HK40078634A

HK40078634A - Recombinant silk solids and films

Info

Publication number: HK40078634A
Application number: HK62023068947.8A
Authority: HK
Inventors: A·A·B·达维贾尼; W·J·安德鲁斯三世
Original assignee: 保尔特纺织品公司
Priority date: 2020-02-12
Filing date: 2021-02-12
Publication date: 2023-03-31

Description

Reconstituted silk solids and membranes

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 62/975,656, filed on 12/2/2020, which is hereby incorporated by reference in its entirety.

Sequence listing

The present application contains a sequence listing that has been filed electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy was created at 12.2.2021 under the name BTT-036WO \.

Technical Field

The present disclosure relates to a composition for a shaped body comprising a recombinant spider silk protein and a plasticizer. Furthermore, the present disclosure relates to a shaped body comprising a recombinant spider silk protein and a plasticizer, and to a method for the preparation of the shaped body.

Background

As an alternative to petroleum-based products, biorenewable and biodegradable materials are receiving increasing attention. To this end, considerable efforts have been made to develop methods for making materials and fibers from molecules derived from plants and animals, including recombinant silk.

However, traditional methods of processing reconstituted filaments (such as wet spinning) use solvents and coagulation baths to produce fibers. This is disadvantageous because the chemicals used as solvents and in the coagulation bath need to be extracted from the fiber after the spinning process and subjected to a closed loop process to provide a sustainable and reliable process. Melt spinning is also used, but high temperatures can lead to degradation of the reconstituted filament fibers, which can negatively impact the properties of the final reconstituted filament material. In addition, it is desirable to make other material forms, such as solids or films, from the reconstituted filaments for various applications.

Thus, there is a need for compositions of recombinant silk polypeptides, including solids and films, that have desirable mechanical and aesthetic properties while minimizing degradation of the recombinant silk. Furthermore, the homogeneity of the recombinant filaments in the overall composition may be important. Therefore, there is also a need for new methods of producing such compositions.

Disclosure of Invention

According to some embodiments, provided herein is a method of making a shaped body comprising: providing a composition comprising reconstituted filaments and a plasticizer, wherein the composition is in a flowable state; placing the composition in a mold; applying heat and pressure to the composition in the mold; cooling the composition to form a shaped body comprising the reconstituted filaments.

In some embodiments, the shaped body is in solid form. In some embodiments, the shaped body is a film.

In some embodiments, the reconstituted silk is reconstituted silk powder distributed in the plasticizer. In some embodiments, the crystallinity of the reconstituted filament prior to molding is similar to or less than the crystallinity of 18B. In some embodiments, the recombinant silk protein is human spider whip silk (nephila spider flagelliform silk) or araneis spiders silk (araneus spider silk). In some embodiments, the recombinant silk is 18B. In some embodiments, the recombinant silk comprises SEQ ID NO 1.

In some embodiments, the plasticizer is selected from the group consisting of: triethanolamine, trimethylene glycol or propylene glycol. In some embodiments, the composition comprises 15% by weight propylene glycol. In some embodiments, the plasticizer comprises 10-50% by weight of the composition.

In some embodiments, the heat is applied at a temperature of 130 ℃. In some embodiments, the pressure is applied in the range of 1,500 to 15,000psi.

In some embodiments, the molded body has a hardness of 100 as measured by a type a durometer. In some embodiments, the shaped body has a hardness of 90 or greater as measured by a type a durometer. In some embodiments, the shaped body has a hardness of 50 or greater, 60 or greater, or 70 or greater as measured by a type D durometer. In some embodiments, the shaped body may be machined, cut or drilled and retain its desired shape.

In some embodiments, a shaped body has at least 50%, 60%, 70%, 80%, or 90% full length 18B monomer as compared to the reconstituted filaments of the composition in the flowable state. In some embodiments, the shaped body has at least 35%, at least 40%, at least 45%, or at least 50% full length recombinant silk monomers. In some embodiments, the shaped body has at least 50% total recombinant silk monomers, recombinant silk aggregates, and high molecular weight intermediates.

In some embodiments, the heat and pressure are applied for minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 10 minutes, or 15 minutes. In some embodiments, the heat and pressure are applied for 5 to 8 minutes.

In some embodiments, the method further comprises exposing the shaped body to a relative humidity of at least 50% for at least 24 hours. In some embodiments, the method further comprises exposing the shaped body to a relative humidity of 65% for 72 hours.

In some embodiments, the pressure is applied by a pressing load of at least 1 metric ton, at least 2 metric ton, at least 3 metric ton, at least 4 metric ton, or at least 5 metric ton. In some embodiments, the pressure is applied with a pressing load of 1 to 5 metric tons or 3 to 5 metric tons.

In some embodiments, the cooling is performed at a rate of about 1 deg.C/min, about 3 deg.C/min, or about 45 deg.C/min.

In some embodiments, the composition has a flexural modulus of 50MPa or greater, 60MPa or greater, 70MPa or greater, 80MPa or greater, 90MPa or greater, 100MPa or greater, 150MPa or greater, 200MPa or greater, 250MPa or greater, or 300MPa or greater. In some embodiments, the composition has a maximum flexural strength of 10MPa or greater, 20MPa or greater, 30MPa or greater, 40MPa or greater, 50MPa or greater, 60MPa or greater, 70MPa or greater, 80MPa or greater, 90MPa or greater, or 100MPa or greater.

In some embodiments, the composition has an elongation at break of 1 to 4%. In some embodiments, the composition has an elongation at break of greater than 20%.

In some embodiments, the composition further comprises ammonium persulfate. In some embodiments, the method further comprises immersing the shaped body in ammonium persulfate. In some embodiments, the shaped body is crosslinked.

In some embodiments, the shaped body is a cosmetic or skin care formulation.

Also provided herein are compositions comprising reconstituted silk and a plasticizer, wherein the composition is in solid form.

In some embodiments, the reconstituted silk is reconstituted silk powder distributed in the plasticizer. In some embodiments, the recombinant silk is 18B. In some embodiments, the recombinant silk comprises SEQ ID NO 1.

In some embodiments, the composition further comprises ammonium persulfate. In some embodiments, the shaped body is crosslinked.

In some embodiments, the shaped body is a cosmetic or skin care formulation.

Drawings

The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings.

Fig. 1 shows an image of additional solvent pressed out of the plasticized powder during pressing.

Fig. 2 shows a pressed solid (i.e., a shaped body) with propylene glycol.

Figure 3 shows a picture of a pressed solid showing a darkening of protein colour over time.

Fig. 4A, 4B and 4C show an analysis of temperature as a function of time. (fig. 4A) slow cooling of the solid inside the mold produced a cooling rate of 0.92 ℃/min (fig. 4B) moderate cooling of the solid in ambient air outside the mold produced a cooling rate of 2.7 ℃/min (fig. 4C) rapid cooling of the solid in dry ice outside the mold produced a cooling rate of 45.2 ℃/min.

Figure 5 shows a force versus distance curve to evaluate the effect of at least 72 hours on the mechanical properties of an 18B solid at 65% RH. Series 1,3, 5, 7 and 9 have been adjusted, while series 2, 4, 6, 8 and 11 have not.

Fig. 6 shows the (L) adjusted 72 hours and (R) unadjusted solid form after a 1 minute hold time in a 65% RH environment. The particle size was comparable, but the conditioned samples had more pronounced amorphous regions between the particles, which may help to increase ductility.

FIG. 7 shows a force versus distance curve to evaluate the effect of cooling rate on the mechanical properties of the 18B solid. 10. The 11 and 12 series correspond to slow, medium and fast cooling rates, respectively.

Fig. 8 shows a comparison between (a) slow cooling (B) moderate cooling and (C) fast cooling of the reconstituted filament shaped body.

Figure 9 shows a force versus distance curve to evaluate the effect of average load on the mechanical properties of an 18B solid. 13. The 14, 15, 16 and 17 series correspond to 1, 2, 3, 4 and 5 metric tons, respectively.

Figure 10 shows an image of a reconstituted silk shaped body with porous voids on the solid surface. Voids are visible on the surface of many solid surfaces to the left of the image. The right side shows the dispersed protein particles.

Fig. 11 shows the effect of average press load on the reformed wire shape. The amount of dispersed protein particles decreased as the average load increased from (a) 1 metric ton to (B) 3 metric ton to (C) 5 metric ton.

Fig. 12 shows a force versus distance curve to evaluate the effect of molding time on the mechanical properties of an 18B solid. Series 2, 4, 6, 8, 11, 18, 19, 20 and 21 correspond to 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 10 minutes and 15 minutes, respectively.

Fig. 13 shows the average flexural modulus (MPa) of the reconstituted filament shaped bodies as a function of retention time. Error bars show the standard deviation of the samples.

Fig. 14 shows the average flexural strength (MPa) of the reconstituted filament shaped bodies as a function of retention time. Error bars show the standard deviation of the samples.

Fig. 15 shows the average elongation at break (%) of the recombinant filament molded bodies as a function of the holding time. Error bars show the standard deviation of the samples.

Figure 16 shows the effect of shaping time on the morphology of the non-conditioned reconstituted silk shaped bodies, which were subjected to various holding times while maintaining equal average loads and cooling rates: 1 minute (B), 3 minutes (C), 5 minutes (D), 8 minutes (E), 10 minutes (F), 15 minutes.

Figure 17 shows the effect of mold time on the morphology of the unregulated, reconstituted silk shapes that underwent 1 minute hold versus 5 minutes hold. The following was visually inspected under high light for (a) solid black surfaces (B, C) between 1 minute holding time and 5 minutes holding time. Longer retention times have less distinct powder clumps and are more transparent.

Figure 18 shows the post-fracture surface of the reconstituted silk shape imaged with a bench top SEM. The surface is imaged across different hold times. (a) 1 minute hold time darkening to achieve greater contrast (B) 5 minute hold time (C) 15 minute hold time.

FIG. 19 shows a crosslinked 18B/TEOA sample of a reconstituted silk shaped body.

Fig. 20A and 20B show APS crosslinked 18B/glycerol films after drying (fig. 20A) or standing in water for 1 hour (fig. 20B). The left membrane was soaked in the crosslinking solution for 10 minutes, while the right membrane was soaked for 1 hour.

Figure 21 shows that the cross-linked 18B solid frame using glutaraldehyde chemistry placed in a water container did not show any structural change over the 30 minute test time.

FIG. 22 shows an 18B/glycerol powder dispersed on a surface.

Figure 23 shows the transparency and drape of recombinant silk/glycerol films.

Fig. 24 shows an example of a laser cut recombinant silk/glycerol film.

Fig. 25 shows an image of 18B powder without plasticizer pressed at 130 ℃.

Fig. 26 shows the formation of flash during the press forming process.

Fig. 27 shows an image of a shaped 18B solid prepared by pressing with 1, 3-propanediol (left) and an image of a solid reprocessed and pressed at 130 ℃ to form a film (right).

Detailed Description

The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description. Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by one of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural, and plural terms shall include the singular. The terms "a" and "an" include plural references unless the context indicates otherwise. Generally, the terms and techniques described herein in connection with biochemistry, enzymology, molecular and cellular biology, microbiology, genetics, protein and nucleic acid chemistry, and hybridization are well known and commonly used in the art.

Definition of

The following terms, unless otherwise indicated, shall be understood to have the following meanings:

the term "polynucleotide" or "nucleic acid molecule" refers to a polymeric form of nucleotides that are at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of the DNA or RNA that contain non-natural nucleotide analogs, non-natural internucleoside linkages, or both. The nucleic acid may be in any topological conformation. For example, the nucleic acid can be single-stranded, double-stranded, triple-stranded, quadruplex, partially double-stranded, branched, hairpin, circular, or in a padlock conformation.

Unless otherwise indicated, and as an example of all sequences described herein in the general format "SEQ ID NO:", a "nucleic acid comprising SEQ ID NO: 1" refers to a nucleic acid at least a portion of which has (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 a nucleic acid is used as a probe, the choice between the two will depend on the requirement that the probe be complementary to the desired target.

An "isolated" RNA, DNA, or mixed polymer is substantially separated from other cellular components that naturally accompany a natural polynucleotide in its native host cell, e.g., substantially separated from ribosomes, polymerases, and genomic sequences with which it is naturally associated.

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

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

An endogenous nucleic acid sequence (or the encoded protein product of that sequence) in the genome of an organism is considered "recombinant" herein if the heterologous sequence is placed adjacent to the endogenous nucleic acid sequence such that the order of expression of the endogenous nucleic acid is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to an endogenous nucleic acid sequence, whether the heterologous sequence itself is endogenous (derived from the same host cell or progeny thereof) or exogenous (derived from a different host cell or progeny thereof). For example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell such that the gene has an altered expression pattern. The gene will now become "recombinant" in that it is separated from at least some of the naturally flanking sequences.

A nucleic acid is also considered "recombinant" if it contains any modifications in the genome that do not naturally occur to the corresponding nucleic acid. For example, an endogenous coding sequence is considered "recombinant" if it contains an artificially introduced insertion, deletion, or point mutation (e.g., by human intervention). "recombinant nucleic acid" also includes nucleic acids that integrate into the host cell chromosome at a heterologous site and nucleic acid constructs that exist as episomes.

The term "peptide" as used herein refers to short polypeptides, e.g., typically less than about 50 amino acids in length, more typically less than about 30 amino acids in length. The term as used herein encompasses analogs and mimetics that mimic structure and thus biological function.

The term "polypeptide" encompasses naturally occurring and non-naturally occurring proteins, as well as fragments, mutants, derivatives and analogs thereof. The peptide may be monomeric or polymeric. In addition, a polypeptide may comprise a number of different domains, each domain having one or more different activities.

The term "isolated protein" or "isolated polypeptide" is a protein or polypeptide that, by virtue of its origin or derivative source, (1) does not associate in its native state with the naturally associated components with which it is associated, (2) exists in a purity not found in nature, where purity can be judged relative to the presence of other cellular material (e.g., does not contain other proteins from the same species) (3) is expressed by cells from a different species, or (4) does not exist in nature (e.g., it is a fragment of a polypeptide found in nature, or it includes amino acid analogs or derivatives not found in nature, or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it is naturally derived will be "isolated" from its naturally associated components. The polypeptide or protein may also be rendered substantially free of naturally associated components by isolation using protein purification techniques well known in the art. As defined herein, "isolated" does not necessarily require that the protein, polypeptide, peptide, or oligopeptide so described has been physically removed from its natural environment.

The term "polypeptide fragment" refers to a polypeptide having a deletion, e.g., an amino-terminal and/or carboxy-terminal deletion, as compared to a full-length polypeptide. In a preferred embodiment, a polypeptide fragment is a contiguous sequence, wherein the amino acid sequence of the fragment is identical to the corresponding position in the naturally occurring sequence. Fragments are typically at least 5,6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45 amino acids long, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

A protein has "homology" or "is homologous" to a second protein if the nucleic acid sequence encoding the protein has a sequence that is similar to the nucleic acid sequence encoding the second protein. Alternatively, if two proteins have "similar" amino acid sequences, the proteins have homology to a second protein. (thus, the term "homologous protein" is defined to mean that two proteins have similar amino acid sequences). As used herein, homology between two regions of an amino acid sequence (particularly with respect to predicted structural similarity) is interpreted to imply functional similarity.

When "homologous" is used to refer to a protein or peptide, it is recognized that residue positions that are not identical typically differ by conservative amino acid substitutions. A "conservative amino acid substitution" is one in which one amino acid residue is substituted with another amino acid residue having a side chain (R group) of similar chemical nature (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not significantly alter the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making such adjustments are well known to those skilled in the art. See, e.g., pearson,1994, methods mol. Biol.24.

The twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (Golub and Gren eds., sinauer Associates, sunderland, mass., 2nd edition, 1991), incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of twenty conventional amino acids, unnatural amino acids such as alpha-, alpha-disubstituted amino acids, N-alkyl amino acids, and other non-conventional amino acids may also be suitable components of the polypeptides of the invention. Examples of unconventional amino acids include: 4-hydroxyproline, gamma-carboxyglutamic acid, epsilon-N, N, N-trimethyllysine, epsilon-N-acetyllysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline). In the polypeptide symbols used herein, the left-hand end corresponds to the amino terminus and the right-hand end corresponds to the carboxy terminus, according to standard usage and convention.

Each of the following six groups comprises amino acids that are conservative substitutions for one another: 1) Serine (S), threonine (T); 2 aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), alanine (a), valine (V), and 6) phenylalanine (F), tyrosine (Y), tryptophan (W).

Sequence homology, sometimes referred to as percent sequence identity, of polypeptides is typically measured using sequence analysis software. See, for example, the Sequence Analysis Software Package of the Genetic Computer Group (GCG), the University of Wisconsin Biotechnology Center,910University Avenue, madison, wis.53705 protein Analysis Software uses homology measurements assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions, to match similar sequences. For example, GCG contains programs such as "Gap" and "Bestfit" that can use default parameters to determine sequence homology or sequence identity between closely related polypeptides (e.g., organisms from different species) or between a wild-type protein and its muteins. See, e.g., GCG version 6.1.

A useful algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al, J.Mol.biol.215:403-410 (1990); gish and States, nature Genet.3:266-272 (1993); madden et al, meth.Enzymol.266:131-141 (1996); altschul et al, nucleic Acids Res.25:3389-3402 (1997); zhang and Madden, genome Res.7:649-656 (1997)), especially blastp or lastbtn (Altschul et al, nucleic Acids Res.25:3389-3402 (1997)).

Preferred parameters for BLASTp are: desired values: 10 (default); a filter: seg (default); initial gap penalty: 11 (default); gap extension penalty: 1 (default); highest alignment: 100 (default); word length: 11 (default); description number: 100 (default); penalty matrix: BLOWSUM62.

Preferred parameters for BLASTp are: desired values: 10 (default); a filter: seg (default); initial gap penalty: 11 (default); gap extension penalty: 1 (default); highest alignment: 100 (default); word length: 11 (default); description number: 100 (default); penalty matrix: BLOWSUM62. The length of polypeptide sequences used for homology comparisons is typically at least about 16 amino acid residues, typically at least about 20 residues, more typically at least about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. In searching databases containing sequences from a large number of different organisms, it is desirable to compare the amino acid sequences. Database searches using amino acid sequences can be measured by algorithms other than blastp known in the art. For example, polypeptide sequences can be compared using FASTA (program GCG version 6.1). FASTA provides alignments and percent sequence identities of the best overlapping regions between query and search sequences. Pearson, methods Enzymol.183:63-98 (1990) (incorporated herein by reference). For example, percent sequence identity between amino acid sequences can be determined using FASTA as provided in GCG version 6.1, with its default parameters (word length 2 and NOPAM coefficient of scoring matrix), which is incorporated herein by reference.

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

The term "shaped body" or "solid" as defined herein refers to a body manufactured by shaping a liquid or flexible raw material using a rigid frame called a mold, for example, molding processes including, but not limited to, extrusion, injection molding, compression molding, blow molding, lamination, matrix molding (matrix molding), rotational molding, spin casting, transfer molding, thermoforming, and the like.

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

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

As used herein, the term "melt transition" refers to the transition of a substance or composition from a rubbery state to a less ordered liquid or flowable state.

As used herein, the term "melting temperature" refers to the temperature range in which a material undergoes a melt transition.

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

As used herein, the term "flowable state" refers to a composition that has substantially the same properties as a liquid (i.e., has transitioned from a rubbery state to a more liquid state).

As used herein, the term "cross-linked" refers to a bond formed between reactive groups on two or more proteins. For example, crosslinking may be performed by enzymatic crosslinking or photocrosslinking. For example, ammonium persulfate and light or ammonium persulfate and heat can be used to crosslink silk or silk-like polypeptides.

Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those skilled in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

SUMMARY

Provided herein is a composition for shaped bodies comprising a recombinant spider silk protein and a plasticizer, wherein the composition comprises desired mechanical properties, such as strength, flexibility, stiffness. Further, in some embodiments, the composition is homogeneous or substantially homogeneous in the molten or flowable state. Furthermore, in some embodiments, the recombinant spider silk protein is not substantially degraded (e.g. degraded in an amount of less than 10% by weight, or typically less than 6% by weight) after it has formed a shaped body. In a preferred embodiment, the recombinant silk protein is in the form of a powder. Also provided herein are methods of producing such compositions comprising placing a composition comprising silk fibroin and a plasticizer in a mold and forming a shaped body by applying pressure and heat to the composition in the mold, then cooling the shaped body and optionally exposing to additional conditions, such as high relative humidity. In a preferred embodiment, the heat is sufficiently low that the heat and time of shaping is sufficiently low that degradation of the recombinant silk protein in the shaped body is minimized to maintain the desired properties resulting from the use of recombinant silk.

Recombinant silk proteins

The present disclosure describes embodiments of the invention that include shaped bodies such as solids and membranes synthesized from synthetic protein copolymers (i.e., recombinant polypeptides) such as silk or silk-like recombinant polypeptides. In some embodiments, the shaped bodies, such as solids or films, form a cosmetic or skin care formulation (e.g., a solution for the skin or hair). The shaped bodies provided herein may comprise various moisturizers, emollients, blocking agents, active agents, and cosmetic adjuvants, depending on the embodiment and the desired efficacy of the formulation.

Suitable protein copolymers are discussed in U.S. patent publication nos. 2016/0222174, 2016, 8, 45, 2018, 4, 26, 2018/0111970, and 2018, 3, 1, 2018/0057548, each of which is incorporated herein by reference in its entirety. In addition, protein copolymers (e.g., spider penis hairline, spider silk, regenerated silk fibroin) having a crystallinity similar to or less than 18B and/or a similar ductility index are suitable for use in the shaped bodies described herein. In some embodiments, other non-silk proteins suitable for forming shaped bodies, such as titin, having similar properties are suitable protein copolymers for forming shaped bodies as described herein.

In some embodiments, the synthetic protein copolymer is made from silk-like polypeptide sequences. In some embodiments, the silk-like polypeptide sequence is 1) a block copolymer polypeptide composition produced by mixing and matching repeating domains derived from the silk polypeptide sequence and/or 2) recombinant expression of a block copolymer polypeptide having a size (about 40 kDa) large enough to form a useful shaped body composition by secretion from an industrially scalable microorganism (industrial scalable microbe). Large (about 40kDa to about 100 kDa) block copolymer polypeptides engineered from silk repeat domain fragments, which include sequences from almost all published spider silk polypeptide amino acid sequences, can be expressed in the modified microorganisms described herein. In some embodiments, the silk polypeptide sequences are matched and designed to produce highly expressed and secreted polypeptides capable of forming shaped bodies.

In some embodiments, the block copolymer is engineered via a combinatorial mixture of silk polypeptide domains spanning the silk polypeptide sequence space. In some embodiments, the block copolymers are prepared by expression and secretion in scalable organisms (e.g., yeast, fungi, and gram positive bacteria). In some embodiments, the block copolymer polypeptide comprises 0 or more N-terminal domains (NTD), 1 or more repeat domains (REP), and 0 or more C-terminal domains (CTD). In some aspects of embodiments, the block copolymer polypeptide is a single polypeptide chain of >100 amino acids. In some embodiments, the block copolymer polypeptide comprises a domain that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of the block copolymer polypeptides disclosed in international publication No. WO/2015/042164 "Compositions and Methods for Synthesizing Improved Silk Fibers" (which is incorporated herein by reference in its entirety).

Several types of natural spider silks have been identified. The mechanical properties of each natural spinning type are believed to be closely related to the molecular composition of the filament. See, e.g., garb, j.e. et al, angling spin silk evolution with spin terminal domains, BMC evol.biol., 10; bittencourt, d. Et al, protein families, natural history and biotechnology aspects of spreader silk, genet. Mol. Res, 11 (2012); rising, A. Et al, spider-silk proteins: recollents advances in recombinant production, structure-function relationships and biological applications, cell. Mol. Life Sci.,68, 2, pg.169-184 (2011); and Humenik, M. et al, spreader silk: understating the structure-function relationship of a natural fiber, prog.mol.biol.Transl.Sci.103, pg.131-85 (2011). For example:

the uveal gland (AcSp) filaments tend to have high tenacity, which is the result of moderately high strength plus moderately high ductility. AcSp filaments are characterized by large block ("bulk repeat") sizes, often incorporating motifs for polyserines and GPX. Tubular gland (TuSp or cylindrical) filaments tend to have large diameters, with moderate strength and high ductility. TuSp silks are characterized by their polyserine and polyserine content, as well as short-strand polyalanine. Major ampullate gland (MaSp) filaments tend to have high strength and moderate ductility. MaSp filaments can be one of two subtypes: maSp1 and MaSp2. The MaSp1 filaments are generally less ductile than the MaSp2 filaments and are characterized by polyalanine, GX and GGX motifs. The MaSp2 filaments are characterized by polypropionic acid, GGX and GPX motifs. Small ampullate gland (MiSp) filaments tend to have moderate strength and moderate ductility. The MiSp filaments are characterized by GGX, GA, and poly A motifs, and typically comprise a spacer element of about 100 amino acids. Flagellar (Flag) filaments tend to have very high ductility and moderate strength. Flag filaments are generally characterized by GPG, GGX and short spacer motifs.

The characteristics of each silk type may vary from species to species, and spiders of different lifestyles (e.g., the podiatric spider and the wandering hunter spider) or the evolutionarily older spiders may produce different silks from those described above (for describing spider diversity and classification, see Hormiga, g. and Griswold, c.e., systems, phylogeny, and evolution of orb-seeking spiders, annu.rev.entol.59, pg.487-512 (2014); and black, t.a. Et al, reconstructing web evolution and spider discovery in molecular era, proc.nat.ad.sci.u.s.a., 106, pg.29-34 (2009). However, synthetic block copolymer polypeptides having sequence similarity and/or amino acid composition similarity to the repeat domains of native silk proteins can be used to manufacture consistent shaped bodies on a commercial scale with properties that replicate the properties of the corresponding shaped bodies made from native silk polypeptides.

In some embodiments, a list of putative silk sequences, such as "spidroin", "fibriin", "MaSp", may be compiled by searching for related terms in GenBank and these sequences may be merged with additional sequences obtained by independent sequencing work. The sequence is then translated into amino acids, the repeated entries are filtered, and manually resolved into domains (NTD, REP, CTD). In some embodiments, the candidate amino acid sequence is reverse translated into a DNA sequence optimized for expression in Pichia (Komagataella) pastoris. The DNA sequences were each cloned into an expression vector and transformed into pichia pastoris. In some embodiments, the various silk domains shown to be successfully expressed and secreted are then assembled in a combinatorial manner to construct a silk molecule capable of forming a shaped body.

The silk polypeptide characteristically consists of a repeat domain (REP) flanked by non-repeat regions (e.g., C-terminal and N-terminal domains). In one embodiment, the C-terminal and N-terminal domains are each between 75-350 amino acids in length. The repeat domain exhibits a hierarchical architecture, as depicted in FIG. 1. The repeat domain comprises a series of blocks (also referred to as repeat units). The blocks repeat throughout the silk repeat domain, sometimes perfectly, and sometimes imperfectly (constituting a quasi-repeat domain). The length and composition of the blocks vary for different silk types and different species. Table 1 lists examples of block sequences from selected species and silk types, further examples are found in Rising, A. Et al, spider batch proteins, recovery enhancements in computational production, structure-function relationships and biological applications, cell mol.Life Sci.,68, pg 2, 169-184 (2011); and Gatesy, J. et al, extreme diversity, conservation, and conservation of spreader batch sequences, science,291, 5513, pg.2603-2605 (2001). In some cases, the blocks may be arranged in a regular pattern, forming larger macroscopic repeats that occur multiple times (typically 2-8 times) in the repeating domains of the silk sequence. Repeating blocks within repeating domains or macro-repeating bodies, and repeating macro-repeating bodies within repeating domains, may be separated by a spacer element. In some embodiments, the block sequence comprises a glycine-rich region followed by a polyA region. In some embodiments, a short (-1-10) amino acid motif occurs multiple times within a block. For the purposes of the present invention, blocks from different native silk polypeptides may be selected without reference to the circular arrangement (i.e., otherwise similar recognition blocks between silk polypeptides may be misaligned due to the circular arrangement). Thus, for example, for the purposes of the present invention, the "block" of SGAGG (SEQ ID NO: 3) is identical to GSGAG (SEQ ID NO: 4) and to GGSGA (SEQ ID NO: 5); they are all merely a cyclic arrangement of one another. The particular arrangement selected for a given silk sequence can be determined to a maximum extent by convenience (usually starting with G). Silk sequences obtained from NCBI databases can be divided into block and non-repeat regions.

Table 1: samples of Block sequences

According to certain embodiments of the present invention, the formation of a block copolymer polypeptide from a block and/or macro-repeat domain of a former is described in international publication No. WO/2015/042164, incorporated by reference. Native silk sequences obtained from protein databases such as GenBank or by de novo sequencing are resolved by domain (N-terminal domain, repeat domain and C-terminal domain). The N-terminal domain and C-terminal domain sequences selected for the purpose of synthesis and assembly into fibers or shaped bodies include the natural amino acid sequence information and other modifications described herein. The repeat domain is broken down into a repetitive sequence containing representative blocks, usually 1-8, depending on the type of silk, which capture critical amino acid information while reducing the size of the DNA encoding the amino acids to a readily synthesizable fragment. In some embodiments, a suitably formed block copolymer polypeptide comprises at least one repeat domain comprising at least 1 repeat sequence, and optionally flanked by an N-terminal domain and/or a C-terminal domain.

In some embodiments, the repeat domain comprises at least one repeat sequence. In some embodiments, the repeat sequence is 150-300 amino acid residues. In some embodiments, the repeating sequence comprises a plurality of blocks. In some embodiments, the repeat sequence comprises a plurality of macroscopic repeats. In some embodiments, a block or a macroscopic repeat is segmented into multiple repeating sequences.

In some embodiments, the repeat 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 of the repeated sequences may be altered compared to the native sequence. In some embodiments, the repeat sequence may be altered, such as by adding a serine to the C-terminus of the polypeptide (to avoid it terminating in F, Y, W, C, H, N, M, or D). In some embodiments, the repeat sequence can be modified by filling an incomplete block with homologous sequences from another block. In some embodiments, the repeat sequence may be modified by rearranging the order of the blocks or macroscopic repeats.

In some embodiments, non-repetitive N-and C-terminal domains can be selected for synthesis. In some embodiments, the N-terminal domain may be achieved by removing, for example, a leader signal sequence as identified by SignalP (Peterson, t.n. et al, signalP 4.0.

In some embodiments of the present invention, the substrate is, the N-terminal domain, repeat sequence or C-terminal domain sequence may be derived from the spider funneling spiders (Agelenopsis aperta), aliatypius gulosus spiders, gossada Rickenella zerumbet spiders (Aphonopelma seemann), araneidae species AS217 (Aptostichus sp.AS217), araneidae species AS220, araneus diades, orthodoides spiders, loxosporus macrobrachiatus, arabia amoena, argiodendron argenteus (Argioides), araneus striata (Argiodendron canescens), araneus contorta, atypoidea tridactyla, atypoidea rivularis, phaseolus tenella (Avicifolia juguensis), hippocampus calis califoriuni (Bothromycum califorum), human, diagnostigma spider (Diagnostigma) spider, or Gossata (Guiedae siella) sequence black fishing spider, euagrus chisoseus, nursery web spider, mastoid flag spider (Gasteracantha mammosa), hypochilus thorelli, kukuulcania hibernalis, black widow spider, megahexura fulvva, metapiera grandiosa, nephila anthropomorphic, nephila pseudoptera, nephila clavata, madagastra newcastle disease, nephila amagasseri, nephila maculariensis, nephila maculans, nephila maculatus crutata, palawegia striata bimacula bistriata, green piceida viridans, original meat spider, poagliflora, poulia japonaria, green spider, or Haematocophila amantanus 34a green spider.

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

In some embodiments, the recombinant spider silk polypeptide is based on a recombinant spider silk protein fragment sequence derived from MaSp2 (such as from a species of rhabdospider). In some embodiments, the shaped bodies comprise protein molecules comprising two to twenty repeat units, wherein each repeat unit has a molecular weight greater than about 20kDa. There are more than about 60 amino acid residues, usually in the range of 60 to 100 amino acids, within each repeat unit of the copolymer, which are organized into a number of "quasi-repeat units". In some embodiments, the repeat units of the polypeptides described in the present disclosure have at least 95% sequence identity to the MaSp2 dragline silk protein sequence.

The repeating units of the protein block copolymer forming the shaped body with good mechanical properties can be synthesized using a part of the silk polypeptide. These polypeptide repeat units comprise an alanine-rich region and a glycine-rich region and are 150 amino acids or longer in length. Some exemplary sequences useful as repeat sequences in the protein block copolymers of the present disclosure are provided in commonly owned PCT publication WO2015/042164, which is incorporated by reference in its entirety and demonstrated to be expressed using the pichia pastoris expression system.

In some embodiments, the spider silk protein comprises: a repeating unit that occurs at least twice, the repeating unit comprising: more than 150 amino acid residues and having a molecular weight of at least 10 kDa; an alanine-rich region having 6 or more contiguous amino acids comprising an alanine content of at least 80%; a glycine-rich region having 12 or more contiguous amino acids comprising a glycine content of at least 40% and an alanine content of less than 30%.

In some embodiments, wherein the recombinant spider silk protein comprises repeat units, wherein each repeat unit has at least 95% sequence identity to a sequence comprising 2 to 20 quasi-repeat units; each quasi-repeat unit comprises { GGY- [ GPG-X [) ₁ ] _n1 -GPS-(A) _n2 (SEQ ID NO: 34), wherein for each quasi-repeat unit; x ₁ Independently selected from the group consisting of: SGGQQ (SEQ ID NO: 35), GAGQQ (SEQ ID NO: 36), GQGPY (SEQ ID NO: 37), AGQQ (SEQ ID NO: 38) and SQ; n1 is from 4 to 8 and n2 is from 6 to 10. The repeat unit is comprised of a plurality of quasi-repeat units.

In some embodiments, 3 "long" quasi repeating units are followed by 3 "short" quasi repeating units. As mentioned above, short quasi-repeat units are those in which n1=4 or 5. Long quasi-repeat units are defined as those in which n1=6, 7 or 8. In some embodiments, all of the short quasi-repeats have the same X at the same position within each quasi-repeat unit of the repeat unit ₁ And (c) a motif. In some embodiments, no more than 3 of the 6 quasi repeat units share the same X ₁ And (3) motif.

In further embodiments, the repeat unit is comprised of quasi-repeat units that use the same X in rows within the repeat unit ₁ Not more than twice. In further embodiments, the repeat unit consists of a quasi repeat unit, wherein at least 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 quasi repeats use the same X in a single quasi repeat unit of the repeat unit ₁ Not more than 2 times.

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

TABLE 2 exemplary polypeptide sequences of recombinant proteins and repeat units

In some embodiments, the structure of the shaped body formed by the recombinant spider silk polypeptide forms a beta-sheet structure, a beta-turn structure or an alpha-helix structure. In some embodiments, the secondary, tertiary, and quaternary protein structures of the formed shaped bodies are described as having nanocrystalline beta-sheet regions, amorphous beta-turn regions, amorphous alpha-helical regions, randomly spatially distributed nanocrystalline regions embedded in an amorphous matrix, or randomly oriented nanocrystalline regions embedded in an amorphous matrix. While not wishing to be bound by theory, it is theorized that the structural properties of the proteins within the spider silk are related to the mechanical properties of the shaped body. Crystalline regions are associated with strength, while amorphous regions are associated with ductility. Compared to flagellated filaments, the Major Ampullate (MA) filaments tend to have higher strength and less ductility than flagellated filaments, and MA filaments have a higher volume fraction of crystalline regions compared to flagellated filaments.

In some embodiments, the molecular weight of the silk protein may range from 20kDa to 2000kDa, or greater than 20kDa, or greater than 10kDa, or greater than 5kDa, or from 5 to 400kDa, or from 5 to 300kDa, or from 5 to 200kDa, or from 5 to 100kDa, or from 5 to 50kDa, or from 5 to 500kDa, or from 5 to 1000kDa, or from 5 to 2000kDa, or from 10 to 400kDa, or from 10 to 300kDa, or from 10 to 200kDa, or from 10 to 100kDa, or from 10 to 50kDa, or from 10 to 500kDa, or from 10 to 1000kDa, or from 10 to 2000kDa, or from 20 to 400kDa, or from 20 to 300kDa, or from 20 to 200kDa, or from 40 to 300kDa, or from 40 to 500kDa, or from 20 to 100, or from 20 to 50kDa, or from 20 to 500kDa, or from 20 to 1000kDa, or from 20 to 2000kDa.

Characterization of recombinant spider silk polypeptide powder impurities and degradation

Based on the strength and stability of the secondary and tertiary structure of protein formation, different recombinant spider silk polypeptides have different physicochemical properties such as melting temperature and glass transition temperature. The silk polypeptide forms a beta sheet structure in monomeric form. In the presence of other monomers, the silk polypeptide forms a three-dimensional lattice of beta sheet structures. The beta sheet structure is separated from and interspersed with amorphous regions of the polypeptide sequence.

The beta-sheet structure is very stable at high temperatures-the melting temperature of the beta-sheet is approximately 257 ℃ as measured by rapid scanning calorimetry. See Cebe et al, coating the Heat-Fast Scanning means Silk Beta Sheet Crystals, nature Scientific Reports 3 (2013). Since the β sheet structure is believed to remain intact above the glass transition temperature of the silk polypeptide, it is hypothesized that the structural transition seen at the glass transition temperature of the recombinant silk polypeptide is due to the increased mobility of the amorphous regions between the β sheets.

Plasticizers lower the glass transition temperature and melting temperature of silk proteins by increasing the mobility of the amorphous regions and potentially disrupting beta sheet formation. Suitable plasticizers for this purpose include, but are not limited to, water and polyols (polyols), such as glycerol, triglycerol, hexaglycerol, and decaglycerol. Other suitable plasticizers include, but are not limited to: dimethyl isosorbide (Dimethyl Isosorbite); bisamides of dimethylaminopropylamine and adipic acid; 2, 2-trifluoroethanol; amides of dimethylaminopropylamine and caprylic/capric acid; DEA acetamide and any combination thereof. Other suitable plasticizers are discussed in Ullsten et al, chapter 5.

Since the hydrophilic part of silk polypeptides can bind to ambient water present in the air as moisture, water is almost always present and the bound ambient water can plasticize the silk polypeptides. In some embodiments, a suitable plasticizer may be glycerin, alone or in combination with water or other plasticizers. Other suitable plasticizers are discussed above.

Furthermore, in the case of recombinant spider silk polypeptides produced by fermentation and recovered therefrom as recombinant spider silk polypeptide powder, there may be impurities in the recombinant spider silk polypeptide powder that act as plasticizers or otherwise inhibit the formation of tertiary structures. For example, residual lipids and sugars can act as plasticizers and thus affect the glass transition temperature of proteins by interfering with the formation of tertiary structures.

Various accepted methods are available for assessing the purity and relative composition of a recombinant spider silk polypeptide powder or composition. Size exclusion chromatography separates molecules based on their relative sizes and can be used to analyze the relative amounts of recombinant spider silk polypeptides in their full-length polymerized and monomeric forms, as well as the amounts of high-, low-, and medium-molecular-weight impurities in the recombinant spider silk polypeptide powder. Similarly, flash high performance liquid chromatography can be used to measure various compounds present in solution, such as recombinant spider silk polypeptides in monomeric form. Ion exchange liquid chromatography can be used to assess the concentration of various trace molecules in solution, including impurities such as lipids and sugars. Other methods of chromatography and quantification of various molecules, such as mass spectrometry, have been recognized in the art.

According to an embodiment, the recombinant spider silk polypeptide may have a purity calculated based on the amount by weight of the recombinant spider silk polypeptide in monomeric form relative to the other components of the recombinant spider silk polypeptide powder. In various instances, the purity may range from 50% by weight to 90% by weight, depending on the type of recombinant spider silk polypeptide and the technique used to recover, isolate and process the recombinant spider silk polypeptide powder.

Both size exclusion chromatography and reverse phase high performance liquid chromatography can be used to measure full length recombinant spider silk polypeptides, making them useful techniques to determine whether a processing step degrades a recombinant spider silk polypeptide by comparing the amount of full length spider silk polypeptide in the composition before and after processing. In various embodiments of the invention, the amount of full length recombinant spider silk polypeptide present in the composition before and after processing may be subject to minimal degradation. The amount of degradation may range from 0.001% by weight to 10% by weight, or 0.01% by weight to 6% by weight, for example less than 10% or 8% or 6% by weight, or less than 5% by weight, less than 3% by weight or less than 1% by weight.

Recombinant silk solid and film compositions and methods of preparation

According to an embodiment, suitable concentration ranges by weight of the recombinant spider silk polypeptide powder in the recombinant spider silk composition are: 1 to 90% by weight, 3 to 80% by weight, 5 to 70% by weight, 10 to 60% by weight, 15 to 50% by weight, 18 to 45% by weight, or 20 to 41% by weight.

In some embodiments, suitable concentration ranges of plasticizer by weight in the recombinant spider silk composition are: 1 to 60% by weight, 10 to 50% by weight, 10 to 40% by weight, 15 to 40% by weight, 10 to 30% by weight, or 15 to 30% by weight. In some embodiments, the plasticizer is glycerin. In some embodiments, the plasticizer is triethanolamine, propylene glycol, or propylene glycol.

In case water is used as plasticizer, suitable weight concentration ranges of water by weight in the recombinant spider silk composition are: 5 to 80% by weight, 15 to 70% by weight, 20 to 60% by weight, 25 to 50% by weight, 19 to 43% by weight, or 19 to 27% by weight. When water is used in combination with another plasticizer, it may be present in a range of 5 to 50% by weight, 15 to 43% by weight, or 19 to 27% by weight.

After the shaped body is formed, the crystallinity of the recombinant protein in the shaped body can be increased, thereby strengthening the shaped body. In some embodiments, the crystallinity index of the shaped body as measured by X-ray crystallography is from 2% to 90%. In some other embodiments, the crystallinity index of the shaped body as measured by X-ray crystallography is at least 3%, at least 4%, at least 5%, at least 6%, or at least 7%.

In some embodiments, various agents may be added to the recombinant spider silk composition to alter the properties of the shaped body, such as hardness, flexural modulus and flexural strength. These include polyethylene glycol (PEG), tween (polysorbate), sodium lauryl sulfate, polyethylene, or any combination thereof. Other suitable reagents are well known in the art.

In some embodiments, a second polymer may be added to form a polymer blend or a two-component fiber with the recombinant spider silk composition. In these cases, it may be useful to include a second polymer whose melting temperature makes it suitable for melting with the recombinant spider silk composition itself without degrading the amorphous regions of the recombinant spider silk polypeptide. In various embodiments, polymers suitable for blending with recombinant spider silk polypeptides will have a melting temperature (Tm) of less than 200 ℃, 180 ℃, 160 ℃, 140 ℃, 120 ℃ or 100 ℃. Typically, the melting temperature of the recombinant spider silk polypeptide will exceed 20 ℃, 25 ℃ or 50 ℃. A non-limiting list of exemplary polymers and melting temperatures is included in table 3 below.

In some embodiments, the water may evaporate during cooling or post-formation conditioning. In some embodiments, the amount of water lost after molding may be 1 to 50% by weight, 3 to 40% by weight, 5 to 30% by weight, 7 to 20% by weight, 8 to 18% by weight, or 10 to 15% by weight, based on the total amount of water. Typically the loss will be less than 15% by weight, in some cases less than 10%, for example 1 to 10%. The evaporation may be intentional or due to the applied treatment. The degree of evaporation can be readily controlled, for example, by selecting the operating temperature, flow rate, and pressure applied, as understood in the art.

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

In various embodiments, the amount of plasticizer may vary depending on the purity and relative composition of the recombinant spider silk polypeptide powder. For example, higher purity powders may have fewer impurities, such as low molecular weight compounds that may act as plasticizers, thus requiring the addition of a higher percentage by weight of plasticizer.

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

Without being limited by theory, in various embodiments of the present invention, the induction of the transition of the recombinant spider silk composition into a flowable state may be used as a pre-processing step in any formulation, in which case it is beneficial to include the recombinant spider silk polypeptide in monomeric form. More specifically, inducing a recombinant spider silk melt composition can be used in applications where it is desirable to prevent aggregation of monomeric recombinant spider silk polypeptides into their crystalline polymer form or to control the conversion of recombinant spider silk polypeptides into their crystalline polymer formation at a later stage of processing. In a specific embodiment, the recombinant spider silk melt composition can be used to prevent aggregation of the recombinant spider silk polypeptide prior to blending the recombinant spider silk polypeptide with the second polymer. In another embodiment, the recombinant spider silk melt composition can be used to produce a matrix for a cosmetic or skin care product, wherein the recombinant spider silk polypeptide is present in its monomeric form in the matrix. In this embodiment, having the recombinant spider silk polypeptide in monomeric form in a matrix allows for the controlled aggregation of the monomers into their crystalline polymeric form upon contact with the skin or by various other chemical reactions.

The cosmetic or skin care product may be applied directly to the skin or hair. In some embodiments, the shaped body has a low melting temperature. In various embodiments, the shaped body has a melting temperature below body temperature (about 34-36 ℃) and melts upon contact with skin.

The cosmetic or skin care products described above may contain various moisturizers, emollients, occlusive agents, active agents and cosmetic adjuvants, depending on the embodiment of the product and the desired efficacy.

As used herein, the term "humectant" refers to a hygroscopic substance that forms a bond with water molecules. Suitable humectants include, but are not limited to, glycerin, propylene glycol, polyethylene glycol, pentylene glycol, tremella extract, sorbitol, diaminonitrile, sodium lactate, hyaluronic acid, aloe vera extract, alpha hydroxy acids, and pyrrolidone carboxylate (NaPCA). As used herein, the term "emollient" refers to a compound that provides a soft or supple appearance to the skin by filling cracks in the skin surface. Suitable emollients include, but are not limited to, shea butter, cocoa butter, squalene, squalane, octyl octanoate, sesame oil, grape seed oil, natural oils containing oleic acid (e.g., sweet almond oil, argan oil, olive oil, avocado oil), oils naturally containing gamma linoleic acid (e.g., evening primrose oil, borage oil), natural oils containing linoleic acid (e.g., safflower oil, sunflower oil), or any combination thereof. The term "occlusive agent" refers to a compound that forms a barrier at the skin surface to retain moisture. In some cases, the emollient or moisturizer may be a blocking agent. Other suitable blocking agents may include, but are not limited to, beeswax, carnuba wax, ceramide (ceramide), vegetable waxes, lecithin, allantoin. Without being limited by theory, the film forming ability of the recombinant spider silk compositions presented herein results in a blocking agent that forms a moisture barrier, as the recombinant spider silk polypeptides function to attract water molecules and also function as a humectant.

The term "active agent" refers to any compound having a known beneficial effect in a skin care formulation or sunscreen. The various active agents may include, but are not limited to, acetic acid (i.e., vitamin C), alpha hydroxy acids, beta hydroxy acids, zinc oxide, titanium dioxide, retinol, niacinamide, other recombinant proteins (as full length sequences or hydrolyzed into subsequences or "peptides"), cuprins, curcuminoids, glycolic acid, hydroquinone, kojic acid, L-ascorbic acid, alpha lipoic acid, azelaic acid, lactic acid, ferulic acid, mandelic acid, dimethylaminoethanol (DMAE), resveratrol, natural extracts containing antioxidants (e.g., green tea extract, pine extract), caffeine, alpha-arbutin, coenzyme Q-10, and salicylic acid. The term "cosmetic adjuvant" refers to various other agents used in the manufacture of cosmetics having commercially desirable characteristics, including, but not limited to, surfactants, emulsifiers, preservatives, and thickeners.

In various embodiments, the temperature to which the recombinant spider silk composition is heated during shaping is minimized to minimize or completely prevent degradation of the recombinant spider silk polypeptide. In specific embodiments, the recombinant spider silk melt will be heated to less than 120 ℃, less than 100 ℃, less than 80 ℃, less than 60 ℃, less than 40 ℃ or less than 20 ℃. Typically, the temperature of the melt during the molding process ranges from 10 ℃ to 120 ℃,10 ℃ to 100 ℃, 15 ℃ to 80 ℃, 15 ℃ to 60 ℃,18 ℃ to 40 ℃, or 18 ℃ to 22 ℃.

In some embodiments of the invention, the recombinant spider silk solid or membrane will be substantially homogeneous, meaning that the material has little or no inclusions or precipitates as examined by light microscopy. In some embodiments, an optical microscope can be used to measure birefringence, which can be used as a proxy for arranging recombinant spider silks into a three-dimensional lattice. Birefringence is an optical property of a material whose refractive index depends on the polarization and propagation of light. In particular, a high axial order as measured by birefringence may be associated with a high tensile strength. In some embodiments, recombinant spider silk solids and membranes will have minimal birefringence.

The amount of degradation of the recombinant spider silk polypeptide can be measured using various techniques. As described above, the amount of degradation of the recombinant spider silk polypeptide can be measured using size exclusion chromatography to measure the amount of full length recombinant spider silk polypeptide present. In various embodiments, the composition degrades in an amount less than 6.0 wt% after forming the shaped body. In another embodiment, the composition degrades after molding in an amount of less than 4.0 wt.%, less than 3.0 wt.%, less than 2.0 wt.%, or less than 1.0 wt.% (such that the amount of degradation can range from 0.001% by weight to 10%, 8%, 6%, 4%, 3%, 2%, or 1%, or from 0.01% by weight to 6%, 4%, 3%, 2%, or 1%). In another embodiment, the recombinant spider silk protein in the melt composition is substantially undegraded.

In some embodiments, the shaped body is crosslinked. For example, in some embodiments, the shaped body is soaked in ammonium persulfate during or after formation of the shaped body to promote cross-linking between proteins in the shaped body. In some embodiments, the crosslinking is enzymatic crosslinking. In some embodiments, the crosslinking is photochemical crosslinking.

In some embodiments, provided herein are crosslinked recombinant silk shaped bodies having desirable mechanical properties and methods of producing the same. The crosslinked shaped body compositions provided herein may be crosslinked to achieve desired mechanical properties, such as flexibility, hardness, or strength, which may be preferred in certain applications. In some embodiments, provided herein are methods of crosslinking a reconstituted silk shape composition to form a crosslinked reconstituted silk solid. In some embodiments, the crosslinking reaction comprises exposing the shaped body to a persulfate salt, such as ammonium persulfate. Heat may be applied to initiate the crosslinking reaction catalyzed by the persulfate. This type of crosslinking reaction does not leave any photoactive or enzymatic compounds in the composition. Furthermore, this crosslinking reaction does not require photo activation and therefore can be efficiently mass produced without the need for light to reach all parts of the crosslinking solution. In some embodiments, crosslinking occurs in the container or mold such that the resulting reconstituted silk shaped body has a specific shape or form.

In some embodiments, the shaped body is formed by 3D printing. Thus, in some embodiments, the shaped body is formed by continuously depositing or forming thin layers of a composition comprising the recombinant filaments and the plasticizer in a flowable state so as to establish the desired 3-D structure. For example, each layer is formed as if it were a layer of printing by moving some type of print head over the workpiece and activating the elements of the print head to produce a "print" of polymerizable liquid material. Thus, in some embodiments, the shaped body is formed layer-by-layer. Each layer comprises a dispersion composition comprising reconstituted filaments in a fluid state and a plasticizer, and the dispersion composition is crosslinked or hardened in the same pattern as a cross-section through the object to be formed. After one layer is complete, the level of the distributed composition will rise over a short distance and the process is repeated. Each polymeric layer should be stable enough to support the next layer.

In another embodiment, a composition comprising reconstituted filaments and a plasticizer is distributed onto a substrate and coalesced according to the cross-sectional shape of the body to be formed. In yet another embodiment, the composition comprising the recombinant filaments and the plasticizer is deposited in the form of droplets deposited in a pattern according to the relevant cross-section of the object to be formed. Yet another method involves dispensing droplets of the composition at an elevated temperature and then solidifying upon contact with a cooler workpiece.

Reformation of reconstituted silk solids and films

In some embodiments of the invention, the method of producing a recombinant spider silk shaped body may additionally comprise reprocessing the shaped body comprising recombinant spider silk (e.g. a solid, film or other shaped article formed from recombinant spider silk).

Without being limited by theory, subjecting the recombinant spider silk polypeptide to heat and pressure in the presence of a plasticizer such as glycerol converts the recombinant spider silk polypeptide into an "open form of the recombinant spider silk polypeptide", wherein the amorphous and non-crystalline fragments of the recombinant spider silk polypeptide unfold and form interactions with the plasticizer. This "open recombinant spider silk polypeptide" is able to shape and form a solid due to interaction with a plasticizer. In particular, the open form of the recombinant spider silk polypeptide is prevented from forming intermolecular interactions to form an irreversible three-dimensional lattice.

Because there is minimal, if any, degradation of the recombinant spider silk polypeptide during shaping, in some embodiments, the recombinant spider silk shaped body is reprocessed by converting the shaped body back into a flowable recombinant spider silk composition, which is then reshaped. In various embodiments, the recombinant spider silk shaped body may be reshaped at least 20 times, at least 10 times, or at least 5 times. In these embodiments, the degradation seen in multiple reforming steps may be as low as 10%. The option of reshaping without degradation allows for the production of a substantially homogeneous composition and also allows for the reuse or redesign of products formed from the composition. For example, molded products of unacceptable quality may be reformed. Scrap product recycling is also a possibility.

Equivalents and ranges

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the invention is not intended to be limited by the above description but rather is as set forth in the appended claims.

In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated to the contrary or otherwise evident from the context. A claim or description that includes one or more members of a group "or" between "one or more members of the group is deemed to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise associated with a given product or process, unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the set is present in, used in, or otherwise associated with a given product or process. The invention includes embodiments in which more than one or all of the members of the group are present in, used in, or otherwise associated with a given product or process.

It should also be noted that the term "comprising" is intended to be open-ended and allows, but does not require, the inclusion of additional elements or steps. When the term "comprising" is used herein, the term "consisting of 8230 \8230; …" is therefore also encompassed and disclosed.

Where ranges are given, endpoints are included. Moreover, it is to be understood that unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values expressed as ranges can assume any specific value or sub-range within the stated ranges in different embodiments of the invention. The lower limit of the range of the present invention is one tenth of the unit, unless the context clearly dictates otherwise.

All sources of citation, such as references, publications, databases, database entries, and technologies cited herein, are incorporated by reference into this application even if not explicitly stated in the citation. The source of the citation is in conflict with the present disclosure, which controls the present disclosure.

The section and table headings are not intended to be limiting.

Examples

The following are examples for carrying out specific embodiments of the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental error and deviation should, of course, be allowed for.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA technology and pharmacology within the skill of the art. These techniques are explained fully in the literature. See, e.g., T.E.Creighton, proteins: structures and Molecular Properties (W.H.Freeman and Company, 1993); l. leininger, biochemistry (Worth Publishers, inc., current addition); sambrook, et al, molecular Cloning: A Laboratory Manual (2nd edition, 1989); methods In Enzymology (s.Colowick and n.kaplan eds., academic Press, inc.); remington's Pharmaceutical Sciences,18th Edition (Easton, pennsylvania: mack Publishing Company, 1990); carey and Sundberg Advanced Organic Chemistry 3rd Ed. (plenum Press) Vols A and B (1992).

Example 1: recombinant silk eggFormation of white solids

Beta sheet plays an important role in the structural integrity of the silk material. They constitute crystalline segments of silk. Typically, a strong chaotropic solvent is required to break the beta sheet as it forms. The melting temperature of the beta sheet is above its degradation point. However, the glass transition temperature is below the degradation temperature and can be further reduced by using a plasticizer.

Sufficient entanglement is required to make the solid. The melting temperature of the beta-sheet is too high, but since most proteins are amorphous, chain mobility can be provided for the amorphous chains to allow sufficient entanglement. The application of heat and a plasticizer can lower the thermal glass transition temperature. The three components required to obtain an 18B solid are heat, pressure and plasticizer.

Recombinant spider silk of the 18B polypeptide sequence (SEQ ID NO: 1) is produced by multi-batch large-scale fermentation, recovered and dried to a powder ("18B powder"). Details of the production of 18B reconstituted Silk powder can be found in PCT publication No. WO2015/042164, "Methods and Compositions for Synthesizing Improved Silk Fibers (Methods and Compositions for Synthesizing Improved Silk Fibers)", which is incorporated herein by reference in its entirety. The reconstituted silk powder was mixed using a household spice mill. Water and plasticizer were added at various ratios to the 18B powder to produce recombinant spider silk compositions with varying ratios of protein powder to plasticizer. The resulting composition is 10-50% by weight Triethanolamine (TEOA), propylene glycol or propylene glycol. The mixture was then pressurized at 130 ℃. The samples were pressed in a mould using a pressure in the range 1500 to 15000 psi.

At 30% by weight of TEOA, some of the TEOA plasticizer was extruded from the die during the pressing process, as shown in fig. 1. This shows that the amount of TEOA can be reduced if it can be distributed uniformly throughout the powder. Pressure is used to compact the powder particles.

The hardness of the solid was measured with a hardness meter. The durometer has an indenter that is pressed into the material. The greater the penetration, the softer the material and the lower the measured hardness value. There are many types of durometer suitable for various hardness ranges. Type a durometer is suitable for soft plastics, and if the value exceeds 90, a type D durometer should be used. The difference between them is in the indenter geometry and the applied force. Since durometer D is suitable for harder plastics, it has a sharper indenter and higher indentation force. The hardness of the solids pressed with TEOA, propylene glycol (1,3 propylene glycol) was 100 when measured with type A, indicating that their hardness exceeded that measurable by type A durometer. The TEOA processed solid has a hardness of 76HD as measured by a type D durometer. The propylene glycol processed solid had a hardness of 71HD as measured by a type D durometer (FIG. 2). By comparison, high Density Polyethylene (HDPE) safety helmets have similar stiffness. Propylene glycol solids have the lowest hardness, as measured by the type D durometer, starting at 55HD and dropping to 30 in 10 seconds. The solids can be machined, cut and drilled into the desired shape because their rigidity prevents the solids from deforming under the force of the tool (fig. 2).

Example 2: degradation of recombinant silk in silk solids

The SEC results of pressed films and solids showed that the low and medium molecular weights were similar between the solid sample, the film sample (see example 5) and the control 18B powder. This indicates that the degradation caused by compaction is minimal or non-existent.

Table 4 SEC data for pressed solids and films and control powders. N =2 mean results and standard deviation. HMWI = high molecular weight impurities; IMWI = medium molecular weight impurities; LMWI = low molecular weight impurities. All samples were from the same batch of 18B powder. The solid was pressed with 30wt% (by weight%) TEOA and the film was pressed with 40wt% glycerol.

Protein degradation data are summarized in table 5. Here, the sample is heated to 130 ℃ and the pressing time is increased. At each time point, the solid was sampled and placed back into the mold where it was heated and pressurized. There was no significant degradation for up to 10 minutes, based on the HMWI, 18B aggregate and 18B monomer, and the IMWI and LMWI values between samples. Starting at 20 minutes, the 18B monomer content decreased, while the medium (IMWI) and Low (LMWI) molecular weight components increased, indicating degradation after 20 minutes. As the solid was pressed longer, it also became darker (fig. 3).

TABLE 5 SEC data for control powder (SLD 33-P), solvent plasticized powder (SLD 33-PH), and pressed solid (SLD 33) for increased pressing time. All samples were from the same batch of 18B powder. The solids were pressed with 15% by weight of 1,3 propanediol.

Example 3: flexural characterization of 18B solids

When sintered by compression molding as described herein (e.g., example 1), 18B protein powders have shown promising ability to be stable protein powders with desirable solid properties. Propylene glycol (TMG or 1, 3-propanediol) was identified as a suitable plasticizer to aid in shaping. To optimize the molding process, the mechanical properties of the 18B-TMG solid need to be further characterized. Lots 18B with 15% by weight TMG solid powder were made and subjected to 3 point bend testing according to ASTM D790.

The mechanical properties of the 18B solid are provided for a range of processing parameters, including form retention time, cooling rate, post-form conditioning and average pressing load, as described below. Processing parameters that are beneficial or detrimental to the mechanical properties of the final solid product have also been found to improve processing efficiency and capacity.

Materials and methods

To test the flexural properties of reconstituted silk solids, the ASTM D790 standard recommends a depth-to-depth ratio as close as possible to 16. For this experiment, the span of the apparatus was fixed at 38.1mm so that the final specimen depth was between 2.25mm and 2.54 mm.

A thickness (in grams) of 0.66mm per final weight was obtained using a 25.4mm x 50.8mm (1:. Times.2 ") compression mold. The preform weight for each specimen was 3.8g to 4.0g to achieve the final sample depth based on an observed weight reduction of about 10% during the forming process.

An 18B/TMG mixture was prepared using 255.16g of 18B powder and 45.347g of TMG, which was mixed five times using a spice mill, resulting in 300.5g of 15.1% by weight TMG/84.9% by weight total masterbatch of 18B. They were divided into 4.0 g-each specimens for molding under prescribed conditions and then tested for flexural properties.

Among the 63 samples used for the test, the average depth-to-depth ratio was 15.72, the standard deviation was 0.35, and the coefficient of variation was 0.022. The Zwick prodine was test configured according to the ASTM D790 test procedure file. The key test parameters were a preload of 0.1MPa, a starting position interval of 3mm and a crosshead speed of 254 mm/min.

The recombinant filament solids preparation conditions tested were forming time, cooling rate, post-forming conditioning and average load during forming.

The molding time is defined as the time (in minutes) during which the mold is pressed at 130 ℃. The molding times were measured at 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 10 minutes, and 15 minutes.

For post-formation conditioning, the conditioned samples were held in a conditioning chamber at 65% Relative Humidity (RH) for at least 72 hours after the formation time. The unconditioned sample was stored on top of the bench at ambient laboratory conditions.

The average load is the load in metric tons that the test specimen is subjected to. Because the specimen size and the mold size are constant, each specimen in the sample set is subjected to nearly equal pressure during the molding process. Average loads of 1 metric ton, 2 metric ton, 3 metric ton, 4 metric ton and 5 metric ton were tested.

Finally, the cooling rate level is defined as slow, medium or fast. Starting from the removal of the solid sample by opening the mold, each level was quantified using an IR thermometer to record the solid surface temperature at 1 minute intervals (slow, medium) or 10 second intervals (fast). The results of the curves shown below produce cooling rates of 0.92 deg.C/min, 2.7 deg.C/min, and 45.2 deg.C/min, slow, medium, and fast, respectively. Although in FIGS. 4A-4C, the samples with the medium cooling rate were at different hold times than the samples with the slow and fast cooling rates, the cooling rate was not significantly different from the hold times. Slow, medium and fast cooling rates as defined above were tested.

Table 6 below shows the conditions used to prepare each sample ID. Each sample ID was performed in triplicate, and a total of 63 18B solid samples were prepared.

Post-forming conditioning

The 18B solid sample was formed as described above using 4.0g of sample and formed at 130 ℃ under an average load of 2 metric tons. The molded samples were cooled at a moderate cooling rate and conditioned with or without exposure to 65% Relative Humidity (RH) for at least 72 hours after formation. The samples were shaped for 1, 2, 3, 4 or 5 minutes. The conditions for evaluating the conditioning effect after molding are based on samples 1 to 9 and 11 provided in table 6.

Fig. 5 shows the stress-strain curves generated from the unconditioned 18B solid sample and the conditioned 18B solid sample. The stress-strain curve was used to determine the mechanical properties of the 18B solid, including elongation at break. Sample IDs 1,3, 5, 7, and 9 were adjusted, while sample IDs 2, 4, 6, 8, and 11 were not adjusted, as shown in tables 6 and 7.

The bend data for conditioned versus unconditioned 18B solid samples are shown in table 7 below. The average values of flexural modulus (MPa), maximum flexural strength (MPa), and elongation at break (%) for each conditioned and unconditioned sample measured in triplicate are provided (along with the Standard Deviation (SD) of the measurements). Note that 20% elongation means no solid break, since 20% is the maximum testable elongation.

Fig. 6 shows the (L) adjusted 72 hours and (R) unadjusted solid form after a 1 minute hold time in a 65% RH environment. The solid had comparable particle size, but the conditioned sample had more pronounced amorphous regions between the particles, which may help to increase ductility.

Macroscopically, it is clear that all 65% RH-conditioned samples are more ductile under load than the unconditioned samples. The presence of two hydroxyl groups in the TMG plasticizer helps to improve water solubility and moisture absorption. As shown in fig. 6, the shorter the forming time, the resulting solid had a powdery morphology and contained more particles.

There is a trade-off between adjusting the stiffness and elongation of the sample based on the effect of the adjustment. Conditioning the sample produces a sample that is neither very strong nor very hard, nor does it break. Safety limitations of the testing equipment require that the test be stopped at a maximum of 20% elongation and that no conditioned sample break if this elongation is reached. There is also more variability in the sample that is not conditioned. For the uncrosslinked 18B solids, the conditioning effect was significant, indicating that they are susceptible to water. Thus, a crosslinked 18B solid will be produced to reduce the response of the 18B solid to water. The mechanical properties of strength, stiffness and elongation of the crosslinked 18B solid will also be maximized.

The conditioned samples did not break because their elongation exceeded the safety measures of Zwick prodine. For this reason, the conditioned sample fracture surface could not be evaluated. Macroscopic (visual) post-fracture observation of the fracture surface of the unadjusted sample shows that almost all bending fractures can be characterized as highly brittle and that the ductility behavior is slightly different depending on the processing. The starting point is typically within 0.5cm of the center of the width of the sample. SEM imaging of three surfaces confirmed these conclusions.

Cooling rate after forming

An 18B solid sample was formed as described above using 4.0g of the sample and formed at 130 ℃ for 5 minutes at an average load of 2 metric tons. The shaped sample is cooled at a slow, medium or fast cooling rate. The method of measuring the cooling rate and the quantitative basis for slow, medium and fast cooling are described in the materials and methods above. The conditions for evaluating the effect of cooling rate were based on samples 10-12 provided in table 6.

FIG. 7 shows stress-strain curves generated from samples 10-12 to evaluate the effect of cooling rate on the mechanical properties of the 18B solid. 10. The 11 and 12 series correspond to slow, medium and fast cooling rates, respectively.

The bend data for the 18B solid samples at slow, medium and fast cooling rates are shown in table 8 below. The average values of flexural modulus (MPa), maximum flexural strength (MPa), and elongation at break (%) of each conditioned and unconditioned sample measured in triplicate are provided (along with the Standard Deviation (SD) of the measurements).

Fig. 8 shows the morphology of 18B solids exposed to (a) slow cooling (B) moderate cooling and (C) fast cooling.

In a structural polymer, increased cooling rates will produce samples that are stronger, stiffer, and have relatively similar elongations. Faster cooling results in smaller crystals and lower crystallinity (more amorphous regions) and therefore one would expect lower stiffness. However, the current results conflict with this assumption. On average, the flexural modulus and maximum strength of slow cooling were 262.72MPa and 5.29MPa, respectively. On average, the flexural modulus and maximum strength of the moderately cooled samples were 309.54MPa and 6.11MPa, respectively. On average, the flexural modulus and maximum strength of the rapidly cooled samples were 292.35MPa and 6.12MPa, respectively. As the cooling rate increases, the variability decreases.

Pressure of formation

The 18B solid sample was shaped as described above using 4.0g of the sample and shaped at 130 ℃ for 5 minutes, then cooled at a moderate cooling rate. The samples were molded at an average load of 1 metric ton, 2 metric ton, 3 metric ton, 4 metric ton or 5 metric ton. The conditions for evaluating the effect of the average load pressure during molding are based on samples 13-17 provided in Table 6.

FIG. 9 shows the stress-strain curves generated from samples 13-17 to evaluate the effect of forming pressure (average load) on the mechanical properties of the 18B solid. 13. The 14, 15, 16 and 17 series correspond to 1, 2, 3, 4 and 5 metric tons, respectively.

The flexural data for the 18B solid samples at different average loads are shown in table 9 below. The average values of flexural modulus (MPa), maximum flexural strength (MPa), and elongation at break (%) of each conditioned and unconditioned sample measured in triplicate are provided (along with the Standard Deviation (SD) of the measurements).

Sample ID #13-17 demonstrates the effect of different press loads on samples pressed for 5 minutes and cooled at a moderate rate. The trend to increase the compression load is an increase in flexural modulus, while the trend to strength and elongation is not determinable due to variability. As the set load increases, the strength is greater at an average load of 1 metric ton (average 5.68 MPa), then decreases at an average load of 2-4 metric tons, and then increases to a maximum of 5.85MPa at an average load of 5 metric tons. Nevertheless, due to variability, the impact of the compaction load on strength is not known. The elongation was between 2.05% and 4.38% according to the average load without any significant, noticeable tendency. To maximize the stiffness of the reconstituted filament solid material, it is preferred to determine an average load of 3-5 metric tons.

The dispersed protein particles appear as black dots, but as the case may be, the surface may be porous voids, as shown in fig. 10. The particles tend to preferentially position themselves in these voids. Increasing the compaction load appears to reduce the number of dispersed particles, but beyond 3 metric tons, the benefit is reduced (as shown in fig. 11). Specifically, fig. 11 shows solid images produced by different average press loads. There is a decrease in the amount of protein particles dispersed as the average load increases from (a) 1 metric ton to (B) 3 metric ton to (C) 5 metric ton.

Time of formation

An 18B solid sample was formed as described above using a 4.0g sample and formed at 130 c under an average load of 2 metric tons. The sample is shaped for 1, 2, 3, 4, 5,6, 8, 10, or 15 minutes. The shaped sample was cooled at a moderate cooling rate and no conditioning was performed. The conditions for evaluating the conditioning effect after molding were based on samples 2, 4, 6, 8, 14, 18, 19, 20, and 21 provided in table 6 and table 10.

Fig. 12 shows the stress-strain curves generated from samples 2, 4, 6, 8, 14, 18, 19, 20, and 21 to evaluate the effect of forming time on the mechanical properties of the 18B solid. 2. The series of 4, 6, 8, 14, 18, 19, 20 and 21 correspond to molding times of 1, 2, 3, 4, 5,6, 8, 10 and 15 minutes, respectively.

The flexural data for the 18B solid samples formed for different lengths of time are shown in table 10 below. The average values of flexural modulus (MPa), maximum flexural strength (MPa), and elongation at break (%) of each conditioned and unconditioned sample measured in triplicate are provided (along with the Standard Deviation (SD) of the measurements).

It was found that increasing the mold retention time only indicated an increase in solid stiffness. There does not appear to be any statistically significant effect on the flexural strength and elongation at break of the solid as the mold time varies. Fig. 13, 14 and 15 support the average flexural modulus, average flexural strength and average elongation at break, respectively. Specifically, fig. 13 shows the average flexural modulus (MPa) of the holding time. As the holding time increases, the average flexural modulus increases. Error bars show the standard deviation of the samples. FIG. 14 shows the average flexural strength (MPa) of the holding time. There appears to be no statistically significant difference in the maximum bending strength over all of the molding times tested. Fig. 15 shows the average elongation at break (%) of the holding time. There does not appear to be any significant relationship between elongation at break and retention time. Error bars are standard deviations of the samples.

The flexural modulus generally increases with increasing retention time. Note that for flexural strength, the nominal value for any given hold time is within the error range for other mold times. Thus, it can be concluded that there appears to be no significant difference in strength based on the molding time. Again, there does not appear to be any significant relationship between retention time and elongation at break. Limiting the testing to 3 specimens per sample set may partially account for relatively large errors and variability due to time constraints. Based on these results, it is recommended to focus future processing on molding times of around 5 to 8 minutes, with an average load of 3 to 5 metric tons and moderate cooling rates. While longer forming times will on average produce harder solids, increasing the forming time too long will result in reduced throughput. Alternatively, a shorter forming time may result in a powdered solid that is not particularly aesthetically pleasing.

Optical microscopy of the specimen surface prior to fracture is intended to reveal the effect of each of the four factors on the solid morphology and to aid in understanding the role of each factor in solid processing. The result of changing only the molding time from 1 minute to 15 minutes is shown in fig. 16. Specifically, fig. 16 shows the morphology of untreated solids subjected to various holding times while maintaining the same average load and cooling rate: 1 minute (B), 3 minutes (C), 5 minutes (D), 8 minutes (E), 10 minutes (F), and 15 minutes. As the molding time was increased from 1 minute to 5 minutes, the particle aggregates were greatly reduced for each one minute increase in molding time.

This conclusion is supported by visual inspection, as shown in fig. 17, where longer mold times result in a more uniform, translucent solid. Specifically, fig. 17 shows a visual inspection of (a) solid black surfaces (B, C) for glare between 1 minute hold time and 5 minutes hold time. Solids with longer retention times produce less distinct lumps of powder and are more translucent. After 5-6 minutes there was a clear lack of significant difference, although particle aggregates were present even at 15 minutes. The recommended forming time is 5 minutes and the thickness is not more than 3mm to avoid long time exposure of the protein to high temperature and minimize obvious particle aggregation.

Figure 18 shows the post-fracture surface of the reconstituted silk shaped bodies imaged with a Benchtop SEM at different shaping times. (a) 1 minute hold time darkening to achieve greater contrast (B) 5 minute molding time (C) 15 minute molding time. A holding time of 5 minutes shows the greatest combination of ductility and brittleness.

Conclusion

The samples with the greatest stiffness were the result of higher forming times and increased press load loads. It is recommended to explore these samples as the best path for the hard solids to travel. The most promising specimens are from sample ID #11, 12 and 17. Due to the height difference between the samples, the strength and elongation trends based on the molding time cannot be reliably distinguished.

The recommended molding time is between 5 and 8 minutes. While longer forming times will on average produce harder solids, increasing the forming time too long will result in reduced yield/productivity and lead to protein degradation. Alternatively, a shorter forming time of less than 5 minutes may result in a powdered solid that is not particularly aesthetically pleasing.

There appears to be no statistically significant difference in modulus, maximum strength and elongation at break between rapid, moderate and slow cooling. The use of moderate and slow cooling is recommended because it is most convenient to implement.

The samples with the greatest elongation at break were conditioned at 65% Relative Humidity (RH) for at least 72 hours, with a percent elongation at break far beyond the capabilities of the Zwick ProLine device.

Example 4: crosslinked reconstituted silk solids

The 18B solid was crosslinked using ammonium persulfate. Ammonium persulfate is soluble in water, but insoluble in TEOA or IPA. Water has a negative impact on the manufacturing of the solid and the solid cannot remain in the water for a long time because it swells and decomposes. However, ammonium persulfate may be dissolved in water and mixed with another solvent.

Two approaches have been attempted to crosslink solids using ammonium persulfate. In the first method, 79.7mg of Ammonium Persulfate (APS) was added to 100.4mg of water and dissolved using a vortex mixer. The solution was added to 7.79g of TEOA and mixed using a vortex mixer. This gave a 50mM ammonium persulfate solution in a 99/1 TEOA/water solution.

This solution was dispersed in 9.518g of 18B to give 55% by weight 18B dispersion. The mixture is placed in a mold and pressed at 130-135 ℃. The solid was left to cure in an oven for 15 hours and then placed in water. The solid expanded and began to disintegrate in water, indicating that no cross-linking occurred.

In another crosslinking method, the 18B compacted solid is immersed in an Ammonium Persulfate (APS) solution. 684mg of APS was dissolved in 1.3mL of DI water. IPA was added to the solution as the solid was over-swollen and disintegrated in pure water. Addition of 11.45mL of IPA resulted in precipitation of APS from the solution. After an additional 3.3mL of water was added, the salt dissolved back into solution to give a 187mM APS solution in 71/29 IPA/water mixture. The weight percentage of ammonium persulfate is 5wt%, the weight percentage of water is 32wt%, and the weight percentage of water is 63wt%.

The TEOA pressed samples were soaked in the crosslinking solution for 1 hour and then stored at 80 ℃ for 3 hours. The resulting solid was water resistant and did not disintegrate in water even after 1 day of water exposure (fig. 19).

The glycerol pressed film was also crosslinked. The films were soaked in APS/IPA/water solution for 10 and 60 minutes and cured overnight. Films with longer soak times are more opaque, especially when wet. After curing in the oven, the dried film was hard and brittle (fig. 20A). After less than a short soak in water, the water diffuses through the structure, resulting in rubbery behavior (fig. 20B).

In addition to water resistance, crosslinking also solves another problem of solid materials. Since plasticizers are all hygroscopic, solids absorb water and lose dimensional stability. The solid pressed samples, kept at high humidity levels, became soft and tough, similar to the glycerol pressed films. Crosslinking helps to maintain the structural integrity of the material. The solid pressed with 10wt% propylene glycol at 130 ℃ was cross-linked using two chemicals, glutaraldehyde and ammonium persulfate.

The glutaraldehyde chemical consists of 10wt% glutaraldehyde, 10wt% water, 1.5wt% aluminum chloride hexahydrate, and 78.5wt% isopropyl alcohol. The solid was soaked in the crosslinking solution for 12 hours and then placed in a 125 ℃ hot oven for 5 minutes to cure.

The ammonium persulfate chemistry consisted of 5wt% ammonium persulfate, 25wt% water, and 73wt% isopropanol. The solid was left to cure by standing in the chemistry for 1 hour and at 60 ℃ for 3 hours.

After crosslinking with either chemical, the solid became water resistant and maintained its shape when immersed in water (fig. 21).

Example 5: forming membranes from recombinant silk proteins

Film pressing

Solvated 18B powder was also dispersed as a plasticizer on the surface in 30-50% by weight glycerol (fig. 22) and pressed with glycerol between two parallel plates. Films pressed with glycerin are easily bent and can conform to surfaces, while other solvents form hard, brittle films. The drapability increases with decreasing film thickness. These flexible films are optically transparent (fig. 23). These films can be cut using a laser cutter or die (fig. 24).

As a control, 18B was pressed at 130 ℃ without any solvent to give a brittle film, a white film (fig. 25), where the powder was simply flattened and compacted into a film.

Film extrudate

Solvated 18B was extruded into 18B film extrudates. During pressing to form the 18B solid/film described in examples 1 and 2, the coating flowed between the flush surfaces, referred to as flash, and formed a thin flexible film (fig. 26). Thus, film formation was performed by extrusion.

Example 6: reshaping of reconstituted silk solids

The shaped 18B solid prepared by pressing with 1,3 propylene glycol as described in example 1 was reprocessed and pressed at 130 ℃ to form a film. A photograph of the reworked film is shown in fig. 27. Specifically, the original 18B solid prepared by compression with 1,3 propylene glycol is on the left and the reprocessed film is on the right. This result indicates that the reconstituted silk solids described herein can be reprocessed using the methods described herein to form different shaped bodies.

Other embodiments

It is to be understood that the words which have been used are words of description rather than limitation, and that changes may be made within the scope of the appended claims without departing from the true scope and spirit of the invention in its broader aspects.

Although the present invention has been described with a certain length and a certain specificity with respect to several described embodiments, the present disclosure is not intended to be limited to any such details or embodiments or any particular embodiments, but rather should be construed with reference to the appended claims in order to provide as broad an interpretation of such claims as is practicable in view of the prior art and, therefore, to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the section headings, materials, methods, and examples are illustrative only and not intended to be limiting.

Sequence listing

<110> Baote textiles Co (BOLT THREADS, INC.)

<120> recombinant silk solid and membrane

<130> BTT-036WO

<140>

<141>

<150> 62/975,656

<151> 2020-02-12

<160> 38

<170> patent version 3.5

<210> 1

<211> 945

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 1

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805 810 815

Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly

820 825 830

Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly

835 840 845

Pro Tyr Gly Pro Gly Ala Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr

850 855 860

Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser

865 870 875 880

Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala

885 890 895

Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln

900 905 910

Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro

915 920 925

Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala

930 935 940

Ala

945

<210> 2

<211> 315

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<400> 2

Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gly Gly

1 5 10 15

Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Ser Gly Gln Gln Gly

20 25 30

Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly

35 40 45

Pro Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro

50 55 60

Gly Ala Gly Gln Gln Gly Pro Gly Gly Ala Gly Gln Gln Gly Pro Gly

65 70 75 80

Ser Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln

85 90 95

Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro

100 105 110

Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala

115 120 125

Ala Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Arg Ser Gln Gly Pro

130 135 140

Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly

145 150 155 160

Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly

165 170 175

Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr

180 185 190

Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser

195 200 205

Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr Gly Pro Gly Ala

210 215 220

Ala Ala Ala Ala Ala Ala Val Gly Gly Tyr Gly Pro Gly Ala Gly Gln

225 230 235 240

Gln Gly Pro Gly Ser Gln Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro

245 250 255

Gly Gly Gln Gly Pro Tyr Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala

260 265 270

Ala Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln

275 280 285

Gly Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Gln Gly Pro Tyr

290 295 300

Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala

305 310 315

<210> 3

<211> 5

<212> PRT

<213> unknown

<220>

<223> description unknown: silk polypeptide block sequences

<400> 3

Ser Gly Ala Gly Gly

1 5

<210> 4

<211> 5

<212> PRT

<213> unknown

<220>

<223> description unknown: silk polypeptide block sequences

<400> 4

Gly Ser Gly Ala Gly

1 5

<210> 5

<211> 5

<212> PRT

<213> unknown

<220>

<223> description unknown: silk polypeptide block sequences

<400> 5

Gly Gly Ser Gly Ala

1 5

<210> 6

<211> 181

<212> PRT

<213> Aliatypus gulosus

<400> 6

Gly Ala Ala Ser Ser Ser Ser Thr Ile Ile Thr Thr Lys Ser Ala Ser

1 5 10 15

Ala Ser Ala Ala Ala Asp Ala Ser Ala Ala Ala Thr Ala Ser Ala Ala

20 25 30

Ser Arg Ser Ser Ala Asn Ala Ala Ala Ser Ala Phe Ala Gln Ser Phe

35 40 45

Ser Ser Ile Leu Leu Glu Ser Gly Tyr Phe Cys Ser Ile Phe Gly Ser

50 55 60

Ser Ile Ser Ser Ser Tyr Ala Ala Ala Ile Ala Ser Ala Ala Ser Arg

65 70 75 80

Ala Ala Ala Glu Ser Asn Gly Tyr Thr Thr His Ala Tyr Ala Cys Ala

85 90 95

Lys Ala Val Ala Ser Ala Val Glu Arg Val Thr Ser Gly Ala Asp Ala

100 105 110

Tyr Ala Tyr Ala Gln Ala Ile Ser Asp Ala Leu Ser His Ala Leu Leu

115 120 125

Tyr Thr Gly Arg Leu Asn Thr Ala Asn Ala Asn Ser Leu Ala Ser Ala

130 135 140

Phe Ala Tyr Ala Phe Ala Asn Ala Ala Ala Gln Ala Ser Ala Ser Ser

145 150 155 160

Ala Ser Ala Gly Ala Ala Ser Ala Ser Gly Ala Ala Ser Ala Ser Gly

165 170 175

Ala Gly Ser Ala Ser

180

<210> 7

<211> 126

<212> PRT

<213> original carnivorous spider (Plectreurys tristis)

<400> 7

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

1 5 10 15

Gly Ser Gly Ala Ser Thr Ser Val Ser Thr Ser Ser Ser Ser Gly Ser

20 25 30

Gly Ala Gly Ala Gly Ala Gly Ser Gly Ala Gly Ser Gly Ala Gly Ala

35 40 45

Gly Ser Gly Ala Gly Ala Gly Ala Gly Ala Gly Gly Ala Gly Ala Gly

50 55 60

Phe Gly Ser Gly Leu Gly Leu Gly Tyr Gly Val Gly Leu Ser Ser Ala

65 70 75 80

Gln Ala Gln Ala Gln Ala Gln Ala Ala Ala Gln Ala Gln Ala Gln Ala

85 90 95

Gln Ala Gln Ala Tyr Ala Ala Ala Gln Ala Gln Ala Gln Ala Gln Ala

100 105 110

Gln Ala Gln Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

115 120 125

<210> 8

<211> 239

<212> PRT

<213> original carnivorous spider (Plectreurys tristis)

<400> 8

Gly Ala Ala Gln Lys Gln Pro Ser Gly Glu Ser Ser Val Ala Thr Ala

1 5 10 15

Ser Ala Ala Ala Thr Ser Val Thr Ser Gly Gly Ala Pro Val Gly Lys

20 25 30

Pro Gly Val Pro Ala Pro Ile Phe Tyr Pro Gln Gly Pro Leu Gln Gln

35 40 45

Gly Pro Ala Pro Gly Pro Ser Asn Val Gln Pro Gly Thr Ser Gln Gln

50 55 60

Gly Pro Ile Gly Gly Val Gly Gly Ser Asn Ala Phe Ser Ser Ser Phe

65 70 75 80

Ala Ser Ala Leu Ser Leu Asn Arg Gly Phe Thr Glu Val Ile Ser Ser

85 90 95

Ala Ser Ala Thr Ala Val Ala Ser Ala Phe Gln Lys Gly Leu Ala Pro

100 105 110

Tyr Gly Thr Ala Phe Ala Leu Ser Ala Ala Ser Ala Ala Ala Asp Ala

115 120 125

Tyr Asn Ser Ile Gly Ser Gly Ala Asn Ala Phe Ala Tyr Ala Gln Ala

130 135 140

Phe Ala Arg Val Leu Tyr Pro Leu Val Gln Gln Tyr Gly Leu Ser Ser

145 150 155 160

Ser Ala Lys Ala Ser Ala Phe Ala Ser Ala Ile Ala Ser Ser Phe Ser

165 170 175

Ser Gly Thr Ser Gly Gln Gly Pro Ser Ile Gly Gln Gln Gln Pro Pro

180 185 190

Val Thr Ile Ser Ala Ala Ser Ala Ser Ala Gly Ala Ser Ala Ala Ala

195 200 205

Val Gly Gly Gly Gln Val Gly Gln Gly Pro Tyr Gly Gly Gln Gln Gln

210 215 220

Ser Thr Ala Ala Ser Ala Ser Ala Ala Ala Ala Thr Ala Thr Ser

225 230 235

<210> 9

<211> 182

<212> PRT

<213> Cat face spider (Araneeus gemfibrodes)

<400> 9

Gly Asn Val Gly Tyr Gln Leu Gly Leu Lys Val Ala Asn Ser Leu Gly

1 5 10 15

Leu Gly Asn Ala Gln Ala Leu Ala Ser Ser Leu Ser Gln Ala Val Ser

20 25 30

Ala Val Gly Val Gly Ala Ser Ser Asn Ala Tyr Ala Asn Ala Val Ser

35 40 45

Asn Ala Val Gly Gln Val Leu Ala Gly Gln Gly Ile Leu Asn Ala Ala

50 55 60

Asn Ala Gly Ser Leu Ala Ser Ser Phe Ala Ser Ala Leu Ser Ser Ser

65 70 75 80

Ala Ala Ser Val Ala Ser Gln Ser Ala Ser Gln Ser Gln Ala Ala Ser

85 90 95

Gln Ser Gln Ala Ala Ala Ser Ala Phe Arg Gln Ala Ala Ser Gln Ser

100 105 110

Ala Ser Gln Ser Asp Ser Arg Ala Gly Ser Gln Ser Ser Thr Lys Thr

115 120 125

Thr Ser Thr Ser Thr Ser Gly Ser Gln Ala Asp Ser Arg Ser Ala Ser

130 135 140

Ser Ser Ala Ser Gln Ala Ser Ala Ser Ala Phe Ala Gln Gln Ser Ser

145 150 155 160

Ala Ser Leu Ser Ser Ser Ser Ser Phe Ser Ser Ala Phe Ser Ser Ala

165 170 175

Thr Ser Ile Ser Ala Val

180

<210> 10

<211> 180

<212> PRT

<213> yellow spider (Argiope aurantia)

<400> 10

Gly Ser Leu Ala Ser Ser Phe Ala Ser Ala Leu Ser Ala Ser Ala Ala

1 5 10 15

Ser Val Ala Ser Ser Ala Ala Ala Gln Ala Ala Ser Gln Ser Gln Ala

20 25 30

Ala Ala Ser Ala Phe Ser Arg Ala Ala Ser Gln Ser Ala Ser Gln Ser

35 40 45

Ala Ala Arg Ser Gly Ala Gln Ser Ile Ser Thr Thr Thr Thr Thr Ser

50 55 60

Thr Ala Gly Ser Gln Ala Ala Ser Gln Ser Ala Ser Ser Ala Ala Ser

65 70 75 80

Gln Ala Ser Ala Ser Ser Phe Ala Arg Ala Ser Ser Ala Ser Leu Ala

85 90 95

Ala Ser Ser Ser Phe Ser Ser Ala Phe Ser Ser Ala Asn Ser Leu Ser

100 105 110

Ala Leu Gly Asn Val Gly Tyr Gln Leu Gly Phe Asn Val Ala Asn Asn

115 120 125

Leu Gly Ile Gly Asn Ala Ala Gly Leu Gly Asn Ala Leu Ser Gln Ala

130 135 140

Val Ser Ser Val Gly Val Gly Ala Ser Ser Ser Thr Tyr Ala Asn Ala

145 150 155 160

Val Ser Asn Ala Val Gly Gln Phe Leu Ala Gly Gln Gly Ile Leu Asn

165 170 175

Ala Ala Asn Ala

180

<210> 11

<211> 199

<212> PRT

<213> predator magic face spider (deinopsis spinosa)

<400> 11

Gly Ala Ser Ala Ser Ala Tyr Ala Ser Ala Ile Ser Asn Ala Val Gly

1 5 10 15

Pro Tyr Leu Tyr Gly Leu Gly Leu Phe Asn Gln Ala Asn Ala Ala Ser

20 25 30

Phe Ala Ser Ser Phe Ala Ser Ala Val Ser Ser Ala Val Ala Ser Ala

35 40 45

Ser Ala Ser Ala Ala Ser Ser Ala Tyr Ala Gln Ser Ala Ala Ala Gln

50 55 60

Ala Gln Ala Ala Ser Ser Ala Phe Ser Gln Ala Ala Ala Gln Ser Ala

65 70 75 80

Ala Ala Ala Ser Ala Gly Ala Ser Ala Gly Ala Gly Ala Ser Ala Gly

85 90 95

Ala Gly Ala Val Ala Gly Ala Gly Ala Val Ala Gly Ala Gly Ala Val

100 105 110

Ala Gly Ala Ser Ala Ala Ala Ala Ser Gln Ala Ala Ala Ser Ser Ser

115 120 125

Ala Ser Ala Val Ala Ser Ala Phe Ala Gln Ser Ala Ser Tyr Ala Leu

130 135 140

Ala Ser Ser Ser Ala Phe Ala Asn Ala Phe Ala Ser Ala Thr Ser Ala

145 150 155 160

Gly Tyr Leu Gly Ser Leu Ala Tyr Gln Leu Gly Leu Thr Thr Ala Tyr

165 170 175

Asn Leu Gly Leu Ser Asn Ala Gln Ala Phe Ala Ser Thr Leu Ser Gln

180 185 190

Ala Val Thr Gly Val Gly Leu

195

<210> 12

<211> 171

<212> PRT

<213> Nephila clavipes)

<400> 12

Gly Ala Thr Ala Ala Ser Tyr Gly Asn Ala Leu Ser Thr Ala Ala Ala

1 5 10 15

Gln Phe Phe Ala Thr Ala Gly Leu Leu Asn Ala Gly Asn Ala Ser Ala

20 25 30

Leu Ala Ser Ser Phe Ala Arg Ala Phe Ser Ala Ser Ala Glu Ser Gln

35 40 45

Ser Phe Ala Gln Ser Gln Ala Phe Gln Gln Ala Ser Ala Phe Gln Gln

50 55 60

Ala Ala Ser Arg Ser Ala Ser Gln Ser Ala Ala Glu Ala Gly Ser Thr

65 70 75 80

Ser Ser Ser Thr Thr Thr Thr Thr Ser Ala Ala Arg Ser Gln Ala Ala

85 90 95

Ser Gln Ser Ala Ser Ser Ser Tyr Ser Ser Ala Phe Ala Gln Ala Ala

100 105 110

Ser Ser Ser Leu Ala Thr Ser Ser Ala Leu Ser Arg Ala Phe Ser Ser

115 120 125

Val Ser Ser Ala Ser Ala Ala Ser Ser Leu Ala Tyr Ser Ile Gly Leu

130 135 140

Ser Ala Ala Arg Ser Leu Gly Ile Ala Asp Ala Ala Gly Leu Ala Gly

145 150 155 160

Val Leu Ala Arg Ala Ala Gly Ala Leu Gly Gln

165 170

<210> 13

<211> 268

<212> PRT

<213> three-belt spider (Argiope trifasciata)

<400> 13

Gly Gly Ala Pro Gly Gly Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala

1 5 10 15

Gly Phe Gly Pro Gly Gly Gly Ala Gly Phe Gly Pro Gly Gly Gly Ala

20 25 30

Gly Phe Gly Pro Gly Gly Ala Ala Gly Gly Pro Gly Gly Pro Gly Gly

35 40 45

Pro Gly Gly Pro Gly Gly Ala Gly Gly Tyr Gly Pro Gly Gly Ala Gly

50 55 60

Gly Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Tyr Gly

65 70 75 80

Pro Gly Gly Ala Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly

85 90 95

Ala Gly Pro Gly Gly Ala Gly Gly Glu Gly Pro Val Thr Val Asp Val

100 105 110

Asp Val Thr Val Gly Pro Glu Gly Val Gly Gly Gly Pro Gly Gly Ala

115 120 125

Gly Pro Gly Gly Ala Gly Phe Gly Pro Gly Gly Gly Ala Gly Phe Gly

130 135 140

Pro Gly Gly Ala Pro Gly Ala Pro Gly Gly Pro Gly Gly Pro Gly Gly

145 150 155 160

Pro Gly Gly Pro Gly Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly

165 170 175

Gly Tyr Gly Pro Gly Gly Ala Gly Gly Val Gly Pro Ala Gly Thr Gly

180 185 190

Gly Phe Gly Pro Gly Gly Ala Gly Gly Phe Gly Pro Gly Gly Ala Gly

195 200 205

Gly Phe Gly Pro Gly Gly Ala Gly Gly Phe Gly Pro Ala Gly Ala Gly

210 215 220

Gly Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Phe Gly

225 230 235 240

Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly

245 250 255

Glu Gly Pro Val Thr Val Asp Val Asp Val Ser Val

260 265

<210> 14

<211> 420

<212> PRT

<213> Nephila clavipes)

<400> 14

Gly Val Ser Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly

1 5 10 15

Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro Gly Gly Ala Gly Gly Pro

20 25 30

Tyr Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr

35 40 45

Gly Pro Gly Gly Ala Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly

50 55 60

Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro

65 70 75 80

Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly

85 90 95

Gly Tyr Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro Gly Gly

100 105 110

Ser Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Thr

115 120 125

Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly

130 135 140

Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly Gly Ser Gly Pro

145 150 155 160

Gly Gly Phe Gly Pro Gly Gly Ser Gly Pro Gly Gly Tyr Gly Pro Gly

165 170 175

Gly Ser Gly Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly

180 185 190

Phe Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Ala Pro Gly Gly Ala

195 200 205

Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly

210 215 220

Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Pro Gly Gly Ala Gly Gly

225 230 235 240

Ala Gly Gly Ala Gly Gly Ser Gly Gly Ala Gly Gly Ser Gly Gly Thr

245 250 255

Thr Ile Ile Glu Asp Leu Asp Ile Thr Ile Asp Gly Ala Asp Gly Pro

260 265 270

Ile Thr Ile Ser Glu Glu Leu Pro Ile Ser Gly Ala Gly Gly Ser Gly

275 280 285

Pro Gly Gly Ala Gly Pro Gly Gly Val Gly Pro Gly Gly Ser Gly Pro

290 295 300

Gly Gly Val Gly Pro Gly Gly Ser Gly Pro Gly Gly Val Gly Pro Gly

305 310 315 320

Gly Ser Gly Pro Gly Gly Val Gly Pro Gly Gly Ala Gly Gly Pro Tyr

325 330 335

Gly Pro Gly Gly Ser Gly Pro Gly Gly Ala Gly Gly Ala Gly Gly Pro

340 345 350

Gly Gly Ala Tyr Gly Pro Gly Gly Ser Tyr Gly Pro Gly Gly Ser Gly

355 360 365

Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Pro

370 375 380

Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Ala Gly Gly Pro Tyr

385 390 395 400

Gly Pro Gly Gly Ala Gly Gly Pro Tyr Gly Pro Gly Gly Glu Gly Gly

405 410 415

Pro Tyr Gly Pro

420

<210> 15

<211> 376

<212> PRT

<213> Black widow spider (Latrodectus hesperus)

<400> 15

Gly Ile Asn Val Asp Ser Asp Ile Gly Ser Val Thr Ser Leu Ile Leu

1 5 10 15

Ser Gly Ser Thr Leu Gln Met Thr Ile Pro Ala Gly Gly Asp Asp Leu

20 25 30

Ser Gly Gly Tyr Pro Gly Gly Phe Pro Ala Gly Ala Gln Pro Ser Gly

35 40 45

Gly Ala Pro Val Asp Phe Gly Gly Pro Ser Ala Gly Gly Asp Val Ala

50 55 60

Ala Lys Leu Ala Arg Ser Leu Ala Ser Thr Leu Ala Ser Ser Gly Val

65 70 75 80

Phe Arg Ala Ala Phe Asn Ser Arg Val Ser Thr Pro Val Ala Val Gln

85 90 95

Leu Thr Asp Ala Leu Val Gln Lys Ile Ala Ser Asn Leu Gly Leu Asp

100 105 110

Tyr Ala Thr Ala Ser Lys Leu Arg Lys Ala Ser Gln Ala Val Ser Lys

115 120 125

Val Arg Met Gly Ser Asp Thr Asn Ala Tyr Ala Leu Ala Ile Ser Ser

130 135 140

Ala Leu Ala Glu Val Leu Ser Ser Ser Gly Lys Val Ala Asp Ala Asn

145 150 155 160

Ile Asn Gln Ile Ala Pro Gln Leu Ala Ser Gly Ile Val Leu Gly Val

165 170 175

Ser Thr Thr Ala Pro Gln Phe Gly Val Asp Leu Ser Ser Ile Asn Val

180 185 190

Asn Leu Asp Ile Ser Asn Val Ala Arg Asn Met Gln Ala Ser Ile Gln

195 200 205

Gly Gly Pro Ala Pro Ile Thr Ala Glu Gly Pro Asp Phe Gly Ala Gly

210 215 220

Tyr Pro Gly Gly Ala Pro Thr Asp Leu Ser Gly Leu Asp Met Gly Ala

225 230 235 240

Pro Ser Asp Gly Ser Arg Gly Gly Asp Ala Thr Ala Lys Leu Leu Gln

245 250 255

Ala Leu Val Pro Ala Leu Leu Lys Ser Asp Val Phe Arg Ala Ile Tyr

260 265 270

Lys Arg Gly Thr Arg Lys Gln Val Val Gln Tyr Val Thr Asn Ser Ala

275 280 285

Leu Gln Gln Ala Ala Ser Ser Leu Gly Leu Asp Ala Ser Thr Ile Ser

290 295 300

Gln Leu Gln Thr Lys Ala Thr Gln Ala Leu Ser Ser Val Ser Ala Asp

305 310 315 320

Ser Asp Ser Thr Ala Tyr Ala Lys Ala Phe Gly Leu Ala Ile Ala Gln

325 330 335

Val Leu Gly Thr Ser Gly Gln Val Asn Asp Ala Asn Val Asn Gln Ile

340 345 350

Gly Ala Lys Leu Ala Thr Gly Ile Leu Arg Gly Ser Ser Ala Val Ala

355 360 365

Pro Arg Leu Gly Ile Asp Leu Ser

370 375

<210> 16

<211> 200

<212> PRT

<213> three-belt spider (Argiope trifasciata)

<400> 16

Gly Ala Gly Tyr Thr Gly Pro Ser Gly Pro Ser Thr Gly Pro Ser Gly

1 5 10 15

Tyr Pro Gly Pro Leu Gly Gly Gly Ala Pro Phe Gly Gln Ser Gly Phe

20 25 30

Gly Gly Ser Ala Gly Pro Gln Gly Gly Phe Gly Ala Thr Gly Gly Ala

35 40 45

Ser Ala Gly Leu Ile Ser Arg Val Ala Asn Ala Leu Ala Asn Thr Ser

50 55 60

Thr Leu Arg Thr Val Leu Arg Thr Gly Val Ser Gln Gln Ile Ala Ser

65 70 75 80

Ser Val Val Gln Arg Ala Ala Gln Ser Leu Ala Ser Thr Leu Gly Val

85 90 95

Asp Gly Asn Asn Leu Ala Arg Phe Ala Val Gln Ala Val Ser Arg Leu

100 105 110

Pro Ala Gly Ser Asp Thr Ser Ala Tyr Ala Gln Ala Phe Ser Ser Ala

115 120 125

Leu Phe Asn Ala Gly Val Leu Asn Ala Ser Asn Ile Asp Thr Leu Gly

130 135 140

Ser Arg Val Leu Ser Ala Leu Leu Asn Gly Val Ser Ser Ala Ala Gln

145 150 155 160

Gly Leu Gly Ile Asn Val Asp Ser Gly Ser Val Gln Ser Asp Ile Ser

165 170 175

Ser Ser Ser Ser Phe Leu Ser Thr Ser Ser Ser Ser Ala Ser Tyr Ser

180 185 190

Gln Ala Ser Ala Ser Ser Thr Ser

195 200

<210> 17

<211> 357

<212> PRT

<213> use steel \34801s (Uloborus diversus)

<400> 17

Gly Ala Ser Ala Ala Asp Ile Ala Thr Ala Ile Ala Ala Ser Val Ala

1 5 10 15

Thr Ser Leu Gln Ser Asn Gly Val Leu Thr Ala Ser Asn Val Ser Gln

20 25 30

Leu Ser Asn Gln Leu Ala Ser Tyr Val Ser Ser Gly Leu Ser Ser Thr

35 40 45

Ala Ser Ser Leu Gly Ile Gln Leu Gly Ala Ser Leu Gly Ala Gly Phe

50 55 60

Gly Ala Ser Ala Gly Leu Ser Ala Ser Thr Asp Ile Ser Ser Ser Val

65 70 75 80

Glu Ala Thr Ser Ala Ser Thr Leu Ser Ser Ser Ala Ser Ser Thr Ser

85 90 95

Val Val Ser Ser Ile Asn Ala Gln Leu Val Pro Ala Leu Ala Gln Thr

100 105 110

Ala Val Leu Asn Ala Ala Phe Ser Asn Ile Asn Thr Gln Asn Ala Ile

115 120 125

Arg Ile Ala Glu Leu Leu Thr Gln Gln Val Gly Arg Gln Tyr Gly Leu

130 135 140

Ser Gly Ser Asp Val Ala Thr Ala Ser Ser Gln Ile Arg Ser Ala Leu

145 150 155 160

Tyr Ser Val Gln Gln Gly Ser Ala Ser Ser Ala Tyr Val Ser Ala Ile

165 170 175

Val Gly Pro Leu Ile Thr Ala Leu Ser Ser Arg Gly Val Val Asn Ala

180 185 190

Ser Asn Ser Ser Gln Ile Ala Ser Ser Leu Ala Thr Ala Ile Leu Gln

195 200 205

Phe Thr Ala Asn Val Ala Pro Gln Phe Gly Ile Ser Ile Pro Thr Ser

210 215 220

Ala Val Gln Ser Asp Leu Ser Thr Ile Ser Gln Ser Leu Thr Ala Ile

225 230 235 240

Ser Ser Gln Thr Ser Ser Ser Val Asp Ser Ser Thr Ser Ala Phe Gly

245 250 255

Gly Ile Ser Gly Pro Ser Gly Pro Ser Pro Tyr Gly Pro Gln Pro Ser

260 265 270

Gly Pro Thr Phe Gly Pro Gly Pro Ser Leu Ser Gly Leu Thr Gly Phe

275 280 285

Thr Ala Thr Phe Ala Ser Ser Phe Lys Ser Thr Leu Ala Ser Ser Thr

290 295 300

Gln Phe Gln Leu Ile Ala Gln Ser Asn Leu Asp Val Gln Thr Arg Ser

305 310 315 320

Ser Leu Ile Ser Lys Val Leu Ile Asn Ala Leu Ser Ser Leu Gly Ile

325 330 335

Ser Ala Ser Val Ala Ser Ser Ile Ala Ala Ser Ser Ser Gln Ser Leu

340 345 350

Leu Ser Val Ser Ala

355

<210> 18

<211> 32

<212> PRT

<213> Nursery network spider (Euprosthenops australis)

<400> 18

Gly Gly Gln Gly Gly Gln Gly Gln Gly Arg Tyr Gly Gln Gly Ala Gly

1 5 10 15

Ser Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

20 25 30

<210> 19

<211> 42

<212> PRT

<213> Phalaenopsis amabilis green paw (Tetragnatha kauaiensis)

<400> 19

Gly Gly Leu Gly Gly Gly Gln Gly Ala Gly Gln Gly Gly Gln Gln Gly

1 5 10 15

Ala Gly Gln Gly Gly Tyr Gly Ser Gly Leu Gly Gly Ala Gly Gln Gly

20 25 30

Ala Ser Ala Ala Ala Ala Ala Ala Ala Ala

35 40

<210> 20

<211> 42

<212> PRT

<213> yellow spider (Argiope aurantia)

<400> 20

Gly Gly Tyr Gly Pro Gly Ala Gly Gln Gln Gly Pro Gly Ser Gln Gly

1 5 10 15

Pro Gly Ser Gly Gly Gln Gln Gly Pro Gly Gly Leu Gly Pro Tyr Gly

20 25 30

Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala

35 40

<210> 21

<211> 46

<212> PRT

<213> predator magic face spider (deinopsis spinosa)

<400> 21

Gly Pro Gly Gly Tyr Gly Gly Pro Gly Gln Gln Gly Pro Gly Gln Gly

1 5 10 15

Gln Tyr Gly Pro Gly Thr Gly Gln Gln Gly Gln Gly Pro Ser Gly Gln

20 25 30

Gln Gly Pro Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala Ala

35 40 45

<210> 22

<211> 42

<212> PRT

<213> Nephila clavata

<400> 22

Gly Pro Gly Gly Tyr Gly Leu Gly Gln Gln Gly Pro Gly Gln Gln Gly

1 5 10 15

Pro Gly Gln Gln Gly Pro Ala Gly Tyr Gly Pro Ser Gly Leu Ser Gly

20 25 30

Pro Gly Gly Ala Ala Ala Ala Ala Ala Ala

35 40

<210> 23

<211> 174

<212> PRT

<213> predator magic face spider (deinopsis spinosa)

<400> 23

Gly Ala Gly Tyr Gly Ala Gly Ala Gly Ala Gly Gly Gly Ala Gly Ala

1 5 10 15

Gly Thr Gly Tyr Gly Gly Gly Ala Gly Tyr Gly Thr Gly Ser Gly Ala

20 25 30

Gly Tyr Gly Ala Gly Val Gly Tyr Gly Ala Gly Ala Gly Ala Gly Gly

35 40 45

Gly Ala Gly Ala Gly Ala Gly Gly Gly Thr Gly Ala Gly Ala Gly Gly

50 55 60

Gly Ala Gly Ala Gly Tyr Gly Ala Gly Thr Gly Tyr Gly Ala Gly Ala

65 70 75 80

Gly Ala Gly Gly Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

85 90 95

Gly Ala Gly Ala Gly Ser Gly Ala Gly Ala Gly Tyr Gly Ala Gly Ala

100 105 110

Gly Tyr Gly Ala Gly Ala Gly Ala Gly Gly Val Ala Gly Ala Gly Ala

115 120 125

Ala Gly Gly Ala Gly Ala Ala Gly Gly Ala Gly Ala Ala Gly Gly Ala

130 135 140

Gly Ala Ala Gly Gly Ala Gly Ala Gly Ala Gly Ala Gly Ser Gly Ala

145 150 155 160

Gly Ala Gly Ala Gly Gly Gly Ala Arg Ala Gly Ala Gly Gly

165 170

<210> 24

<211> 149

<212> PRT

<213> Black widow spider (Latridectus hesperus)

<400> 24

Gly Gly Gly Tyr Gly Arg Gly Gln Gly Ala Gly Ala Gly Val Gly Ala

1 5 10 15

Gly Ala Gly Ala Ala Ala Gly Ala Ala Ala Ile Ala Arg Ala Gly Gly

20 25 30

Tyr Gly Gln Gly Ala Gly Gly Tyr Gly Gln Gly Gln Gly Ala Gly Ala

35 40 45

Ala Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Tyr Gly Gln Gly

50 55 60

Ala Gly Gly Tyr Gly Arg Gly Gln Gly Ala Gly Ala Gly Ala Gly Ala

65 70 75 80

Gly Ala Gly Ala Arg Gly Tyr Gly Gln Gly Ala Gly Ala Gly Ala Ala

85 90 95

Ala Gly Ala Ala Ala Ser Ala Gly Ala Gly Gly Tyr Gly Gln Gly Ala

100 105 110

Gly Gly Tyr Gly Gln Gly Gln Gly Ala Gly Ala Ala Ala Gly Ala Ala

115 120 125

Ala Ser Ala Gly Ala Gly Gly Tyr Gly Gln Gly Ala Gly Gly Tyr Gly

130 135 140

Gln Gly Gln Gly Ala

145

<210> 25

<211> 161

<212> PRT

<213> Nephila clavipes)

<400> 25

Gly Ala Gly Ala Gly Gly Ala Gly Tyr Gly Arg Gly Ala Gly Ala Gly

1 5 10 15

Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly

20 25 30

Ala Gly Ala Gly Gly Tyr Gly Gly Gln Gly Gly Tyr Gly Ala Gly Ala

35 40 45

Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Ala

50 55 60

Gly Tyr Ser Arg Gly Gly Arg Ala Gly Ala Ala Gly Ala Gly Ala Gly

65 70 75 80

Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Gly Tyr Gly Gly Gln Gly

85 90 95

Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ala

100 105 110

Gly Ser Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala

115 120 125

Ala Ala Gly Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly Ala Gly Gly

130 135 140

Tyr Gly Gly Gln Gly Gly Tyr Gly Ala Gly Ala Gly Ala Ala Ala Ala

145 150 155 160

Ala

<210> 26

<211> 186

<212> PRT

<213> Nephilengys cruentata spider

<400> 26

Gly Ala Gly Ala Gly Val Gly Gly Ala Gly Gly Tyr Gly Ser Gly Ala

1 5 10 15

Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Ala Ser Gly Ala Ala Ala

20 25 30

Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Thr

35 40 45

Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala

50 55 60

Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala

65 70 75 80

Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala

85 90 95

Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Asp Gly Ala Gly Ala

100 105 110

Gly Gly Ala Gly Gly Tyr Gly Arg Gly Ala Gly Ala Gly Ala Gly Ala

115 120 125

Gly Ala Ala Ala Gly Ala Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala

130 135 140

Gly Gln Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Ala Ala Gly Ala

145 150 155 160

Gly Ala Gly Gly Ala Gly Gly Tyr Gly Ala Gly Gln Gly Tyr Gly Ala

165 170 175

Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala

180 185

<210> 27

<211> 132

<212> PRT

<213> use steel \34801s (Uloborus diversus)

<400> 27

Gly Ser Gly Ala Gly Ala Gly Ser Gly Tyr Gly Ala Gly Ala Gly Ala

1 5 10 15

Gly Ala Gly Ser Gly Tyr Gly Ala Gly Ser Ser Ala Ser Ala Gly Ser

20 25 30

Ala Ile Asn Thr Gln Thr Val Thr Ser Ser Thr Thr Thr Ser Ser Gln

35 40 45

Ser Ser Ala Ala Ala Thr Gly Ala Gly Tyr Gly Thr Gly Ala Gly Thr

50 55 60

Gly Ala Ser Ala Gly Ala Ala Ala Ser Gly Ala Gly Ala Gly Tyr Gly

65 70 75 80

Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Arg Ala Ala

85 90 95

Gly Ser Gly Tyr Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Gly Ser

100 105 110

Gly Tyr Gly Ala Gly Ala Gly Ala Gly Ala Gly Ser Gly Tyr Gly Ala

115 120 125

Gly Ala Ala Ala

130

<210> 28

<211> 198

<212> PRT

<213> total different velocity 34801s spider (uloborius diversus)

<400> 28

Gly Ala Gly Ala Gly Tyr Arg Gly Gln Ala Gly Tyr Ile Gln Gly Ala

1 5 10 15

Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Val Gly Tyr Gly

20 25 30

Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala

35 40 45

Ala Ala Ala Gly Ala Gly Ala Gly Arg Gln Ala Gly Tyr Gly Gln Gly

50 55 60

Ala Gly Ala Ser Ala Gly Ala Ala Ala Ala Gly Ala Gly Ala Gly Arg

65 70 75 80

Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala

85 90 95

Ala Gly Ala Asp Ala Gly Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly

100 105 110

Ala Gly Ala Ser Ala Gly Ala Ala Ala Ser Gly Ala Gly Ala Gly Tyr

115 120 125

Gly Gly Gln Ala Gly Tyr Gly Gln Gly Ala Gly Ala Ser Ala Gly Ala

130 135 140

Ala Ala Ala Gly Ala Gly Ala Gly Tyr Leu Gly Gln Ala Gly Tyr Gly

145 150 155 160

Gln Gly Ala Gly Ala Ser Ala Gly Ala Ala Ala Gly Ala Gly Ala Gly

165 170 175

Tyr Gly Gly Gln Ala Gly Tyr Gly Gln Gly Thr Gly Ala Ala Ala Ser

180 185 190

Ala Ala Ala Ser Ser Ala

195

<210> 29

<211> 190

<212> PRT

<213> big-belly orbrachus (araneeus ventricosus)

<400> 29

Gly Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gln Gly

1 5 10 15

Ala Gly Gln Gly Gly Tyr Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala

20 25 30

Ala Ala Ala Gly Gly Ala Gly Gly Ala Gly Arg Gly Gly Leu Gly Ala

35 40 45

Gly Gly Ala Gly Gln Gly Tyr Gly Ala Gly Leu Gly Gly Gln Gly Gly

50 55 60

Ala Gly Gln Ala Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gly Ala

65 70 75 80

Arg Gln Gly Gly Leu Gly Ala Gly Gly Ala Gly Gln Gly Tyr Gly Ala

85 90 95

Gly Leu Gly Gly Gln Gly Gly Ala Gly Gln Gly Gly Ala Ala Ala Ala

100 105 110

Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gly Tyr Gly Gly Leu

115 120 125

Gly Ser Gln Gly Ala Gly Gln Gly Gly Tyr Gly Ala Gly Gln Gly Gly

130 135 140

Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Gln Gly Gly Gln Gly Gly

145 150 155 160

Tyr Gly Gly Leu Gly Ser Gln Gly Ala Gly Gln Gly Gly Tyr Gly Gly

165 170 175

Arg Gln Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala Ala

180 185 190

<210> 30

<211> 166

<212> PRT

<213> Black fishing spider (Dolomedes tenebrosus)

<400> 30

Gly Gly Ala Gly Ala Gly Gln Gly Ser Tyr Gly Gly Gln Gly Gly Tyr

1 5 10 15

Gly Gln Gly Gly Ala Gly Ala Ala Thr Ala Thr Ala Ala Ala Ala Gly

20 25 30

Gly Ala Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Gly Leu Gly

35 40 45

Gly Tyr Gly Gln Gly Ala Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala

50 55 60

Ala Ala Gly Gly Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly

65 70 75 80

Gly Gln Gly Gly Tyr Gly Gln Gly Ala Gly Ala Gly Ala Ala Ala Ala

85 90 95

Ala Ala Gly Gly Ala Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly

100 105 110

Gly Tyr Gly Gln Gly Gly Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala

115 120 125

Ala Ser Gly Gly Ser Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly

130 135 140

Gly Leu Gly Gly Tyr Gly Gln Gly Ala Gly Ala Gly Ala Gly Ala Ala

145 150 155 160

Ala Ser Ala Ala Ala Ala

165

<210> 31

<211> 177

<212> PRT

<213> Nephilengys cruentata spider

<400> 31

Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala

1 5 10 15

Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly

20 25 30

Gly Gln Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala Ala Ser Gly Ala

35 40 45

Gly Gln Gly Gly Tyr Glu Gly Pro Gly Ala Gly Gln Gly Ala Gly Ala

50 55 60

Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu

65 70 75 80

Gly Gly Gln Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala

85 90 95

Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala

100 105 110

Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln

115 120 125

Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala

130 135 140

Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Ser Gly Gln

145 150 155 160

Gly Gly Tyr Gly Arg Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala Ala

165 170 175

Ala

<210> 32

<211> 174

<212> PRT

<213> Nephilengys cruentata spider

<400> 32

Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly Gln Gly Ala

1 5 10 15

Gly Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly

20 25 30

Gly Gln Gly Ala Gly Gln Gly Ala Ala Ala Ala Ala Ala Ser Gly Ala

35 40 45

Gly Gln Gly Gly Tyr Gly Gly Pro Gly Ala Gly Gln Gly Ala Gly Ala

50 55 60

Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu

65 70 75 80

Gly Gly Gln Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala

85 90 95

Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Gln Gly

100 105 110

Ala Ala Ala Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly

115 120 125

Leu Gly Ser Gly Gln Gly Gly Tyr Gly Gly Gln Gly Ala Gly Ala Ala

130 135 140

Ala Ala Ala Gly Gly Ala Gly Gln Gly Gly Tyr Gly Gly Leu Gly Gly

145 150 155 160

Gln Gly Ala Gly Gln Gly Ala Gly Ala Ala Ala Ala Ala Ala

165 170

<210> 33

<211> 8

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic His tag

<220>

<221> site

<222> (1)..(8)

<223> this sequence can encompass 6-8 residues

<400> 33

His His His His His His His His

1 5

<210> 34

<211> 1600

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic polypeptides

<220>

<221> site

<222> (7)..(11)

<223> this area may encompass "SGGQQ", "GAGQQ", "GQGPY", "AGQQ", or "SQ", where some locations may not exist

<220>

<221> site

<222> (15)..(19)

<220>

<221> site

<222> (23)..(27)

<220>

<221> site

<222> (31)..(35)

<220>

<221> site

<222> (39)..(43)

<220>

<221> site

<222> (47)..(51)

<220>

<221> site

<222> (55)..(59)

<220>

<221> site

<222> (63)..(67)

<220>

<221> site

<222> (4)..(67)

<223> this region can encompass 4-8 repeating "GPG-X1" repeat units, where X1 is "SGGQQ", "GAGQQ", "GQGPY", "AGQQ", or "SQ", and some locations are not present

<220>

<221> site

<222> (71)..(80)

<223> this sequence can encompass 6-10 residues

<220>

<221> site

<222> (87)..(91)

<220>

<221> site

<222> (95)..(99)

<220>

<221> site

<222> (103)..(107)

<220>

<221> site

<222> (111)..(115)

<220>

<221> site

<222> (119)..(123)

<220>

<221> site

<222> (127)..(131)

<220>

<221> site

<222> (135)..(139)

<220>

<221> site

<222> (143)..(147)

<220>

<221> site

<222> (84)..(147)

<220>

<221> site

<222> (151)..(160)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (167)..(171)

<220>

<221> site

<222> (175)..(179)

<220>

<221> site

<222> (183)..(187)

<220>

<221> site

<222> (191)..(195)

<220>

<221> site

<222> (199)..(203)

<220>

<221> site

<222> (207)..(211)

<220>

<221> site

<222> (215)..(219)

<220>

<221> site

<222> (223)..(227)

<220>

<221> site

<222> (164)..(227)

<220>

<221> site

<222> (231)..(240)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (247)..(251)

<220>

<221> site

<222> (255)..(259)

<220>

<221> site

<222> (263)..(267)

<220>

<221> site

<222> (271)..(275)

<220>

<221> site

<222> (279)..(283)

<220>

<221> site

<222> (287)..(291)

<220>

<221> site

<222> (295)..(299)

<220>

<221> site

<222> (303)..(307)

<220>

<221> site

<222> (244)..(307)

<223> this area can encompass 4-8 repeating "GPG-X1" repeat units, where X1 is "SGGQQ", "GAGQQ", "GQGPY", "AGQQ", or "SQ", and some locations do not exist

<220>

<221> site

<222> (311)..(320)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (327)..(331)

<220>

<221> site

<222> (335)..(339)

<220>

<221> site

<222> (343)..(347)

<220>

<221> site

<222> (351)..(355)

<220>

<221> site

<222> (359)..(363)

<220>

<221> site

<222> (367)..(371)

<220>

<221> site

<222> (375)..(379)

<220>

<221> site

<222> (383)..(387)

<220>

<221> site

<222> (324)..(387)

<220>

<221> site

<222> (391)..(400)

<223> this sequence can encompass 6-10 residues

<220>

<221> site

<222> (407)..(411)

<220>

<221> site

<222> (415)..(419)

<220>

<221> site

<222> (423)..(427)

<220>

<221> site

<222> (431)..(435)

<220>

<221> site

<222> (439)..(443)

<220>

<221> site

<222> (447)..(451)

<220>

<221> site

<222> (455)..(459)

<220>

<221> site

<222> (463)..(467)

<220>

<221> site

<222> (404)..(467)

<220>

<221> site

<222> (471)..(480)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (487)..(491)

<220>

<221> site

<222> (495)..(499)

<220>

<221> site

<222> (503)..(507)

<220>

<221> site

<222> (511)..(515)

<220>

<221> site

<222> (519)..(523)

<220>

<221> site

<222> (527)..(531)

<220>

<221> site

<222> (535)..(539)

<220>

<221> site

<222> (543)..(547)

<220>

<221> site

<222> (484)..(547)

<220>

<221> site

<222> (551)..(560)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (567)..(571)

<220>

<221> site

<222> (575)..(579)

<220>

<221> site

<222> (583)..(587)

<220>

<221> site

<222> (591)..(595)

<220>

<221> site

<222> (599)..(603)

<220>

<221> site

<222> (607)..(611)

<220>

<221> site

<222> (615)..(619)

<220>

<221> site

<222> (623)..(627)

<220>

<221> site

<222> (564)..(627)

<220>

<221> site

<222> (631)..(640)

<223> this sequence can encompass 6-10 residues

<220>

<221> site

<222> (647)..(651)

<220>

<221> site

<222> (655)..(659)

<220>

<221> site

<222> (663)..(667)

<220>

<221> site

<222> (671)..(675)

<220>

<221> site

<222> (679)..(683)

<220>

<221> site

<222> (687)..(691)

<220>

<221> site

<222> (695)..(699)

<220>

<221> site

<222> (703)..(707)

<220>

<221> site

<222> (644)..(707)

<220>

<221> site

<222> (711)..(720)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (727)..(731)

<220>

<221> site

<222> (735)..(739)

<220>

<221> site

<222> (743)..(747)

<220>

<221> site

<222> (751)..(755)

<220>

<221> site

<222> (759)..(763)

<220>

<221> site

<222> (767)..(771)

<220>

<221> site

<222> (775)..(779)

<220>

<221> site

<222> (783)..(787)

<220>

<221> site

<222> (724)..(787)

<220>

<221> site

<222> (791)..(800)

<223> this sequence can encompass 6-10 residues

<220>

<221> site

<222> (807)..(811)

<220>

<221> site

<222> (815)..(819)

<220>

<221> site

<222> (823)..(827)

<220>

<221> site

<222> (831)..(835)

<220>

<221> site

<222> (839)..(843)

<220>

<221> site

<222> (847)..(851)

<220>

<221> site

<222> (855)..(859)

<220>

<221> site

<222> (863)..(867)

<220>

<221> site

<222> (804)..(867)

<220>

<221> site

<222> (871)..(880)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (887)..(891)

<220>

<221> site

<222> (895)..(899)

<220>

<221> site

<222> (903)..(907)

<220>

<221> site

<222> (911)..(915)

<220>

<221> site

<222> (919)..(923)

<220>

<221> site

<222> (927)..(931)

<220>

<221> site

<222> (935)..(939)

<220>

<221> site

<222> (943)..(947)

<220>

<221> site

<222> (884)..(947)

<220>

<221> site

<222> (951)..(960)

<223> this sequence can encompass 6-10 residues

<220>

<221> site

<222> (967)..(971)

<220>

<221> site

<222> (975)..(979)

<220>

<221> site

<222> (983)..(987)

<220>

<221> site

<222> (991)..(995)

<220>

<221> site

<222> (999)..(1003)

<220>

<221> site

<222> (1007)..(1011)

<220>

<221> site

<222> (1015)..(1019)

<220>

<221> site

<222> (1023)..(1027)

<220>

<221> site

<222> (964)..(1027)

<220>

<221> site

<222> (1031)..(1040)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (1047)..(1051)

<220>

<221> site

<222> (1055)..(1059)

<220>

<221> site

<222> (1063)..(1067)

<220>

<221> site

<222> (1071)..(1075)

<220>

<221> site

<222> (1079)..(1083)

<220>

<221> site

<222> (1087)..(1091)

<220>

<221> site

<222> (1095)..(1099)

<220>

<221> site

<222> (1103)..(1107)

<220>

<221> site

<222> (1044)..(1107)

<220>

<221> site

<222> (1111)..(1120)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (1127)..(1131)

<220>

<221> site

<222> (1135)..(1139)

<220>

<221> site

<222> (1143)..(1147)

<220>

<221> site

<222> (1151)..(1155)

<220>

<221> site

<222> (1159)..(1163)

<220>

<221> site

<222> (1167)..(1171)

<220>

<221> site

<222> (1175)..(1179)

<220>

<221> site

<222> (1183)..(1187)

<220>

<221> site

<222> (1124)..(1187)

<220>

<221> site

<222> (1191)..(1200)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (1207)..(1211)

<220>

<221> site

<222> (1215)..(1219)

<220>

<221> site

<222> (1223)..(1227)

<220>

<221> site

<222> (1231)..(1235)

<220>

<221> site

<222> (1239)..(1243)

<220>

<221> site

<222> (1247)..(1251)

<220>

<221> site

<222> (1255)..(1259)

<220>

<221> site

<222> (1263)..(1267)

<220>

<221> site

<222> (1204)..(1267)

<220>

<221> site

<222> (1271)..(1280)

<223> this sequence can encompass 6-10 residues

<220>

<221> site

<222> (1287)..(1291)

<220>

<221> site

<222> (1295)..(1299)

<220>

<221> site

<222> (1303)..(1307)

<220>

<221> site

<222> (1311)..(1315)

<220>

<221> site

<222> (1319)..(1323)

<220>

<221> site

<222> (1327)..(1331)

<220>

<221> site

<222> (1335)..(1339)

<220>

<221> site

<222> (1343)..(1347)

<220>

<221> site

<222> (1284)..(1347)

<220>

<221> site

<222> (1351)..(1360)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (1367)..(1371)

<220>

<221> site

<222> (1375)..(1379)

<220>

<221> site

<222> (1383)..(1387)

<220>

<221> site

<222> (1391)..(1395)

<220>

<221> site

<222> (1399)..(1403)

<220>

<221> site

<222> (1407)..(1411)

<220>

<221> site

<222> (1415)..(1419)

<220>

<221> site

<222> (1423)..(1427)

<220>

<221> site

<222> (1364)..(1427)

<220>

<221> site

<222> (1431)..(1440)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (1447)..(1451)

<220>

<221> site

<222> (1455)..(1459)

<220>

<221> site

<222> (1463)..(1467)

<220>

<221> site

<222> (1471)..(1475)

<220>

<221> site

<222> (1479)..(1483)

<220>

<221> site

<222> (1487)..(1491)

<220>

<221> site

<222> (1495)..(1499)

<220>

<221> site

<222> (1503)..(1507)

<220>

<221> site

<222> (1444)..(1507)

<220>

<221> site

<222> (1511)..(1520)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (1527)..(1531)

<220>

<221> site

<222> (1535)..(1539)

<220>

<221> site

<222> (1543)..(1547)

<220>

<221> site

<222> (1551)..(1555)

<220>

<221> site

<222> (1559)..(1563)

<220>

<221> site

<222> (1567)..(1571)

<220>

<221> site

<222> (1575)..(1579)

<220>

<221> site

<222> (1583)..(1587)

<220>

<221> site

<222> (1524)..(1587)

<220>

<221> site

<222> (1591)..(1600)

<223> this sequence can cover 6-10 residues

<220>

<221> site

<222> (1)..(1600)

<223> this sequence can encompass 2-20 "GGY- [ GPG-X1] n1-GPS- (A) n2" repeat units, where X1 is "SGGQQ", "GAGQQ", "GQGPY", "AGQQ", or "SQ", n1 is 4-8 and n2 is 6-10 and some positions may not be present.

<400> 34

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

20 25 30

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

35 40 45

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

50 55 60

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

65 70 75 80

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

85 90 95

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

100 105 110

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

115 120 125

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

130 135 140

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

145 150 155 160

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

165 170 175

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

180 185 190

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

195 200 205

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

210 215 220

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

225 230 235 240

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

245 250 255

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

260 265 270

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

275 280 285

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

290 295 300

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

305 310 315 320

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

325 330 335

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

340 345 350

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

355 360 365

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

370 375 380

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

385 390 395 400

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

405 410 415

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

420 425 430

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

435 440 445

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

450 455 460

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

465 470 475 480

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

485 490 495

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

500 505 510

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

515 520 525

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

530 535 540

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

545 550 555 560

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

565 570 575

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

580 585 590

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

595 600 605

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

610 615 620

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

625 630 635 640

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

645 650 655

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

660 665 670

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

675 680 685

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

690 695 700

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

705 710 715 720

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

725 730 735

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

740 745 750

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

755 760 765

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

770 775 780

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

785 790 795 800

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

805 810 815

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

820 825 830

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

835 840 845

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

850 855 860

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

865 870 875 880

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

885 890 895

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

900 905 910

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

915 920 925

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

930 935 940

Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala

945 950 955 960

Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

965 970 975

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

980 985 990

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

995 1000 1005

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa

1010 1015 1020

Xaa Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala

1025 1030 1035

Ala Ala Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro

1040 1045 1050

Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly

1055 1060 1065

Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa

1070 1075 1080

Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa

1085 1090 1095

Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala

1100 1105 1110

Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Xaa Xaa

1115 1120 1125

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa

1130 1135 1140

Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly

1145 1150 1155

Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro

1160 1165 1170

Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly

1175 1180 1185

Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr

1190 1195 1200

Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa

1205 1210 1215

Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa

1220 1225 1230

Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

1235 1240 1245

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa

1250 1255 1260

Xaa Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala

1265 1270 1275

Ala Ala Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro

1280 1285 1290

Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly

1295 1300 1305

Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa

1310 1315 1320

Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa

1325 1330 1335

Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala

1340 1345 1350

Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr Gly Pro Gly Xaa Xaa

1355 1360 1365

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa

1370 1375 1380

Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly

1385 1390 1395

Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro

1400 1405 1410

Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly

1415 1420 1425

Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala Ala Ala Gly Gly Tyr

1430 1435 1440

Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa

1445 1450 1455

Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa

1460 1465 1470

Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa

1475 1480 1485

Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa

1490 1495 1500

Xaa Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala Ala Ala Ala Ala Ala

1505 1510 1515

Ala Ala Gly Gly Tyr Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro

1520 1525 1530

Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly

1535 1540 1545

Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa

1550 1555 1560

Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Gly Xaa Xaa Xaa Xaa

1565 1570 1575

Xaa Gly Pro Gly Xaa Xaa Xaa Xaa Xaa Gly Pro Ser Ala Ala Ala

1580 1585 1590

Ala Ala Ala Ala Ala Ala Ala

1595 1600

<210> 35

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

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Ser Gly Gly Gln Gln

1 5

<210> 36

<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

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Gly Ala Gly Gln Gln

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<211> 5

<212> PRT

<213> Artificial sequence

<220>

<223> description of artificial sequences: synthetic peptides

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Gly Gln Gly Pro Tyr

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Ala Gly Gln Gln

1

Claims

1. A method of making a shaped body comprising:

a. providing a composition comprising recombinant filaments and a plasticizer, wherein the composition is in a flowable state;

b. placing the composition in a mold;

c. applying heat and pressure to the composition in the mold; and

d. cooling the composition to form a shaped body comprising the reconstituted filaments.

2. The method of claim 1, wherein the shaped body is in solid form.

3. The method of claim 1, wherein the shaped body is a film.

4. The method of claim 1, wherein the reconstituted silk is reconstituted silk powder dispersed in the plasticizer.

5. The method of claim 1, wherein, prior to forming, the crystallinity of the reconstituted filament is similar to or less than the crystallinity of 18B.

6. The method of claim 1, wherein the recombinant silk protein is human facial spider penis or large web spider silk.

7. The method of claim 1, wherein the recombinant silk is 18B.

8. The method of claim 1, wherein the recombinant silk comprises SEQ ID NO 1.

9. The method of claim 1, wherein the plasticizer is selected from the group consisting of: triethanolamine, propylene glycol or propylene glycol.

10. The method of claim 1, wherein the composition comprises 15% by weight propylene glycol.

11. The method of claim 1, wherein the plasticizer comprises 10-50% by weight of the composition.

12. The method of claim 1, wherein the heat is applied at a temperature of 130 ℃.

13. The method of claim 1, wherein the pressure is applied in the range of 1,500 to 15,000psi.

14. The method of claim 1, wherein the shaped body has a hardness of 100 as measured by a type a durometer.

15. The method of claim 1, wherein the molded body has a hardness of 90 or greater as measured by a type a durometer.

16. The method according to claim 1, wherein the molded body has a hardness of 50 or more, 60 or more, or 70 or more as measured by a type D durometer.

17. The method of claim 1, wherein the shaped body can be machined, cut or drilled and retain its desired shape.

18. The method of claim 1, wherein the shaped body has at least 50%, 60%, 70%, 80%, or 90% full length 18B monomer as compared to a reconstituted filament of the composition in the flowable state.

19. The method of claim 1, wherein the shaped body has at least 35%, at least 40%, at least 45%, or at least 50% full length recombinant silk monomers.

20. The method of claim 1, wherein the shaped body has at least 50% total combined weight of recombinant silk monomers, recombinant silk aggregates, and high molecular weight intermediates.

21. The method of claim 1, wherein the heat and pressure are applied for minutes, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 10 minutes, or 15 minutes.

22. The method of claim 1, wherein the heat and pressure are applied for 5 to 8 minutes.

23. The method of claim 1, further comprising exposing the shaped body to a relative humidity of at least 50% for at least 24 hours.

24. The method of claim 1, further comprising exposing the shaped body to a relative humidity of 65% for 72 hours.

25. The method of claim 1, wherein the pressure is applied with a pressing load of at least 1 metric ton, at least 2 metric ton, at least 3 metric ton, at least 4 metric ton, or at least 5 metric ton.

26. The method of claim 1, wherein the pressure is applied with a pressing load of 1 to 5 metric tons, or 3 to 5 metric tons.

27. The method of claim 1, wherein the cooling is performed at a rate of about 1 ℃/min, about 3 ℃/min, or about 45 ℃/min.

28. The method of claim 1, wherein the composition has a flexural modulus of 50MPa or greater, 60MPa or greater, 70MPa or greater, 80MPa or greater, 90MPa or greater, 100MPa or greater, 150MPa or greater, 200MPa or greater, 250MPa or greater, or 300MPa or greater.

29. The method of claim 1, wherein the composition has a maximum flexural strength of 10MPa or greater, 20MPa or greater, 30MPa or greater, 40MPa or greater, 50MPa or greater, 60MPa or greater, 70MPa or greater, 80MPa or greater, 90MPa or greater, or 100MPa or greater.

30. The method of claim 1, wherein the composition has an elongation at break of 1 to 4%.

31. The method of claim 1, wherein the composition has an elongation at break of greater than 20%.

32. The method of claim 1, wherein the composition further comprises ammonium persulfate.

33. The method of claim 1, further comprising immersing the shaped body in ammonium persulfate.

34. The method of claim 1, wherein the shaped body is crosslinked.

35. The method according to claim 1, wherein the shaped body is a cosmetic or skin care preparation.

36. A composition comprising recombinant silk and a plasticizer, wherein the composition is in solid form.

37. The composition of claim 36, wherein the shaped body is in solid form.

38. The composition of claim 36, wherein the shaped body is a film.

39. The composition of claim 36, wherein the reconstituted silk is reconstituted silk powder dispersed in the plasticizer.

40. The composition of claim 36, wherein the recombinant silk is 18B.

41. The composition of claim 36, wherein the recombinant silk comprises SEQ ID No. 1.

42. The composition of claim 36, wherein the plasticizer is selected from the group consisting of: triethanolamine, propylene glycol or propylene glycol.

43. The composition of claim 36, wherein the composition comprises 15% by weight propylene glycol.

44. The composition of claim 36, wherein the plasticizer comprises 10-50% by weight of the composition.

45. The composition of claim 36, wherein the shaped body has a hardness of 100 as measured by a type a durometer.

46. The composition of claim 36, wherein the shaped body has a hardness of 90 or greater as measured by a type a durometer.

47. The composition of claim 36, wherein the shaped body has a hardness of 50 or more, 60 or more, or 70 or more as measured by a type D durometer.

48. The composition of claim 36, wherein the shaped body can be machined, cut or drilled and retain its desired shape.

49. The composition of claim 36, wherein the shaped body has at least 50%, 60%, 70%, 80%, or 90% full length 18B monomer as compared to a reconstituted filament of the composition in the flowable state.

50. The composition of claim 36, wherein the shaped body has at least 35%, at least 40%, at least 45%, or at least 50% full length recombinant silk monomers.

51. The composition of claim 36, wherein the shaped body has at least 50% total combined weight of recombinant silk monomers, recombinant silk aggregates, and high molecular weight intermediates.

52. The composition of claim 36, wherein the composition has a flexural modulus of 50MPa or greater, 60MPa or greater, 70MPa or greater, 80MPa or greater, 90MPa or greater, 100MPa or greater, 150MPa or greater, 200MPa or greater, 250MPa or greater, or 300MPa or greater.

53. The composition of claim 36, wherein the composition has a maximum flexural strength of 10MPa or greater, 20MPa or greater, 30MPa or greater, 40MPa or greater, 50MPa or greater, 60MPa or greater, 70MPa or greater, 80MPa or greater, 90MPa or greater, or 100MPa or greater.

54. The composition of claim 36, wherein the composition has an elongation at break of 1 to 4%.

55. The composition of claim 36, wherein the composition has an elongation at break of greater than 20%.

56. The composition of claim 36, wherein the composition further comprises ammonium persulfate.

57. The composition of claim 36, wherein the shaped body is crosslinked.

58. The composition of claim 36, wherein the shaped body is a cosmetic or skin care formulation.