the mechanical properties of each primitive spinning type are closely related to the molecular composition of the silk. See, for example, Garb, JE et al., Untangling spider silk evolution with spidroin terminal domains, BMC Evol. Biol., 10:243 (2010); Bittencourt, D. et al., Protein families, natural history and biotechnological aspects of spider silk, Genet. Mol. Res., 11:3 (2012); Rising, A. et al., Spider silk proteins: recent advances in recombinant production, structure-function relationships and biomedical applications, Cell. Mol. Life Sci., 68:2, pp. 169-184 (2011); and Humenik, M. et al., Spider silk: understanding the structure-function relationship of a natural fiber, Prog. Mol. Biol. Transl. Sci., 103, pp. 131-85 (2011). For example:

[0084] Gracilaria (AcSp) filaments tend to have high toughness, a result of a combination of moderately high strength and moderately high ductility. AcSp filaments are characterized by large block ("overall repeat") sizes, often doped with polyserine and GPX motifs. Tubular gland (TuSp or cylindrical) filaments tend to have large diameters, moderate strength, and high ductility. TuSp filaments are characterized by their polyserine and polythreonine content, as well as short bundles of polyalanine. Macroambulatory gland (MaSp) filaments tend to have high strength and moderate ductility. MaSp filaments can be of two subtypes: MaSp1 and MaSp2. MaSp1 filaments are generally less ductile than MaSp2 filaments, and MaSp1 filaments are characterized by polyalanine, GX, and GGX motifs. MaSp2 filaments are characterized by polyalanine, GGX, and GPX motifs. Microambulatory gland (MiSp) filaments tend to have moderate strength and moderate ductility. MiSp filaments are characterized by GGX, GA, and poly A motifs, and often contain spacer elements of about 100 amino acids. Flag filaments tend to have high extensibility and moderate strength. Flag filaments are typically characterized by GPG, GGX, and short spacer motifs.

[0085] The properties of each silk type can vary from species to species, and spiders with different lifestyles (e.g., sedentary web spinners versus vagabond hunters) or that are evolutionarily older can produce silk with properties different from those described above (for a description of spider diversity and taxonomy, see Hormiga, G. and Griswold, CE, Systems, phylogeny, and evolution of orb-weaving spiders, Annu. Rev. Entomol. 59, pp. 487–512 (2014); and Blackedge, TA 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 peptides with sequence similarity and / or amino acid composition similarity to the repeating domains of the original silk proteins can be used to manufacture consistent filamentous fibers that reproduce the properties of the corresponding natural silk fibers on a commercial scale.

[0086] Silk nucleotide and peptide sequences

[0087] A list of probable filament sequences can be compiled by searching relevant terms in GenBank, such as “spidroin,” “fibroin,” and “MaSp,” and these sequences can be combined with other sequences obtained through independent sequencing work. The sequences are then translated into amino acids, duplicate entries are filtered out, and the sequences are manually broken down into domains (NTD, REP, CTD). In some embodiments, candidate amino acid sequences are reverse-translated into DNA sequences optimized for microbial expression, for example, in *P. pastoris* or *E. coli*. The DNA sequences are then cloned into expression vectors and transformed into microorganisms, such as *P. pastoris* or *E. coli*. In some embodiments, the various filament domains that demonstrate successful expression and secretion are subsequently assembled in a combinatorial manner to construct filament molecules capable of forming fibers.

[0088] Silk polypeptides are characterized by repeating domains (REPs) flanked by non-repetitive regions (e.g., C-terminal and N-terminal domains). These repeating domains exhibit a hierarchical structure. Each repeating domain contains a series of blocks (also called repeating units). These blocks are repeated throughout the silk repeating domain, sometimes perfectly repeated, sometimes imperfectly repeated (forming quasi-repetitive domains). The length and composition of the blocks vary between different silk types and between different species. Table 1 lists examples of block sequences from selected species and silk types, with further examples provided in the following literature: 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 can be arranged in a regular pattern, forming larger macroscopic repeats that appear multiple times (typically 2 to 8 times) within the repeating domains of the silk sequence. Repeating blocks within repeating domains or macroscopic repeats, as well as macroscopic repeats within repeating domains, can be separated by spacer elements. Block sequences may contain glycine-rich regions followed by poly A regions. Short (approximately 1-10) amino acid motifs can appear multiple times within a block. Figure 1 The diagram depicts a subset of commonly observed motifs. For the purposes of this invention, blocks from different natural silk polypeptides can be selected without reference to circular arrangements (i.e., blocks identified as otherwise similar between silk polypeptides may not align due to circular arrangements). Thus, for example, for the purposes of this invention, the “block” of SGAGG (SEQ ID NO:42) is identical to GSGAG (SEQ ID NO:43) and identical to GGGSA (SEQ ID NO:44); they are all simply circular arrangements of each other. The specific arrangement chosen for a given silk sequence may be determined particularly by convenience (usually starting with G). Silk sequences obtained from the NCBI database can be divided into blocks and non-repetitive regions.

[0089] Table 1: Samples of Patch Sequences

[0090]

[0091]

[0092]

[0093]

[0094]

[0095] Fiber-forming block copolymer peptides derived from block and / or macroscopic repeating domains according to certain embodiments of the present invention are described in International Publication No. WO / 2015 / 042164 (incorporated by reference). Natural filament sequences obtained from protein databases (e.g., GenBank) or by de novo sequencing are disassembled according to domains (N-terminal domain, repeating domain, and C-terminal domain). The N-terminal and C-terminal domain sequences selected for synthesis and assembly into filaments include natural amino acid sequence information and other modifications described herein. Repeating domains are disassembled into repeating sequences containing representative blocks, typically 1 to 8 blocks depending on the type of filament, which capture key amino acid information while reducing the size of the DNA encoding the amino acids to easily synthesized fragments. In some embodiments, suitably formed block copolymer peptides comprise at least one repeating domain containing at least one repeating sequence, and optionally side-mounted with an N-terminal domain and / or a C-terminal domain.

[0096] In some embodiments, the repeating domain comprises at least one repeating sequence. In some embodiments, the repeating sequence is 150 to 300 amino acid residues. In some embodiments, the repeating sequence comprises multiple blocks. In some embodiments, the repeating sequence comprises multiple macrorepeaters. In some embodiments, blocks or macrorepeaters are segmented into multiple repeating sequences.

[0097] In some embodiments, the repetitive sequence begins 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 parts of the repetitive sequence can be altered compared to the original sequence. In some embodiments, the repetitive sequence can be modified, for example, by adding a serine residue to the C-terminus of the polypeptide (to avoid termination at F, Y, W, C, H, N, M, or D). In some embodiments, the repetitive sequence can be modified by filling in an incomplete block with a homologous sequence from another block. In some embodiments, the repetitive sequence can be modified by rearranging the order of blocks or macrorepetitive structures.

[0098] In some embodiments, non-repetitive N-terminal and C-terminal domains may be selected for synthesis. In some embodiments, the N-terminal domain may be obtained by removing, for example, the leader signal sequence identified by SignalP (Peterson, TN et al., SignalP 4.0: discriminating signal peptides from transmembrane regions, Nat. Methods, 8:10, pp. 785-786 (2011)).

[0099] In some implementations, the N-terminal domain, repeating sequence, or C-terminal domain sequence may be derived from funnel-web spiders (Agelenopsis aperta), Aliatipus gulosus, Costa Rican zebra-foot spiders (Aphonopelmaseemanni), certain species of short-toothed spiders (Aptostichus sp. AS217), and certain species of short-toothed spiders (Aptostichus sp.).AS220), Cross-shaped Spider (Araneus diadematus), Cat-faced Spider (Araneus gemmoides), Large-bellied Orb-weaver Spider (Araneus ventricosus), Beautiful Golden Spider (Argiope amoena), Silver Golden Spider (Argiope argentata), Striped Golden Spider (Argiope bruennichi), Three-banded Golden Spider (Argiope trifasciata), Atypoides riversi, Brazilian Yellow-spotted Pink Spider (Avicularia juruensis), California Trap Spider (Bothriocyrtum californicum), Giant-eyed Spider (Deinopis Spinosa), Gray Diguetia Canities, Black Fishing Spider (Dolomedestenebrosus), Black Fishing Spider (Euagrus chisoseus), Nursery Web Spider (Euprosthenops australis), Papillary Spinosa (Gasteracantha mammosa), Hypochilus thorelli, Kukulcania hibernalis, Black Widow Spider (Latrodectus) The following spiders are listed: *Nephila hesperus*, *Megahexura fulva*, *Metepeira grandiosa*, *Nephila antipodiana*, *Nephila clavata*, *Nephila clavipes*, *Nephila madagascariensis*, *Nephila pilipes*, *Nephilengys cruentata*, *Parawixia bistriata*, *Peucetia viridans*, *Plectreurys tristis*, *Poecilotheria regalis*, *Tetragnatha kauaiensis*, or *Uloborus diversus*.

[0100] In some embodiments, the silk polypeptide nucleotide-coding sequence can be operatively linked to the α-mating factor nucleotide-coding sequence. In some embodiments, the silk polypeptide nucleotide-coding sequence can be operatively linked to another endogenous or heterologous secretion signal coding sequence. In some embodiments, the silk polypeptide nucleotide-coding sequence can be operatively linked to the 3X FLAG nucleotide-coding sequence. In some embodiments, the silk polypeptide nucleotide-coding sequence is operatively linked to other affinity markers such as 6 to 8 His residues (SEQ ID NO: 45).

[0101] secretion signal

[0102] The amount of protein secreted from a cell varies significantly among proteins and is partly dependent on a secretion signal operatively linked to the protein in its nascent state. Many secretion signals are known in the art, some commonly used for the production of secreted recombinant proteins, including those from *Pichia pastoris* and *Saccharomyces cerevisiae*. Among these, the secretion signal of the α-mating factor (αMF) in *Saccharomyces cerevisiae* stands out, consisting of a 19-amino acid signal peptide at the N-terminus (also referred to herein as pre-αMF(sc)) followed by a 70-amino acid leader peptide (also referred to herein as pro-αMF(sc)). The inclusion of pro-αMF(sc) in the *Saccharomyces cerevisiae* αMF secretion signal (also referred to herein as pre-αMF(sc) / pro-αMF(sc)) has been shown to be crucial for achieving high protein secretion yields. Adding pro-αMF(sc) or its functional variants to signal peptides other than pre-αMF(sc) has also been explored as a means to achieve recombinant protein secretion, but has shown varying degrees of effectiveness, increasing the secretion of certain recombinant proteins in some recombinant host cells, but having no effect on or reducing the secretion of other recombinant proteins.

[0103] As described in U.S. Patent Application 15 / 724,196, the use of multiple different secretion signals can improve the secretion yield of recombinant proteins produced in host cells such as *Pichia pastoris*. Recombinant host cells containing the same number of polynucleotide sequences encoding recombinant proteins operatively linked to only one secretion signal (e.g., pre-αMF(sc) / pro-αMF(sc)) produce higher recombinant protein secretion yields compared to recombinant host cells containing multiple polynucleotide sequences encoding recombinant proteins operatively linked to at least two different secretion signals. Without being bound by theory, the use of at least two different secretion signals may allow recombinant host cells to engage in different cellular secretion pathways to achieve efficient secretion of recombinant proteins, thereby preventing oversaturation of any single secretion pathway.

[0104] At least one distinct secretion signal comprises a signal peptide selectable from Table 2 or Table 3, or a functional variant having at least 80% amino acid sequence identity with 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 containing 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 with a signal peptide selected from Table 2 or 3. In some embodiments, the signal peptide mediates the translocation of the nascent recombinant protein to the ER post-translationally (i.e., protein synthesis precedes translocation, such that the nascent recombinant protein is present in the cytoplasm before translocation to the ER). In other embodiments, the signal peptide mediates the translocation of the nascent recombinant protein to the ER during co-translation (i.e., protein synthesis and translocation to the ER occur simultaneously). The advantage of using a signal peptide that mediates co-translational translocation to the ER is that it prevents readily foldable recombinant proteins from presenting a conformation that hinders translocation to the ER and thus inhibits secretion.

[0105] Table 2 - Secretion Signals

[0106]

[0107] Table 3 - Recombinant secretion signals

[0108]

[0109]

[0110] expression carrier

[0111] The expression vectors of this invention can be produced in accordance with the teachings of this specification using techniques known in the art. Sequences (e.g., vector sequences or sequences encoding transgenes) are commercially available from companies such as Integrated DNA Technologies, Coralville, IA, or Atum, Menlo Park, CA. Examples of expression vectors guiding high-level expression of chimeric polypeptides are illustrated herein.

[0112] Another standard source of polynucleotides used in this invention is polynucleotides isolated from organisms (e.g., bacteria), cells, or selected tissues. Nucleic acids from the selected source can be isolated using a standard procedure, typically involving sequential phenol and phenol / chloroform extraction followed by ethanol precipitation. After precipitation, the polynucleotides can be treated with a restriction endonuclease, which cleaves the nucleic acid molecules into fragments. Fragments of selected sizes can be separated using a variety of techniques, including agarose or polyacrylamide gel electrophoresis or pulsed-field gel electrophoresis (Care et al. (1984) Nuc. Acid Res. 12:5647-5664; Chu et al. (1986) Science 234:1582; ​​Smith et al. (1987) Methods in Enzymology 151:461), to provide starting material of appropriate size for cloning.

[0113] Another method for obtaining the nucleotide components of expression vectors or constructs is PCR. MacPherson et al. taught the general procedure for PCR in PCR: A PRACTICAL APPROACH (IRL Press at Oxford University Press, (1991)). The PCR conditions for each application reaction can be determined empirically. Many 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 deoxyribonuclease. Exemplary primers are described below in the examples. After amplification, the resulting fragments can be detected by agarose gel electrophoresis followed by ethidium bromide staining and UV irradiation.

[0114] Another method for obtaining polynucleotides is through enzymatic digestion. For example, nucleotide sequences can be generated by digesting a suitable vector with an appropriate recognition restriction endonuclease. The restriction cleavage fragments can be blunt-ended by treating with Escherichia coli DNA polymerase I (Klenow) in the presence of four deoxyribonucleotide triphosphates (dNTPs) using standard techniques.

[0115] Polynucleotides are inserted into a suitable backbone, such as a plasmid, using methods well-known in the art. For example, the insert fragment and vector DNA may be contacted with a restriction endonuclease under suitable conditions to produce complementary or blunt ends on each molecule that can pair with each other and be joined by a ligase. Alternatively, synthetic nucleic acid adapters may be attached to the ends of the polynucleotides. These synthetic adapters may contain nucleic acid sequences corresponding to specific restriction sites in the vector DNA. Other methods are known and available in the art. Component polynucleotides can be sourced from a variety of sources.

[0116] In some embodiments, an expression vector containing R, N, or C sequences is transformed into a host organism for expression and secretion. In some embodiments, the expression vector includes a secretion signal. In some embodiments, the expression vector includes a terminator signal. In some embodiments, the expression vector is designed to integrate into the host cell genome and includes: a homologous region of the target genome, a promoter, a secretion signal, a tag (e.g., a FLAG tag), a termination / polyA signal, a selectable marker for Pichia pastoris, a selectable marker for Escherichia coli, an origin of replication for E. coli, and a restriction site for releasing the fragment of interest.

[0117] Host cell transformant

[0118] Host cells transformed with nucleic acid molecules or vectors expressing spider silk polypeptides and their progeny are provided. These cells may also carry the nucleic acid sequence of the present invention on a vector, which may be, but does not necessarily have to be, a freely replicating vector. In other embodiments of the invention, the nucleic acid has been integrated into the genome of the host cell.

[0119] In some embodiments, the microorganisms or host cells capable of producing the block copolymer peptides of the present invention on a large scale include a combination of the following: 1) the ability to produce large (>40 kDa) peptides; 2) the ability to secrete peptides extracellularly and circumvent costly downstream intracellular purification; 3) the ability to resist contaminants (such as viral and bacterial contaminants) on a large scale; and / or 4) the prior art for growing and processing organisms is a large-scale (1-2000 m3) bioreactor.

[0120] A variety of host organisms can be engineered / converted into polypeptide expression systems containing block copolymers. Preferred organisms for expressing recombinant silk polypeptides include yeasts, fungi, Gram-negative bacteria, and Gram-positive bacteria. In some implementations, the host organism is *Arxula adeninivorans*, *Aspergillus aculeatus*, *Aspergillus awamori*, *Aspergillus ficuum*, *Aspergillus fumigatus*, *Aspergillus japonicus*, *Aspergillus nidulans*, *Aspergillus niger*, *Aspergillus oryzae*, *Aspergillus sojae*, *Aspergillus tubigensis*, *Bacillus alkalophilus*, *Bacillus amyloliquefaciens*, *Bacillus anthracis*, or *Bacillus brevis*. Bacillus brevis, Bacillus circulans, Bacillus coagulans, Bacillus lautus, Bacillus lentus, Bacillus licheniformis, Bacillus methanolicus, Bacillus stearothermophilus, Bacillus subtilis, Bacillus thuringiensis, Candida boidinii, Chrysosporium lucknowense, Escherichia coli, Fusarium graminearum, Fusarium venenatum, Kluyveromyces lactis, Kluyveromyces maculae *Marxianus*, *Myceliopthora thermophila*, *Neurospora**Penicillium crassa*, *Ogataea polymorpha*, *Penicillium camemberti*, *Penicillium canescens*, *Penicillium chrysogenum*, *Penicillium emersonii*, *Penicillium funiculosum*, *Penicillium griseoroseum*, *Penicillium purpurogenum*, *Penicillium roqueforti*, *Phanerochaete chrysosporium*, *Pichia angusta*, *Pichia methanolica*, *Pichia pastoriensis*, *Pichia polymorpha*, *Pichia stipitis*, *Rhizomucor miehei*, *Rhizomucor microphylla* The following are listed: *Pusillus*, *Rhizopusarrhizus*, *Streptomyces lividans*, *Saccharomyces cerevisiae*, *Schwanniomyces occidentalis*, *Trichoderma harzianum*, *Trichoderma reesei*, or *Yarrowia lipolytica*.

[0121] In a preferred aspect, the method involves culturing host cells to allow them to directly secrete the product for easy recovery without the need for biomass extraction. In some embodiments, the block copolymer peptide is secreted directly into a culture medium for collection and processing.

[0122] Engineered host cell lines

[0123] Any suitable host cell line can be used to produce recombinant proteins. The methyltrophic yeast *Pichia pastoris* is widely used for recombinant protein production. *Pichia pastoris* grows to high cell density, providing tightly controlled methanol-induced transgene expression and efficiently secreting heterologous proteins in defined media. However, during the cultivation of *Pichia pastoris* strains, recombinant expressed proteins may be degraded before collection, resulting in a mixture of proteins containing fragments of the recombinant expressed protein and a reduced yield of full-length recombinant proteins. Another widely used cell line for recombinant protein production is the bacterium *Escherichia coli*.

[0124] In some embodiments, the modified strains described herein recombinantly express filamentous polypeptide sequences with reduced protease activity. In some embodiments, the filamentous polypeptide sequence is 1) a block copolymer polypeptide composition produced by mixing and matching repeating domains derived from filamentous polypeptide sequences, and / or 2) recombinant expression of a block copolymer polypeptide of sufficiently large size (about 40 kDa) secreted by industrially scalable microorganisms for the formation of useful fibers. Large (about 40 kDa to about 100 kDa) block copolymer polypeptides (including sequences comprising virtually all published amino acid sequences from spider silk polypeptides) engineered from filament repeating domain fragments can be expressed in the modified microorganisms described herein. In some embodiments, the filamentous polypeptide sequence is matched and engineered for the production of highly expressed and secreted polypeptides capable of forming fibers. In some embodiments, knockout of protease genes or reduction of protease activity in the host modified strain reduces the degradation of the filamentous polypeptide.

[0125] In some implementations, to reduce the protease activity in *Pichia pastoris*, the genes encoding these enzymes are inactivated or mutated to reduce or eliminate their activity. This can be accomplished by mutating or inserting the gene itself or by modifying gene regulatory elements. This can be achieved using standard yeast genetics techniques. Examples of such techniques include gene substitution via double homologous recombination, in which a homologous region flanked by an inactivated gene is cloned into a vector flanked by an optional marker gene (e.g., an antibiotic resistance gene or a gene supplementing the auxotrophic form of the yeast strain).

[0126] Alternatively, homologous regions can be amplified by PCR and ligated to a selectable marker gene via overlap PCR. Such DNA fragments are then converted to *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 blotting. In alternative experiments, gene inactivation can be achieved through single homologous recombination, in which case, for example, the 5' end of the ORF of the gene is cloned into a promoter-free vector that also contains the selectable marker gene. Such vectors are converted to *Pichia pastoris* after being linearized by digestion with restriction enzymes that cleave only the homologous fragment of the target gene. Integration at the target gene site is confirmed by PCR on genomic DNA or Southern blotting. In this way, replication of the gene fragment cloned on the vector is achieved in the genome, generating two copies of the target gene locus: a first copy in which the ORF is incomplete, resulting in the expression of a shortened, inactive protein (if any); and a second copy without a promoter to drive transcription.

[0127] Alternatively, transposon mutagenesis can be used to inactivate the target gene. Libraries of such mutants can be screened by PCR targeting insertion events in the target gene.

[0128] The functional phenotype (i.e., defects) of engineered / knockout strains can be assessed using techniques known in the art. For example, defects in protease activity in engineered strains can be detected using any of a variety of methods known in the art, such as the determination of hydrolytic activity of chromogenic protease substrates, band shifts of substrate proteins of selected proteases, etc.

[0129] The reduction in protease activity described herein can be achieved through mechanisms other than knockout mutations. For example, the desired protease can be reduced by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, downregulating, expressing interfering RNA, ribozymes, or antisense sequences targeting the gene of interest, or any other technique known in the art. In preferred strains, the protease activity of the proteases encoded at PAS_chr4_0584 (YPS1-1) and PAS_chr3_1157 (YPS1-2) is reduced by any of the methods described above. In some aspects, the present invention relates to methyltrophic yeast strains, particularly *Pichia pastoris* strains in which the YPS1-1 and YPS1-2 genes are inactivated. In some embodiments, additional protease-encoding genes 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.

[0130] In some embodiments, the *P. pastoris* strains disclosed herein have been modified to express filamentous polypeptides. WO 2015 / 042164, particularly paragraphs 114 to 134 (incorporated herein by reference), provides a method for producing filamentous polypeptides according to preferred embodiments. This document discloses synthetic protein copolymers based on recombinant spider silk protein fragment sequences derived, for example, from the species *Argiope bruennichi*. Filamentous polypeptides are described comprising two to twenty repeating units, each repeating unit having a molecular weight greater than about 20 kDa. Within each repeating unit of the copolymer are more than about 60 amino acid residues organized into numerous “quasi-repeatable units.” In some embodiments, the repeating units of the polypeptides described herein have at least 95% sequence identity with the *MaSp2* drag filament protein sequence.

[0131] Methods for producing and purifying recombinant proteins

[0132] The method described herein involves fermenting an inoculum of recombinant host cells provided herein under suitable fermentation conditions in a suitable fermentation broth and a suitable fermentation vessel to produce recombinant proteins with desired cumulative yield and / or cumulative titer and / or cumulative productivity.

[0133] In some embodiments, the recombinant host cell secretes the recombinant protein. In various embodiments, the recombinant host cell may be a prokaryote that does not secrete the recombinant protein. In a specific embodiment, the recombinant host cell is Escherichia coli.

[0134] In various embodiments, the recombinant host cell may be a eukaryote or prokaryote that secretes the recombinant protein, such as Gram-negative or Gram-positive bacteria. In some embodiments, the recombinant host cell is *Pichia pastoris*. In specific embodiments, the recombinant host cell is a *Pichia pastoris* strain that has lost the activity of one or more proteases (e.g., through functional knockout). Furthermore, specific embodiments discussed below are suitable for producing recombinant hydrophobic or partially hydrophobic proteins, such as silk proteins.

[0135] By reference to U.S. Patent 9,963,554, “Methods and Compositions for Synthesizing Improved Silk Fibers,” incorporated herein by reference, compositions for synthesizing block copolymers, recombinant microorganisms for their production, and synthetic fibers comprising said proteins are disclosed. By reference to U.S. Patent Application 15 / 724,196, “Modified Strains for the Production of Recombinant Silk,” incorporated herein by reference, engineered Pasteurella multocida cells selected or genetically engineered to reduce the degradation of recombinant proteins expressed by yeast cells, and methods for culturing yeast cells for the production of useful compounds. Other suitable microbial strains, including Escherichia coli, can be cultured and used for the production of useful compounds.

[0136] Fermentation

[0137] In some embodiments, the inoculum for the recombinant host cells may be derived from a seed strain (i.e., a series of ferments that increase in volume to produce an appropriate number of recombinant host cells). According to this embodiment, the number of seeds may range from 2-7, 3-7, 3-6, or 3-5.

[0138] In some embodiments, the stem cell weight (DCW) per liter of culture medium from the recombinant host cell inoculum is at least 0.2 g / L, at least 0.5 g / L, at least 0.7 g / L, at least 0.8 g / L, at least 1 g / L, at least 2 g / L, at least 3 g / L, at least 4 g / L, or at least 5 g / L; between 0.2 g / L and 3 g / L, between 0.2 g / L and 2 g / L, or between 0.2 g / L and 1 g / L; between 0.5 g / L and 3 g / L, between 0.5 g / L and 2 g / L, or between 0.5 g / L and 1 g / L; between 1 g / L and 3 g / L, between 1 g / L and 2 g / L, or between 0.5 g / L and 1 g / L; or between 3 g / L and 1 g / L. DCW can be determined using a biophotometer (e.g., Eppendorf BioPhotometer D30).

[0139] In most embodiments, the amount of inoculum will depend on the size of the fermentation vessel. In embodiments with fermentation vessels smaller than 150L, ​​the DCW can be in the range of 0.1 g / L to 0.5 g / L. In embodiments with fermentation vessels larger than 150L, ​​the DCW can be in the range of 2 to 4 g / L.

[0140] According to a specific implementation, a suitable fermented broth is any fermented broth in which the recombinant host cells can survive (i.e., maintain growth and / or vitality). Non-limiting examples of suitable fermented broths include aqueous culture media containing nutrients necessary for the growth and / or vitality of the recombinant host cells. Non-limiting examples of such nutrients include carbon sources, nitrogen sources, phosphate sources, salts, minerals, bases, acids, vitamins (e.g., biotin), amino acids, and metals (e.g., iron, zinc, calcium, copper, sodium, potassium, cobalt, magnesium, manganese).

[0141] In some implementations, any of the above nutrients may be restricted to inhibit cell growth and improve the productivity, yield, or titer of the recombinant protein. The carbon source may be any carbon source capable of being fermented by the recombinant host cell. Non-limiting examples of suitable carbon sources include monosaccharides, disaccharides, polysaccharides, acetates, ethanol, methanol, methane, and combinations thereof. Non-limiting examples of monosaccharides include dextrose (glucose), fructose, galactose, xylose, arabinose, and combinations thereof. Non-limiting examples of disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of polysaccharides include starch, glycogen, cellulose, and combinations thereof.

[0142] The nitrogen source can be any nitrogen source that can be assimilated (i.e., metabolized) by the recombinant host cells. Non-limiting examples of suitable nitrogen sources include anhydrous ammonia rich in air or oxygen, ammonium sulfate, ammonium nitrate, diammonium phosphate, monoammonium phosphate, ammonium polyphosphate, sodium nitrate, urea, peptone, protein hydrolysate, yeast extract, and any one of the above.

[0143] In some implementations, any or all nutrients may be sterilized using heat or ozonation before being added to the fermented broth, with the aim of reducing or eliminating microbial contamination. For example, carbon sources may be caramelized or sterilized using heat before being added to the fermented broth. Similarly, carbon sources may be ozonated before being added to the fermented broth. Suitable methods for ozonation are discussed in Dziugan et al., “Ozonation as an effective way to stabilize new kinds of fermentation media used in biotechnological production of liquid fuel additives,” Biotechnology for Biofuels, 9:150 (2016).

[0144] Fermented broth may contain acid or alkali to adjust and / or maintain pH. In some such embodiments, pH is between 4.0 and 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, 5.0, or 4.5; between 4.5 and 8.0, 7.5, 7.0, 6.5, 6.0, 5.5, or 5.0; between 5.0 and 8.0, 7.5, 7.0, 6.5, 6.0, or 5.5; between 5.5 and 8.0, 7.5, 7.0, 6.5, or 6.0; between 6.0 and 8.0, 7.5, 7.0, or 6.5; between 6.5 and 8.0, 7.5, or 7.0; between 7.0 and 8.0 or 7.5; or between 7.5 and 8.0.

[0145] Non-limiting examples of suitable acids include aspartic acid, acetic acid, hydrochloric acid, and sulfuric acid. Non-limiting examples of suitable bases include sodium hydroxide, potassium hydroxide, calcium hydroxide, ammonium hydroxide, calcium carbonate, ammonia, and diammonium phosphate. In some embodiments, strong acids or strong bases are used to limit the dilution of the fermentation broth.

[0146] In some implementations, the fermented broth contains such nutrients or such amounts of such nutrients to enable and / or maintain the desired oxygen uptake rate (OUR). In some such embodiments, the OUR is expected to be at least 40 mmol O2 / L / hr, at least 80 mmol O2 / L / hr, at least 100 mmol O2 / L / hr, at least 105 mmol O2 / L / h, at least 110 mmol O2 / L / h, at least 115 mmol O2 / L / h, at least 120 mmol O2 / L / hr, or at least 140 mmol O2 / L / hr, at least 160 mmol O2 / L / hr, at least 180 mmol O2 / L / hr, at least 200 mmol O2 / L / hr, or at least 220 mmol O2 / L / hr; between 40 mmol O2 / L / hr and 220 mmol O2 / L / hr, between 60 mmol O2 / L / hr and 220 mmol O2 / L / hr, between 80 mmol O2 / L / hr and 220 mmol O2 / L / hr, or between 100 mmol O2 / L / hr and 220 mmol O2 / L / hr. O2 / L / hr; between 100 mmol O2 / L / hr and 140 mmol O2 / L / hr, between 100 mmol O2 / L / hr and 135 mmol O2 / L / hr, between 100 mmol O2 / L / hr and 130 mmol O2 / L / hr, or between 100 mmol O2 / L / hr and 125 mmol O2 / L / hr; between 110 mmol O2 / L / hr and 125 mmol O2 / L / hr, or between 110 mmol O2 / L / hr and 120 mmol O2 / L / hr; or between 115 mmol O2 / L / hr and 120 mmol O2 / L / hr. OUR can be calculated by a person skilled in the art using the direct method described in Bioreaction Engineering Principles, 3rd Edition, 2011, Spring Science + Business Media, page 449.

[0147] In some embodiments, the fermented broth contains such nutrients or such amounts of nutrients that increase the yield of recombinant proteins from the recombinant host cells relative to the yield of byproducts. Non-limiting examples of such byproducts include ethanol. In some embodiments, during 72 hours of fermentation, the cumulative yield of ethanol produced by the recombinant host cells is less than 0.1 g / L, less than 1 g / L, less than 5 g / L, less than 10 g / L, or less than 15 g / L; between 0.1 g / L and 15 g / L, between 1 g / L and 15 g / L, between 5 g / L and 15 g / L, between 10 g / L and 15 g / L, or between 0.5 g / L and 15 g / L; or between 0.1 g / L and 1.5 g / L, between 0.2 g / L and 1.5 g / L, between 0.5 g / L and 1.5 g / L, between 0.7 g / L and 1.5 g / L, or between 1.0 g / L and 1.5 g / L.

[0148] In some embodiments, the fermented broth contains such nutrients or such amounts of such nutrients to achieve and / or maintain a desired dissolved oxygen (DO) content. In some such embodiments, the desired DO content is at least 2%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 100%; or between 2% and 40%, between 2% and 5%, between 5% and 40%, between 2% and 20%, between 5% and 20%, between 2% and 15%, or between 5% and 15%.

[0149] In some embodiments, the fermented broth contains such nutrients or such amounts of such nutrients to achieve and / or maintain a desired respiratory quotient (RQ; i.e., the ratio of carbon dioxide produced to oxygen consumed). In some such embodiments, the desired RQ is less than 2, less than 1.75, less than 1.5, or less than 1.25; or between 1 and 1.1, between 1 and 1.2, between 1 and 1.3, between 1 and 1.4, or between 1 and 1.5.

[0150] In some embodiments, the fermented broth contains such nutrients or such amounts of such nutrients to achieve and / or maintain the desired recombinant host cell doubling time. In some such embodiments, the desired doubling time is at least 4 hours, 8 hours, 12 hours, 16 hours, 18 hours, 22 hours, 26 hours, 30 hours, 34 hours, or 36 hours; or between 4 and 12 hours, between 4 and 10 hours, between 4 and 8 hours, between 6 and 12 hours, between 6 and 10 hours, or between 6 and 8 hours.

[0151] In some embodiments, the fermented broth contains one or more supplemental proteins. In embodiments where recombinant host cells secrete recombinant proteins, the addition of such supplemental proteins can facilitate the transfer of protease activity from the recombinant proteins produced by the recombinant host cells. Non-limiting examples of supplemental proteins include bovine serum albumin (BSA) and casein amino acids. Other supplemental proteins are well known in the art.

[0152] Nutrients can be added to the fermented broth in large, incremental, or continuous doses. In embodiments where nutrients are added continuously, they can be added at a rapid, slow, or exponential rate.

[0153] In embodiments where nutrients are continuously added to fermented broth, nutrients can be added by continuously adding a nutrient-containing culture medium. In these embodiments, an equal volume of aqueous culture medium from the fermented broth can be removed from the fermentation to maintain a constant total volume of the fermented broth. In some embodiments, recombinant host cells can be removed from the fermented broth and re-added to a culture medium containing the nutrients, and then added back to the fermented broth.

[0154] A suitable fermentation vessel is any fermentation vessel in which the recombinant host cells can survive (maintain growth and / or viability). Non-limiting examples of suitable fermentation vessels include culture plates, vials, flasks, or fermenters. Non-limiting examples of suitable fermenters include stirred tank fermenters, airlift fermenters, bubble column reactors, fixed-bed bioreactors, and any combination thereof.

[0155] Suitable fermentation conditions are any conditions under which recombinant host cells can survive (maintain growth and / or viability). Non-limiting examples of such fermentation conditions include a suitable volume of fermentation broth, a suitable pH of fermentation broth, a suitable dissolved oxygen (DO) content in the fermentation broth, a suitable temperature, suitable oxygenation, suitable agitation of the recombinant host cells, and a suitable fermentation duration.

[0156] In various embodiments, a suitable temperature can be any temperature suitable for the growth and / or viability of recombinant host cells and / or the production of recombinant proteins. In some embodiments, the temperature is at least 15°C, 20°C, 25°C, 30°C, or 35°C; between 15°C and 35°C, between 15°C and 25°C, between 15°C and 20°C, between 20°C and 35°C, between 20°C and 30°C, between 20°C and 25°C, between 25°C and 35°C, or between 25°C and 30°C.

[0157] Suitable oxygenation can be any oxygenation suitable for the growth and / or viability of the recombinant host cells and / or the production of the recombinant host cells. Such oxygenation can be achieved by providing suitable aeration and / or suitable agitation to the fermentation vessel and / or fermentation broth. In some embodiments, the suitable aeration is at least 1.5 vvm, at least 1.6 vvm, at least 1.7 vvm, at least 1.8 vvm, at least 1.9 vvm, or at least 2 vvm; between 1.5 vvm and 2 vvm, between 1.5 vvm and 1.9 vvm, between 1.5 vvm and 1.8 vvm, between 1.5 vvm and 1.7 vvm, between 1.5 vvm and 1.6 vvm, between 1.6 vvm and 2 vvm, between 1.7 vvm and 2 vvm, between 1.8 vvm and 2 vvm, or between 1.7 vvm and 1.9 vvm.

[0158] Depending on the implementation plan and the type of fermentation, the appropriate stirring of recombinant host proteins in fermented broth can be varied.

[0159] According to the implementation scheme, a bubble column can be used for aeration. The complexity of the bubble column can vary depending on the specific implementation scheme (e.g., it can be single-phase or multi-phase) and can provide a variety of gas velocities. Non-limiting examples of suitable gas velocities include, but are not limited to, 0.003–0.08 m / s. For non-limiting examples of bubble reactors, see Kantarci et al., “Bubble Column Reactors, Process Biochemistry 40:2263–2283 (2005)”.

[0160] In some embodiments, the fermented broth contains an agent (“defoamer”) for reducing foaming during fermentation. Foam, as defined herein, is a dispersion of gas in a continuous liquid phase located at or near the top of the fermentation vessel. Depending on the embodiment, the defoamer may be selected and optimized to reduce interaction with any recombinant protein product. Non-limiting examples of defoamers include silicone-based oils, emulsions, and polymers; polypropylene glycol; polyethylene glycol-based defoamers; polyalkylene glycol-based defoamers; difunctional ethylene oxide / propylene oxide (EO / PO) block copolymers; fatty acid-based defoamers; polyester-based defoamers; oil-based defoamers; and any combination thereof. Suitable defoamers are discussed in Junker, “Foam and its Mitigation in Fermentation Systems, Biotechnol. Prog., 23:767-784 (2007)”. In embodiments where the recombinant protein is a hydrophobic protein (such as silk fibroin), the defoamer may be selected such that it solubilizes or does not solubilize the hydrophobic protein.

[0161] The expected cumulative yield of recombinant protein can be any cumulative yield that contributes to low production costs. As used herein, cumulative yield is calculated as the percentage of the mass of recombinant protein produced relative to the mass of carbon source metabolized by the recombinant host cells during fermentation (i.e., the mass of carbon source provided minus the mass of carbon source remaining in the fermentation broth; for example, if 100 g of glucose is provided to the recombinant host cells and 25 g of recombinant protein is produced at the end of fermentation with 10 g of glucose remaining, the cumulative yield of recombinant protein is 27.7%). Assuming all other metrics are equal, a higher cumulative yield provides lower production costs compared to a lower cumulative yield. In some embodiments, the cumulative yield of recombinant silk protein after 72 hours of fermentation is at least 1%, at least 5%, at least 30%, or at least 100% based on carbon source; between 1% and 5%, between 5% and 10%, between 10% and 35%, between 35% and 50%, or between 50% and 100%.

[0162] The desired cumulative titer of recombinant protein can be any cumulative titer that contributes to low production costs. As used herein, the cumulative titer is calculated as grams of recombinant protein produced per liter of fermented broth during fermentation (i.e., g / L). Assuming all other parameters are equal, a higher cumulative titer provides lower production costs compared to a lower cumulative titer. In some embodiments, after 72 hours of fermentation, the cumulative titer of recombinant protein is at least 2 g / L, at least 5 g / L, at least 15 g / L, or at least 30 g / L; between 1 g / L and 100 g / L, 5 g / L, 15 g / L, or 30 g / L; between 10 g / L and 100 g / L, 80 g / L, or 75 g / L; or between 5 g / L and 30 g / L.

[0163] The expected cumulative productivity of recombinant protein can be any cumulative productivity that contributes to low production costs. As used herein, cumulative productivity is calculated as grams of recombinant protein produced per liter of fermented broth per hour during fermentation (i.e., g / L / hr). Assuming all other metrics are equal, a higher cumulative productivity provides lower production costs compared to a lower cumulative productivity. In some embodiments, the cumulative productivity of recombinant protein is at least 0.001 g / L / hr, at least 0.025 g / L / hr, at least 0.05 g / L / hr, at least 0.1 g / L / hr, or at least 0.2 g / L / hr; between 0.001 g / L / hr and 0.5 g / L / hr.

[0164] The methods provided herein can be performed at any fermentation scale and / or according to any fermentation procedure known in the art. The fermentation procedure can be batch-fed, batch, continuous, or any combination thereof. In some embodiments, the method begins with one or more batch fermentations, followed by one or more continuous fermentations, wherein the inoculum of recombinant host cells, suitable fermentation broth, suitable fermentation vessel, and / or one or more suitable fermentation conditions may differ between the one or more batch fermentations and / or one or more continuous fermentations. In some embodiments, the temperature of the batch fermentation is higher than that of the continuous fermentation. In some such embodiments, the temperature of the batch fermentation is above 27°C, while the temperature of the continuous fermentation is below 27°C.

[0165] In some embodiments, fermentation is carried out in stages. Such stages may include a growth stage, a production stage, and / or a recovery stage. In some embodiments, the stages differ from one another in terms of the inoculum for recombinant host cells, a suitable fermentation broth, a suitable fermentation vessel, and / or one or more suitable fermentation conditions.

[0166] Methods for isolating recombinant proteins

[0167] According to the implementation scheme, various methods can be used to isolate and recover the recombinant protein of interest. As discussed above, some, but not all, of these methods are specific to recombinant host cells that secrete the recombinant protein of interest. Furthermore, some of these methods are specific to hydrophobic recombinant proteins of interest.

[0168] Figure 1 A process flow for isolating recombinant proteins according to one embodiment of the present invention is described. Those skilled in the art will understand that... Figure 1 Some of the steps shown may be performed alternately in sequence and / or repeatedly. Those skilled in the art will recognize that the disclosed embodiments are not intended to limit the scope of the methods provided herein, and that the methods may vary based on the recombinant host cells used, the desired cumulative yield, cumulative titer and / or cumulative productivity, or other factors.

[0169] In optional step A02, biomass (i.e., intact or destroyed recombinant host cells and cell debris) is removed from the fermentation broth containing recombinant host cells. In various embodiments, biomass removal may also include the removal of insoluble fermentation impurities (such as, for example, antifoaming agents and other fermentation broth components that may have precipitated during protein solubilization).

[0170] In various embodiments, biomass removal can be performed based on size, weight, density, or a combination thereof. Size-based biomass removal can be accomplished by filtration, such as using a filter press, candle filter, or other industrially used filtration system with a molecular weight cutoff smaller than the size of the recombinant host cells. Weight- or density-based biomass removal can be accomplished by gravity settling or centrifugation, such as using a settling tank, a low-g-force decanter centrifuge, a disk stack separator, a two-phase nozzle centrifuge, a solid jet centrifuge, or a hydrocyclone. As disclosed herein, biomass removal produces a centrifuged liquid containing proteins (i.e., a light phase or clear cell broth) and a solids (heavy phase) containing biomass and insoluble fermentation impurities. Suitable conditions for biomass removal (e.g., g-force, settling time, centrifugation time, solids percentage in centrifuge input, centrifuge feed rate) can be determined using methods known in the art, with the aim of minimizing biomass and insoluble fermentation impurities in the clear cell broth. In some embodiments, biomass removal provides a clear cell broth with a wet-filled solids volume of less than 5%, less than 1%, less than 0.5%, or less than 0.1%. In some embodiments, biomass removal provides a clear cell broth containing protein at concentrations between 1 g / L and 50 g / L. In some embodiments, the clear cell broth is subjected to precision centrifugation to remove residual solids. In some embodiments, the solids obtained from biomass removal are subjected to at least one more round of protein solubilization and biomass removal, wherein all centrifuged liquids are ultimately combined for further processing according to the methods provided herein.

[0171] According to the implementation scheme, step A02 may be performed before and / or after step A04. Step A02 may be performed several times. For example, several rounds of centrifugation and / or filtration may be performed before and / or after step A04 to remove biomass.

[0172] In step A04, the recombinant protein is solubilized. In some embodiments where step A02 is not performed, the recombinant protein may be separated from the recombinant host cell prior to solubilization by centrifuging the recombinant host cell and the recombinant protein associated with the recombinant host cell into biomass clumps (hereinafter referred to as "cell clumps") and discarding the supernatant. This step may be advantageous when the recombinant protein cannot dissolve and / or aggregates with itself and / or adheres to the surface of the recombinant host cell. In other embodiments, the recombinant protein is solubilized in whole-cell broth. In some embodiments, the recombinant protein is solubilized in a clear cell broth produced by performing step A02.

[0173] In some embodiments, solubilization of recombinant proteins can be accomplished by adding a solubilizer to whole-cell broth, clear cell broth, or cell clumps. Non-limiting examples of suitable solubilizers include surfactants, co-solubilizers, SDS, urea, cysteine, guanidine thiocyanate, enzymes that hydrolyze polysaccharides (e.g., dextranase, lysozyme, mannosinase, chitosanase), high-pH water (H₂O at pH 11-12), or other known dissociative agents. Different types of recombinant proteins may require different solubilizers. Suitable conditions for protein solubilization (e.g., type and amount of extractant, temperature, incubation time, agitation, and pH) can be determined using methods known in the art, aimed at maximizing the yield of the recombinant protein and minimizing the lysis of recombinant host cells and the solubilization of impurities. As discussed above, in certain embodiments where the recombinant protein cannot dissolve and / or aggregates with itself and / or aggregates in or near the recombinant host cells, the recombinant host cells may be centrifuged and the supernatant may be discarded before adding the solubilizer to the clumps.

[0174] In some embodiments, various techniques can be used to perforate or permeabilize the cell membrane of the recombinant host cells to remove excess protein from the cell membrane before solubilization and / or precipitation. Such methods include chemical disruption, mechanical disruption, or sonication. Mechanical disruption of the cell membrane includes homogenization, shearing, freezing / thawing, heating, pressurization, sonication, and filtration. Chemical disruption includes detergents (such as triton, sodium dodecyl sulfate) or dissociation agents (such as urea and guanidine). Other methods are well known in the art.

[0175] In specific embodiments, urea is used as a solubilizer to solubilize the recombinant protein and prevent damage to the recombinant host cells. The urea concentration can be varied to prevent damage to the recombinant host cells. According to embodiments, the amount of urea concentration can be in the range of 4M to 10M. In various embodiments, the recombinant host cells and recombinant protein can be incubated with urea for 1-2 hours, 1-3 hours, or 1-4 hours. According to embodiments, other known dissociation agents such as guanidine thiocyanate are used to solubilize the recombinant protein.

[0176] In specific implementations, high-pH H2O or aqueous buffer solutions are used to solubilize the recombinant protein and prevent damage to the recombinant host cells. The pH of the high-pH H2O or aqueous buffer solution can be varied to prevent damage to the recombinant host cells. According to the implementation, the pH range of the high-pH H2O may be pH 10 to pH 12.5, pH 10.5 to pH 12.5, pH 11 to pH 12.5, pH 12 to pH 12.5, pH 10 to pH 12, pH 10.5 to pH 11.0, pH 10.5 to pH 11.5, pH 10.5 to pH 12, pH 10.5 to pH 12.5, pH 11 to pH 11.5, pH 11 to pH 12, pH 11.5 to pH 12.5, or pH 12 to pH 12.5. In various implementation schemes, the recombinant host cells and recombinant proteins may be incubated with high pH H2O for at least 10 minutes, at least 15 minutes, at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 75 minutes, at least 90 minutes, at least 115 minutes, or at least 120 minutes.

[0177] In specific implementations, homogenization is used to lyse host cells. Homogenization pressure (psi) can be between 5,000-100,000 psi, 5,000-10,000 psi, 10,000-20,000 psi, 20,000-30,000 psi, 30,000-40,000 psi, 40,000-50,000 psi, 50,000-60,000 psi, 60,000-70,000 psi, 70,000-80,000 psi, 80,000-90,000 psi, or 90,000-100,000 psi. Homogenization can be a single pass or multiple passes. In some implementations, homogenization is performed in one, two, three, four, or five passes.

[0178] In step A06, impurities are removed from the fermentation product. Step A06 may be performed before and / or after steps A04 and / or A08. Step A06 may be repeated any number of times. Removal of impurities from the fermentation product can be accomplished by filtration, absorption (e.g., carbon absorption or solid-state absorption), dialysis, and phase separation induced by coagulation or the use of various chemicals. In embodiments where phase separation is induced by coagulation, coagulation can be induced by cooling the fermentation product to a temperature sufficient to induce phase separation. In other embodiments, phase separation can be chemically induced by adding a structure-building agent (cosmotrope) and / or a compound used to precipitate proteins from solution. Detailed embodiments for removing impurities using phase separation are described below with reference to Figure C. In some embodiments where the recombinant protein has thermal stability, other proteins can be removed by subjecting the fermentation product to high temperatures to denature the other proteins and centrifuging to separate the denatured proteins from the proteins in solution.

[0179] In some implementations, filtration, microfiltration, dialysis filtration, and / or ultrafiltration (e.g., for deionized water) are used to remove impurities. Membranes suitable for microfiltration may range from 0.1 μM to 1 μM. Non-limiting examples of suitable membranes for ultrafiltration include 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 yields a recombinant protein slurry in water as a retention and a permeate containing impurities. Suitable ultrafiltration conditions (e.g., membrane, temperature, volume displacement) are determined using methods known in the art, with the aim of maximizing the permeate density. In some embodiments, ultrafiltration provides a retention with a density between 1 g / mL and 30 g / mL. In some embodiments, ultrafiltration includes a concentration step (which produces a concentrated retention) followed by a dialysis filtration step (which removes impurities and produces a suspended protein slurry in water). In some such embodiments, the concentrated retention has a concentration factor that reduces the volume relative to the initial volume by between 2 and 12 times. In some embodiments, dialysis filtration provides a constant volume displacement between 3 and 10 times.

[0180] Depending on the implementation scheme and the type of impurity to be removed, the method for removing impurities may vary. Removal of lipid impurities from isolated recombinant proteins can be accomplished by methods known in the art. Non-limiting examples of such methods include absorption by char or other absorption media that specifically bind lipids. Removal of polysaccharide impurities from isolated recombinant proteins can be accomplished by methods known in the art. Non-limiting examples of such methods include enzymatic treatment with hydrolysates followed by removal of small sugars produced by ultrafiltration. Non-limiting examples of such enzymes include dextranases, lysozymes, mannoses, and chitosanases.

[0181] In step A08, the solubilized recombinant protein is separated. The recombinant protein is solubilized. The solubilized recombinant protein can be separated using a variety of different methods, including extraction buffers, size exclusion chromatography, gel filtration, ultrasonic protein extraction, and ion exchange chromatography. In some embodiments where biomass is not removed in optional step A02, the recombinant protein can be separated together with the recombinant host cell.

[0182] In some embodiments, the recombinant protein is precipitated as a single separation step or as a single separation step plus other separation steps. Precipitation of the solubilized recombinant protein can be accomplished by adding a precipitant to the fermentation broth. Non-limiting examples of such precipitants include sulfate ions (e.g., ammonium sulfate, sodium sulfate, sulfuric acid) or citrate ions (e.g., sodium citrate). In some embodiments, the precipitant is an acid. In some embodiments, the precipitant is a salt. In one embodiment, the precipitant is H₂SO₄.

[0183] The pH of the solution containing the solubilized recombinant protein can be adjusted or altered using any suitable acid. Suitable acids include mineral acids such as hydrochloric acid (HCl), sulfuric acid (H₂SO₄), nitric acid (HNO₃), boric acid (H₃BO₃), phosphoric acid (H₃PO₄), hydrofluoric acid (HF), hydrobromic acid (HBr), perchloric acid (HClO₄), and hydroiodic acid (HI); organic acids such as citric acid, formic acid, acetic acid, propionic acid, butyric acid, valeric acid, capriocic acid, oxalic acid, lactic acid, malic acid, benzoic acid, carbonic acid, uric acid, taurine, p-toluenesulfonic acid, trifluoromethanesulfonic acid, aminomethylphosphonic acid, and 2,2,2,-trichloroacetic acid (TCA); or any combination thereof or other suitable acids known in the art. Salts of any of the acids disclosed above may also be used.

[0184] In some embodiments, the recombinant protein is precipitated at pH 4-10. In some embodiments, precipitation is carried out at pH 4, 5, 6, 7, 8, 9, or 10. In some embodiments, precipitation is carried out at at least pH 4, at least pH 4.5, at least pH 5, at least pH 5.5, at least pH 6, at least pH 6.5, at least pH 7, at least pH 7.5, at least pH 8, at least pH 8.5, at least pH 9, at least pH 9.5, or at least pH 10. In one embodiment, precipitation is carried out at pH 7. In some embodiments, precipitation is carried out at pH 4-5, pH 5-6, pH 6-7, pH 7-8, pH 8-9, or pH 9-10.

[0185] The precipitation process can be repeated once, twice, or multiple times as needed. In some embodiments, more than one precipitation step is performed, and the pH value is the same for each precipitation. In other embodiments, more than one precipitation step is performed, and the pH value is different for each precipitation. For example, the first precipitation may be performed at pH 4, and then the second precipitation may be performed at pH 7.

[0186] As disclosed herein, the separation of precipitated recombinant proteins can be accomplished based on size, weight, density, or a combination thereof. In some embodiments, such separation provides a suspension of recombinant protein slurry as a retainer and a permeate containing waste. Suitable conditions for precipitating the recombinant protein (e.g., dilution before adding divalent anions, the type and amount of divalent anions, incubation temperature, incubation time) and separating the precipitated recombinant protein can be determined using methods known in the art, with the aim of maximizing the yield of recombinant protein in the suspension slurry. In some embodiments, the yield of precipitated recombinant protein in the suspension slurry is between 20% and 99%. In some embodiments, the suspension slurry has a wet filler solids content between 30% and 65%. In some embodiments, the suspension slurry contains silk protein at a concentration between 10 g / L and 50 g / L. In some embodiments, the steps of precipitating silk protein and separating the precipitated silk protein are repeated at least once (using the same or different process conditions) to further wash away water-soluble impurities.

[0187] In optional step A10, the isolated recombinant protein is concentrated. Concentration of the isolated recombinant protein can be accomplished by evaporation at high temperature and / or reduced pressure (e.g., partial vacuum). Suitable conditions (e.g., temperature, pressure, duration) for concentrating the isolated recombinant protein can be determined using methods known in the art, with the aim of obtaining isolated recombinant protein with an increased dry solids content. In some embodiments, concentration provides a volume reduction of between 20% and 70% of the original volume. In some embodiments, concentration provides a concentrated isolated recombinant protein containing between 3% and 20% dry solids.

[0188] In optional step A12, the separated recombinant protein is dried. Drying the suspended silk protein slurry to obtain silk protein powder can be accomplished by spray drying, drum dryer, freeze-drying, or fluidized bed drying. In some embodiments, the powder has a moisture content of less than 10%, less than 9%, less than 8%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1%.

[0189] Figure 2 A process flow for isolating recombinant proteins according to one embodiment of the present invention is described. Those skilled in the art will understand that... Figure 2 Some of the steps shown may be performed in an alternating order and / or repeated. Those skilled in the art will recognize that the disclosed embodiments are not intended to limit the scope of the methods provided herein, and that the methods may vary based on the recombinant host cells used, the desired cumulative yield, cumulative titer and / or cumulative productivity, or other factors.

[0190] In step B05, the recombinant host cells are lysed and / or otherwise destroyed to release their contents into the fermentation product. Depending on the implementation, the recombinant host cells can be destroyed using various different methods. Suitable methods for lysing and / or destroying host cells include: using heat such as high-temperature short-time (HTST) methods, high-shear cell destruction, physical homogenization, and chemical homogenization.

[0191] In optional step B04, the recombinant protein is solubilized as described above for step A04. Step B04 may be performed before or after step B05. In some embodiments, step B04 may be performed both before and after step B05.

[0192] In optional step B02, biomass is removed as described above for step A02. Furthermore, in the case of solubilization of recombinant proteins, other methods for removing biomass from lysed and / or damaged cells may include centrifugation and filtration.

[0193] In optional step B06, impurities are removed as described above for step A06. Steps B02 and B06 may be performed before or after other steps and may be repeated. In some embodiments, step B06 may be performed before and after step B08.

[0194] In step B08, the recombinant protein is isolated. A suitable method for isolating the recombinant protein has been described above for step A08. Furthermore, the method for isolating the recombinant protein may also include the use of an additional membrane in filtration and / or degumming to remove phospholipids.

[0195] In optional step B10, the recombinant protein is concentrated as described above for step A10. In optional step B12, the recombinant protein is dried as described above for step B10.

[0196] Figure 3 A process flow for purifying recombinant proteins according to one embodiment of the present invention is described. Those skilled in the art will understand that... Figure 3 Some of the steps shown may be performed in an alternating order and / or repeated. Those skilled in the art will recognize that the disclosed embodiments are not intended to limit the scope of the methods provided herein, and that the methods may be varied based on various factors.

[0197] In step CO2, an aqueous two-phase solution is prepared by denaturing the recombinant protein using a strong dissociation agent. Suitable dissociation agents include, but are not limited to: guanidine thiocyanate (GD-SCN), guanidine hydrochloride (GD-HCl), guanidine iodide, urea, lithium perchlorate, lithium acetate, magnesium chloride, sodium dodecyl sulfate (SDS), potassium iodide (KI), or any combination thereof. According to the embodiment, the dissociation agent and the protein may be heated to promote protein denaturation.

[0198] In some embodiments, a kosmotrope (also referred to herein as a "precipitant") is added to the solution to facilitate phase separation. Suitable kosmotropes include the precipitants mentioned above. In other embodiments, a high initial concentration of the dissociation agent is used to denature the recombinant protein, and then the concentration of the dissociation agent is slowly diluted to achieve phase separation.

[0199] In step C04, a phase-separated viscous layer is obtained. Depending on the type of phase separation, various methods can be used to obtain the viscous layer, such as decantation / extraction of the non-viscous layer or extraction of the viscous layer using a Hamilton needle or pipette. Other methods will be known to those skilled in the art.

[0200] As step C06, the phase-separated viscous layer is further processed to remove impurities. Suitable dialysis agents include double-distilled H2O or low-concentration GD-SCN. Depending on the embodiment, various dialysis methods can be performed, including cassette dialysis or other suitable methods known in the art. In some embodiments, tangential flow filtration (TFF) is used to dialyze the viscous layer.

[0201] In some embodiments, the isolated recombinant spider silk protein is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% full-length recombinant spider silk protein.

[0202] In some embodiments, the purity of the isolated recombinant spider silk protein is 5-10%, 10-15%, 15-20%, 20-25%, 25-30%, 30-35%, 35-40%, 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-85%, 85-90%, 90-95%, or 95-100%. In some embodiments, the purity of the isolated recombinant spider silk protein is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%.

[0203] In some embodiments, the full-length recombinant spider silk protein was measured or quantified. Any suitable method can be used to measure or quantify the amount of the full-length recombinant protein, including but not limited to size exclusion chromatography (SEC), SDS-PAGE, Western blotting (protein blotting), high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), or rapid protein liquid chromatography (FPLC), or any other suitable method known in the art, or any combination thereof. In one embodiment, Western blotting was used to determine the amount of the full-length recombinant spider silk protein. In another embodiment, size exclusion chromatography (SEC) was used to measure the amount of the full-length recombinant spider silk protein.

[0204] Example

[0205] The following are embodiments for carrying out specific implementations of the invention. These embodiments are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Efforts have been made to ensure accuracy for the figures used (e.g., quantities, temperatures, etc.), but some experimental error and deviation should certainly be allowed.

[0206] Unless otherwise specified, the practice of this invention will utilize conventional methods of protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology within the scope of the art. Such techniques are fully explained in the literature. See, for example, Tetreighton, Proteins: Structures and Molecular Properties (WH Freeman and Company, 1993); Allehninger, Biochemistry (Worth Publishers, Inc., current edition); Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd edition, 1989); Methods in Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry, 3rd edition (Plenum Press), Volumes A and B (1992).

[0207] Example 1: Purification of 18B using single-step alkaline conditions

[0208] The recombinant protein was solubilized using a high-pH solution without disrupting the host cells secreting the protein. The pH buffer concentration and incubation time were tested to determine the solubility of recombinant spider silk protein from the Argiope bruennichi MaSp2 block (“18B”, SEQ ID NO: 38) expressed in *P. pastoris* with a C-terminal 3x FLAG tag (SEQ ID NO: 40). The FLAG tag was attached to the C-terminus of the 18B peptide sequence using a glycine residue (G) linker.

[0209] Specifically, cell culture fermentation broth was inoculated with *Pichia pastoris* expressing the recombinant 18B protein and incubated to allow for 18B protein expression. The culture was centrifuged to harvest cells, and cell clumps were resuspended in distilled water at a ratio of 1:1 (equal parts cell clumps and water) or 1:3 (one part cell clump and two parts water). The pH of the cell clump suspension was adjusted to a final pH of 11.8–11.9 using 2–10 M NaOH. The cell clump suspension was incubated at room temperature with stirring for 15–30 minutes. During incubation, the pH was adjusted with NaOH to maintain a pH of 11.8–11.9. The cell clump suspension was centrifuged, and the supernatant containing the recombinant protein was collected. The supernatant was lyophilized to concentrate the 18B protein, and the amount of recovered 18B protein was assessed by size exclusion chromatography (SEC) as described below. Figure 4A and 4B ).

[0210] Size exclusion chromatography (SEC) was used to analyze the relative contents of high molecular weight impurities, low molecular weight impurities, medium molecular weight impurities, monomeric 18B, and aggregated 18B. 18B powder was dissolved in 5M guanidine thiocyanate (GdSCN) and injected onto a YarraSEC-3000 SEC-HPLC column to separate components according to molecular weight. Refractive index was used as the detection mode. Quantifications were performed on 18B aggregates, 18B monomers, low molecular weight (1–8 kDa) impurities, medium molecular weight (8–50 kDa) impurities, and high molecular weight (110–150 kDa) impurities. Relevant components are reported as mass % and area % respectively. BSA was used as a general protein standard, assuming that >90% of all proteins showed dn / dc values ​​(refractive index response factors) within approximately 7% of each other. Poly(ethylene oxide) was used as a retention time standard, and BSA calibrators were used as check standards to ensure consistent performance of the method. As a control, samples purified using urea-enriched 18B protein were also evaluated.

[0211] Alkaline extraction of 18B from *Pichia pastoris* cell clumps was performed at pH 11.9, yielding a full-length 18B protein extraction yield normalized to 70-75% of the 18B protein isolated using 5 mg dSCN. Sample purity was calculated using the SEC area % of the extracted 18B protein. The purity of the 18B monomer in the alkaline extract was approximately 35% monomer area, 35% medium molecular weight impurity area, and 28% low molecular weight impurity area %. Figure 4A and 4BIn contrast, solubilizing 18B protein with 10M urea resulted in a lower yield, approximately 26% of the monomer area, 27% of the medium molecular weight impurities area, and 45% of the low molecular weight impurities area. These data indicate that alkaline solubilization and extraction methods lead to a higher yield of 18B and a higher purity of the isolated 18B protein.

[0212] Example 2: Further purification of isolated silk peptides

[0213] To further purify 18B spider protein, ultrafiltration and tangential flow filtration were performed on the 18B sample separated from the above alkaline extraction using a 750 kWh filter and 8 dialysis volumes of water. As previously described, samples containing unfiltered protein, ultrafiltered protein, and protein after 1, 3, 6, and 8 dialysis volumes of water were evaluated by SEC. The SEC% area of ​​18B monomer, medium molecular weight impurities, low molecular weight impurities, and high molecular weight impurities in each sample was [data missing]. Figure 5 The leftmost bar shows the unfiltered protein sample (“Unconditioned Feed”), the second bar from the left shows the ultrafiltered protein sample (“Unconditioned UFR”), and samples of 1, 3, 6, or 8 dialysis volumes are shown in the middle left, middle right, second from the right, and rightmost bars, respectively. Figure 5 The increased dialysis volume during washing resulted in an increase in the percentage area of ​​18B monomers and a decrease in the percentage area of ​​low molecular weight impurities.

[0214] Example 3: Purification of 18B using a two-step alkaline extraction method

[0215] To increase the recovery rate of 18B protein in cells, a two-step extraction process was performed. Using 2M NaOH as the first alkaline extraction step, the pH of whole-cell broth of *P. pastoris* cells expressing 18B was adjusted to pH 11.8 and incubated for 30–60 minutes. A control sample of the whole-cell broth of *P. pastoris* cells expressing 18B was incubated with 5 Mg dSCN for approximately 15 minutes to solubilize and extract the 18B protein. Cells were allowed to clump together, and the supernatant was collected. As a second extraction step, the remaining clumps from the first alkaline extraction step were re-extracted by adding water at pH 11.8 in a clump:water ratio of 1:1, 1:2, or 1:3. The supernatants from the first and second alkaline extractions containing recombinant 18B protein were collected. The supernatants were lyophilized to concentrate the 18B protein, and the samples were evaluated by SEC as described in Example 1. Two separate experimental runs for each extraction condition and the GdSCN control are shown. Figure 6AIncreasing the amount of alkaline water (ratios of 1:2 and 1:3) increased the amount of 18B protein recovered. However, the purity of the 18B monomeric protein obtained from double extraction was highest in a single extraction. The purity of the 18B monomer also increased with increasing amounts of alkaline water relative to clumps used in the second extraction. Figure 6B ).

[0216] Then, as previously described, the extracted sample was purified by ultrafiltration and tangential flow filtration using a 750 kWh filter and up to 8 dialysis volumes of water. The purity of the resulting silk peptide composition was assessed by SEC. Figure 7A and 7B ). Figure 7A The percentage area of ​​18B monomer, medium molecular weight impurities, and low molecular weight impurities is shown. The increase in dialysis volume during tangential flow filtration resulted in an increase in the peak area of ​​18B monomer. Figure 7B The SEC peaks for each sample, starting material (“SM”), ultrafiltration retained product (“UFR”), and tangential flow filtration dialysis volumes 1, 2, 3, 4, 6, and 8 (DF 1, 2, 3, 4, 6, 8) are shown.

[0217] Example 4: Further separation of silk polypeptides from alkaline extract by changing pH value

[0218] The 18B recombinant protein from the alkaline extract was precipitated by adjusting the pH of the extract. In this experiment, the alkaline extraction from whole-cell culture broth was first performed by adjusting the pH of the whole-cell culture broth to a final pH of 11.8-11.9 by adding NaOH, thereby producing an alkaline cell suspension. The cell suspension was incubated at room temperature with stirring for 15-30 minutes. After incubation, the cell suspension was centrifuged, and the alkaline supernatant containing the solubilized 18B protein was collected to produce the 18B alkaline extract.

[0219] Then, the 18B alkaline extract samples were treated with different pH conditions to precipitate the 18B protein. H₂SO₄ was added to the alkaline extract samples to a final pH of 4, 5, 6, 7, 8, 9, or 10. The precipitate containing the 18B recombinant protein was then separated from the alkaline extract. The precipitate samples were evaluated by SEC as previously described. Figure 8 The SEC% area purity of the high molecular weight (HMW) peak, 18B monomer and aggregate peaks, medium molecular weight (IMW) peak, and low molecular weight (LMW) peak is shown for each pH condition. Figure 9 The yield percentage of 18B protein at each tested precipitant pH is shown. Under all conditions, the single-stage precipitation step at pH 7 was found to be most efficient after initial alkaline extraction of the 18B protein, with approximately 70% of the area indicating a purity of approximately 70%. Figure 10The SEC spectrum of the 18B precipitate at pH 6 is shown.

[0220] In addition to dialysis centrifugation, TFF (tangential flow filtration) was performed to separate the basic extract. However, dialysis centrifugation is more effective than TFF in removing impurities and generally achieves 60-70% protein recovery and >70% 18B protein purity.

[0221] The 18B protein precipitate obtained at pH 6 was freeze-dried, wet-spun into fibers, and its toughness was measured. The freeze-dried 18B protein was dissolved in formic acid to a final protein content of 36 wt%. The dissolved protein was extruded at 40 μl / min into a 100% ethanol coagulation bath to produce fibers. The 18B fibers produced by this method had a toughness of 19.4 cN / tex.

[0222] Example 5: Comparison of P0 recovery using alkaline conditions and P0 recovery using salt precipitation

[0223] pH buffer concentration and incubation time were tested to determine their use in solubilizing PO (SEQ ID NO: 39) recombinant silk protein in Escherichia coli cell lysate for extraction from cell cultures.

[0224] Cell culture fermentation broth was inoculated with *Escherichia coli* expressing the recombinant P0 protein with a C-terminal 6x-His tag (SEQ ID NO:46) and incubated to allow P0 protein expression. The culture was centrifuged at 15,000 rcf to form cell clumps. The supernatant was removed, and the cell clumps were resuspended in H2O at a ratio of 1:4 (cell clumps:buffer) or 1:9 (cell clumps:buffer) and incubated for 15–60 min. The pH of the resuspended cell clumps was adjusted to a final pH of 9, 10, 10.5, or 11 using NaOH. As a control, the resuspended cell clump sample was also incubated with 5M guanidine thiocyanate (GdSCN) and sonicated for 1.5 min. The sample was vortexed and homogenized using a rotisserie mixer. The lysate was purified by centrifugation at 15,000 rcf for 5 min, retaining the purified supernatant containing the P0 protein. The supernatant was filtered through a 0.25 μm filter and analyzed by BCA, ELISA, and Western blotting.

[0225] Samples were normalized to a protein concentration of 1 mg / mL, and the amount of solubilized P0 in each sample was assessed by immunoblotting using an anti-His antibody. Figure 11Lane H1 is a control sample lysed by sonication in 5 mg dSCN. Lanes B1-B4 are samples mixed in a 1:4 ratio of cell clumps to buffer (pH 9, pH 10, pH 10.5, and pH 11), and lanes B7-B10 are samples mixed in a 1:9 ratio of cell clumps to buffer (pH 9, pH 10, pH 10.5, and pH 11). Lanes C2-C4 are samples incubated with GdSCN for 15, 30, or 60 minutes.

[0226] In an exemplary method, a cell culture fermentation broth is inoculated with *Escherichia coli* expressing recombinant P0 protein and incubated to induce P0 protein expression. The culture is centrifuged at 15,000 rcf to form cell clumps. The cell clumps are resuspended in H2O at a cell clump:liquid ratio of 1:1 or 1:3, and the cell suspension is homogenized at 10,000 to 40,000 psi to lyse the *E. coli* cells. The lysate is purified by centrifugation, retaining the cell clumps containing insoluble P0. The cell clumps are resuspended in H2O, and the pH of the cell clump suspension is adjusted to a final pH of 11.5 with 2–10 M NaOH. The cell clump suspension is incubated at room temperature with stirring for 15–60 minutes. The pH is adjusted with NaOH to maintain the pH at 11.5 during incubation. After incubation, the cell suspension is centrifuged, and the supernatant containing recombinant P0 protein is collected.

[0227] As an additional method, insoluble PO can be extracted from cell clumps using an alkaline buffer containing 10M urea. After resuspending the cell clumps in H2O, adjust the pH of the cell clump suspension to a final pH of 11.5 with 2-10M NaOH, and add urea to a final concentration of 10M. Incubate the cell clump suspension at room temperature with stirring for 15-60 minutes.

[0228] In all methods, the isolated recombinant P0 protein can be further purified by additional purification steps such as filtration, centrifugation, precipitation, or chromatography.

[0229] Equivalent solution

[0230] Although the invention has been particularly shown and described with reference to preferred embodiments and various alternative embodiments, those skilled in the art will understand that changes in form and detail may be made therein without departing from the spirit and scope of the invention.

[0231] All references, granted patents and patent applications cited in the text of this specification are incorporated herein by reference in their entirety for all purposes.

[0232] Informal sequence list

[0233]

[0234]

Claims

1. A method for isolating recombinant spider silk protein from a host cell culture, comprising: a. Obtaining a cell culture, wherein the cell culture comprises a host cell and a growth medium, wherein the host cell expresses a recombinant spider silk protein, wherein the recombinant spider silk protein comprises a large ampulla 2 (MaSp2) filament, and wherein the cell culture comprises fungal or bacterial cells; b. Collect a portion of the cell culture containing the host cells and the recombinant spider silk protein without separating the host cells from the recombinant spider silk protein; c. Incubate the portion of the cell culture in an aqueous solution for 15-60 minutes under alkaline conditions with a pH range of 11-12, thereby solubilizing the recombinant spider silk protein in the aqueous solution; as well as d. The recombinant spider silk protein is isolated from the aqueous solution to produce an isolated recombinant spider silk protein sample.

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

3. The method of claim 2, wherein the isolated recombinant spider silk protein sample comprises at least 30% of full-length recombinant spider silk protein relative to the total isolated recombinant spider silk protein.

4. The method of claim 3, wherein the percentage of full-length recombinant spider silk protein is measured using Western blotting.

5. The method of claim 3, wherein the percentage of full-length recombinant spider silk protein is measured by size exclusion chromatography.

6. The method of any one of claims 1-3, wherein the purity of the isolated recombinant spider silk protein is at least 30%.

7. The method of any one of claims 1-3, wherein the yield of the isolated recombinant spider silk protein ranges from 50 to 100% relative to the recombinant spider silk isolated by urea or guanidine thiocyanate method.

8. The method of any one of claims 1-3, wherein separating the recombinant spider silk protein comprises precipitating the recombinant spider silk protein by changing the alkaline conditions of the aqueous solution.

9. The method of claim 8, wherein changing the alkaline conditions comprises adjusting the alkaline pH of the portion of the cell culture to a decreased pH value from 4 to 10.

10. The method of claim 9, wherein the reduced pH value is a pH value of 4, 5, 6, 7, 8, 9, or 10.

11. The method of claim 9, wherein the reduced pH value is a pH value from 6 to 7.

12. The method of claim 8, wherein adjusting the alkaline pH value comprises adding an acid to the aqueous solution.

13. The method of claim 12, wherein the acid is H2SO4.

14. The method of any one of claims 1-3, wherein the portion of the cell culture comprises supernatant, whole-cell broth, or cell clumps.

15. The method of any one of claims 1-3, wherein collecting the portion of the cell culture comprises removing the host cells from the growth medium and reconstructing the host cells in the aqueous solution.

16. The method of any one of claims 1-3, wherein collecting the portion of the cell culture comprises lysing the host cells.

17. The method of claim 16, wherein pyrolysis comprises heat treatment, shear failure, physical homogenization, ultrasonic or chemical homogenization.

18. The method of any one of claims 1-3, wherein the portion of the cell culture comprises the host cell from the cell culture and the growth medium.

19. The method of any one of claims 1-3, wherein the aqueous solution comprises a diluted growth medium.

20. The method of any one of claims 1-3, wherein the portion of the cell culture is incubated under alkaline conditions for 15 to 30 minutes.

21. The method of any one of claims 1-3, wherein incubating the portion of the cell culture under alkaline conditions further comprises stirring the portion of the cell culture.

22. The method according to any one of claims 1-3, further comprising removing unsolvated biomass from the aqueous solution under alkaline conditions.

23. The method of claim 22, wherein removing the unsolvated biomass comprises filtration, centrifugation, gravity sedimentation, adsorption, dialysis, or phase separation.

24. The method of claim 23, wherein the filtration is ultrafiltration, microfiltration, or percolation.

25. The method of claim 22, wherein the removal of the unsolvated biomass is repeated at least once.

26. The method of any one of claims 1-3, further comprising removing impurities before or after separating the recombinant spider silk protein.

27. The method of claim 26, wherein removing impurities includes filtration, centrifugation, gravity sedimentation, adsorption, dialysis, or phase separation.

28. The method of claim 27, wherein the filtration is ultrafiltration, microfiltration, or percolation.

29. The method of claim 27, wherein the centrifugation is ultracentrifugation or density gradient centrifugation.

30. The method of claim 27, wherein the adsorption is carbon adsorption.

31. The method of claim 27, wherein the removal of impurities is repeated at least once.

32. The method of any one of claims 1-3, further comprising concentrating the isolated recombinant spider silk protein to produce concentrated spider silk protein.

33. The method of claim 32, wherein concentration comprises precipitation, filtration, ultrafiltration, centrifugation, dialysis, evaporation, or lyophilization.

34. The method of any one of claims 1-3, further comprising drying the isolated recombinant spider silk protein.

35. The method of any one of claims 1-3, further comprising generating silk fibers from the separated reconstituted spider silk.

36. The method of claim 35, wherein the filament has a toughness of at least 19 cN / tex.

37. The method of any one of claims 1-3, wherein the amino acid sequence of the recombinant spider silk protein is as shown in SEQ ID NO.: 38 or SEQ ID NO.:

39.

38. The method of claim 1, wherein the fungal cell is a yeast cell and the yeast cell is a Pasteurella multocida cell.

39. A method for isolating recombinant spider silk protein, the method comprising: a. Obtaining a cell culture, wherein the cell culture comprises a host cell and a growth medium, wherein the host cell expresses a recombinant spider silk protein, wherein the recombinant spider silk protein comprises a large ampulla 2 (MaSp2) filament, and wherein the cell culture comprises fungal or bacterial cells; b. Collect a portion of the cell culture containing the host cells and the recombinant spider silk protein without separating the host cells from the recombinant spider silk protein; c. Incubate the portion of the cell culture in an aqueous solution at a pH range of 11-12 for a time range of 10-60 minutes to solubilize the recombinant spider silk protein in the aqueous solution; d. Adjust the aqueous solution to a non-alkaline pH value in the range of 4-10, thereby precipitating the solubilized recombinant spider silk protein; and e. Isolate the recombinant spider silk protein from the portion of the cell culture to produce the isolated recombinant spider silk protein.

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