WO2013120143A1

WO2013120143A1 - Method of promoting the formation of cross-links between coiled coil silk proteins

Info

Publication number: WO2013120143A1
Application number: PCT/AU2013/000136
Authority: WO
Inventors: Tara Sutherland; John Ramshaw; Michael HUSON; Jeffrey CHURCH; Andrew WARDEN
Original assignee: Commonwealth Scientific And Industrial Research Organisation
Priority date: 2012-02-17
Filing date: 2013-02-15
Publication date: 2013-08-22

Abstract

The present invention relates to the use of dry heat to form cross-links between coiled coil silk proteins in a material comprising the silk proteins. The process produces material with improved toughness and/or solvent stability. The material produced can be used for a variety of purposes such as in the production of personal care products, plastics, textiles, and biomedical products.

Description

METHOD OF PROMOTING THE FORMATION OF CROSS-LINKS BETWEEN COILED COIL SILK PROTEINS

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

The silk produced by honeybees shares many desirable properties with the better known silkworm or spider silks, but has a much more varied amino acid composition giving it a different surface chemistry and a niche for different applications (Sutherland et al., 2006). Natural honeybee silk is composed of four related fibrous proteins (Sutherland et al., 2006) which assemble into a coiled coil structure (Rudall et al., 1962). Honeybee silk proteins can be produced recombinantly in E. coli at high yield (Shi et al., 2008; Weisman et al., 2010) and the four proteins have been shown to assemble to form recombinant protein fibers (Weisman et al., 2010). It has also been shown that a single recombinant honeybee protein will self-assemble to mimic the coiled coil protein structure and mechanical properties of natural honeybee silk (Sutherland et al., 2011).

Recombinant honeybee silk can be manufactured into multiple material forms including fibres and films (Weisman et al., 2010; Sutherland et al., 2011), electrospun mats (Wittmer et al., 2011) and sponges (WO 2011/022771). All forms require post- manufacture treatment of some description to render them water insensitive.

Immersion in methanol (Magoshi et al., 1974; Nazarov et al., 2004; Tsukada et al., 1994) is a common stabilization treatment for regenerated silkworm fibroin materials, resulting in structural change from amorphous or helical conformations to crystalline β- sheet. More recently, stabilization has been effected in regenerated silks by water annealing (Hu et al., 2011). Similarly, recombinant honeybee silk has been stabilized by immersion in 90% methanol solution (Weisman et al., 2010), immersion in 70% methanol solution (Sutherland et al., 2011) and by water annealing (Wittmer et al., 2011), all of which were reported to cause partial structural transition from coiled coil towards β-sheet structure to different degrees. Changes to mechanical properties of honeybee or hornet silk materials due to post-manufacture treatment have not been investigated.

There is a need for further methods to produce silk with suitable toughness and/or solvent stability from recombinantly expressed coiled-coil silk proteins which can be used to manufacture a wide variety of products.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that dry heat can be used to promote the formation of cross-links between coiled coil silk proteins which results in an increase in the toughness and/or solvent stability of material comprising the silk proteins.

Thus, in a first aspect the present invention provides a process for forming cross-links between coiled coil silk proteins in a material comprising the silk proteins, the process comprising

i) obtaining the material in a solid state, and

ii) dry heating the material to at least about 100°C for a sufficient time for the cross-links to form.

In an embodiment, the material is heated to no greater than about 220°C.

In a particularly preferred embodiment, following the process the material has an increased toughness and/or solvent stability when compared to the material before step ii).

In one embodiment, step ii) is conducted under conditions which promote drying of the material. Examples of such conditions include, but are not limited to, a vacuum and/or the presence of a desiccant. In an embodiment, step ii) is performed for at least about 10 hours, more preferably step ii) is performed for about 10 hours to about 100 hours. In a further embodiment, the temperature used in step ii) is between about 100°C and about 150°C, or between about 100°C and about 120°C.

In an alternate embodiment, the temperature used in step ii) is at least about 190°C. In this embodiment, there is no need for the process to be conducted under conditions which promote drying of the material. In an embodiment, the temperature used in step ii) is between about 190°C and about 220°C, or between about 190°C and about 200°C. In an embodiment, step ii) is performed for at least about 20 minutes, or for about 20 minutes to about 120 minutes, or for about 30 minutes to about 60 minutes.

If the material is not sufficiently solid and/or not in a suitably dry state when obtained, the process may further comprise reducing the concentration of water in a material or solution comprising the silk proteins before step ii). In one embodiment, the material or solution is frozen and then freeze-dried. In an alternate embodiment, the silk proteins are precipitated.

In an embodiment, before step ii) the material has a H₂0 content of about 1% to about 10%. In another embodiment, before step ii) the material has a H₂0 content of less than about 5% or less than about 1%.

The process of the invention can be combined with other procedures such as treatment with an alcohol. Thus, in an embodiment, the process further comprises treating the material with a solution comprising at least about 60% alcohol before or after step ii), preferably before. In an embodiment, the solution comprises about 60% to about 80% alcohol. Any suitable alcohol can be used, with preferred examples including methanol and ethanol. In an embodiment, the material is treated for about 24 to about 48 hours.

The present inventors were particularly surprised that the process of the invention did not noticeably disrupt the coiled coil structure of the silk proteins. Thus, in a preferred embodiment, following step ii) silk proteins in the material have a coiled coil structure. In this embodiment, it is preferred that the silk proteins have not been treated with an alcohol before being heated.

In an embodiment, silk proteins have a β-sheet structure. In an alternate embodiment, silk proteins do not have a β-sheet structure. A β-sheet structure is typically present if the material has been heated at higher temperatures such as between about 210°C and about 220°C.

The material can be in any suitable form such as but not limited to, a sponge, particle, fiber or film.

In an embodiment, following the process the material has at least a 75%, or at least a 90% or at least a 100%, reduction in water solubility when compared to the material before step ii).

In another embodiment, following the process the material has at least a 75%, or at least a 90% or at least a 100%, reduction in SDS solubility when compared to the material before step ii).

In a further embodiment, following the process the material has at least about a 10%, at least about a 20%, or at least about a 30%, or at least about a 40%, or at least about a 50%, or at least about a 60%, or at least about a 70%, or at least about a 80%, or at least about a 90%, or about a 100%, increase in solvent stability when compared to the material before step ii). In another embodiment, following the process the material has at least about a 10%, at least about a 20%, or at least about a 30%, or at least about a 40%, or at least about a 50%, or at least about a 60%, or at least about a 70%, or at least about a 80%, or at least about a 90%, or about a 100%, increase in toughness when compared to the material before step ii).

Also provided is a product comprising material produced by the process of the invention.

In another aspect, the present invention provides a solid material comprising coiled coil silk proteins which is insoluble in one or more of 2% SDS, 8M urea or 6M guanadium.

In yet a further aspect, the present invention a solid material comprising coiled coil silk proteins, wherein each silk protein is cross-linked to at least two other silk proteins by an amide and/or ester crosslink.

In a preferred embodiment, the cross-links are amide crosslinks.

In an embodiment, each silk protein is cross-linked to at least two other silk proteins by between about 2 and about 20 cross-links, or between about 2 and about 10 cross-links or between about 2 and about 5 cross-links.

Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.

The present invention is not to be limited in scope by the specific embodiments described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.

Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.

The invention is hereinafter described by way of the following non-limiting Examples and with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE ACCOMPANYING DRAWINGS

Figure 1. Representative 1^st cyclic compression curves of scaffolds from collagen (left) and honey bee silk stabilized by heat treating for 120 min at 190°C (middle) or by immersion in 80% methanol for 48h (right). Figure 2. Representative i ^sl compression curves of honey bee silk scaffolds stabilized by heat treating at 190 °C for 10 min, 30 min, 60 min or 120 min (left to right).

Figure 3. Change in stiffness (triangles, stress at 50% strain) and energy (squares) to compress the honey bee silk scaffolds, as a function of time of heat treatment at 190°C.

Figure 4. Compression tests of heat- or methanol-treated honeybee (HB) silk pTOtein sponges, compared to stabilized collagen sponges. Sponge toughness measured as energy required to compress the sponge to 0.2 N, with three compression and decompression cycles separated by one minute relaxation times.

Figure 5. Raman spectra of honeybee silk sponges. A: Sponges immersed for 48 hours in aqueous methanol solutions containing 60 - 100% methanol. B: Sponges treated by heating at different temperatures for periods of 10 - 120 min. Insets in both panels show the crude ratio of Raman absorbance at 1655 cm^'1 (attributed to coiled coil protein structure) to absorbance at 1666 em^{' 1} (attributed to beta-sheet protein structure) for different samples.

Figure 6. Amide 1 2^nd derivative band overlap analysis of honeybee scaffolds treated with A: 60, B: 80, C: 90 and D: 100% methanol / water for 48 h. Untreated scaffold (black trace), treated scaffold (red trace) and overlap area (shaded region).

Figure 7. Amide I 2^nd derivative band overlap analysis of honeybee scaffolds heat treated at A: 160, B: 180 and C: 190°C for different lengths of time D: 10 minutes, E: 30 minutes, E: 60 minutes and F: 120 minutes. Untreated scaffold (black trace), treated scaffold (red trace) and overlap area (shaded region).

Figure 8v . Amino acid analysis comparing a control untreated honeybee silk sponge (black bars) to a sponge sample heat-treated at 190°C for 60 m (white bars).

Figure 9. Effects of heating with and without vacuum.

Figure 10. Graphic of fibre production set up. Protein solution is injected vertically (via a 30G needle, at 0.06ml/min). into a 90 MeOH bath. The fibre is transported along the coagulation bath, exits and continues over the transfer roller where it is dried. The fibre continues into the rehydration bath of 70% MeOH and over the 3 draw rollers, (running at 2x the speed of the previous roller, total fibre draw 4x original) to

RECTIFIED SHEETS

(Rule 91) ISA AU draw the fibre. The fibre is dried on exiting the bath and collected onto a roller. The fibre continuously travels -4.7 m from the point of injection to the final collection roller. Approximately 200 m of fibre can be collected per hour. Figure 11. Heat treated continuously produced silk fibre on knitting machine.

Figure 12. Heat treated continuously produced silk fibre after knitting on knitting machine. Figure 13. Scanning electron image of fibre knotted when wet.

Figure 14. Enzymatic amino acid analysis of heat treated material. A: Amino acid analysis results obtained by enzymatic digestion, comparing a control (methanol treated) honeybee silk sponge sample to a methanol treated sponge sample heat treated at 190°C for 60 minutes. B: Mole percentage difference for the amino acids lysine, asparagine, aspartic acid, glutamic acid and serine, obtained by enzymatic digestion comparing a control untreated honeybee silk sponge to a sponge sample heat treated at 190°C for 60 minutes. Figure 15. LC-MS/MS detection of the isopeptide crosslink, e-(y-glutamyl)-lysine. A: Standard, B: Heat-Treated and C: Non-Heat Treated.

Figure 16. Comparative behavior of fibres in SDS detergent (Panel A), guanidinium denaturant (Panel B) and stress required to break the fibre (Panel C) after no treatment, various times at 120°C in a vacuum oven, or heat treatment ( 180°C for 1 hr).

KEY TO THE SEQUENCE LISTING

SEQ ID NO.l - Honeybee silk protein termed herein Xenospiral (also termed herein AmelFl) (minus signal peptide).

SEQ ID NO:2 - Honeybee silk protein termed herein Xenospira2 (also termed herein AmelF2) (minus signal peptide).

SEQ ID NO:3 - Honeybee silk protein termed herein Xenospira3 (also termed herein AmelF3) (minus signal peptide).

SEQ ID NO:4 - Honeybee silk protein termed herein Xenospira4 (also termed herein AmelF4) (minus signal peptide).

SEQ ID NO:5 - Bumblebee silk protein termed herein BBF1 (minus signal peptide). SEQ ID NO: 6 - Bumblebee silk protein termed herein BBF2 (minus signal peptide). SEQ ID NO:7 - Bumblebee silk protein termed herein BBF3 (minus signal peptide).

SEQ ID NO:8 - Bumblebee silk protein termed herein BBF4 (minus signal peptide).

SEQ ID NO:9 - Bulldog ant silk protein termed herein BAF1 (minus signal peptide).

SEQ ID NO: 10 - Bulldog ant silk protein termed herein BAF2 (minus signal peptide). SEQ ID NO: 11 - Bulldog ant silk protein termed herein BAF3 (minus signal peptide).

SEQ ID NO: 12 - Bulldog ant silk protein termed herein BAF4 (minus signal peptide).

SEQ ID NO: 13 - Weaver ant silk protein termed herein GAF1 (minus signal peptide).

SEQ ID NO: 14 - Weaver ant silk protein termed herein GAF2 (minus signal peptide).

SEQ ID NO: 15 - Weaver ant silk protein termed herein GAF3 (minus signal peptide). SEQ ID NO: 16 - Weaver ant silk protein termed herein GAF4 (minus signal peptide).

SEQ ID NO: 17 - Hornet silk protein termed herein Vssilk3 (minus signal peptide).

SEQ ID NO: 18 - Hornet silk protein termed herein Vssilk4 (minus signal peptide).

SEQ ID NO: 19 - Hornet silk protein termed herein Vssilk2 (minus signal peptide).

SEQ ID NO:20 - Hornet silk protein termed herein Vssilkl (minus signal peptide). SEQ ID NO:21 - Asiatic honeybee silk protein termed silk protein 1 (also termed

ABS1) (minus signal peptide).

SEQ ID NO:22 - Asiatic honeybee silk protein termed silk protein 2 (also termed ABS2) (minus signal peptide).

SEQ ID NO:23 - Asiatic honeybee silk protein termed silk protein 3 (also termed ABS3) (minus signal peptide).

SEQ ID NO:24 - Asiatic honeybee silk protein termed silk protein 4 (also termed ABS4) (minus signal peptide).

SEQ ID NO:25 - Lacewing silk protein termed herein MalFl (minus signal peptide). SEQ ID NO:26 - Amino acid sequence of the mature form of Tenodera australasiae protein Mantis Fibroin 1.

SEQ ID NO:27 - Amino acid sequence of the mature form of Tenodera australasiae protein Mantis Fibroin 2b.

SEQ ID NO:28 - Amino acid sequence of the mature form of Archimantis monstrosa protein Mantis Fibroin la.

SEQ ID NO:29 - Amino acid sequence of the mature form of Archimantis monstrosa protein Mantis Fibroin 2.

SEQ ID NO:30 - Amino acid sequence of the mature form of Pseudomantis albofimbriata protein Mantis Fibroin 1.

SEQ ID NO:31 - Amino acid sequence of the mature form of Pseudomantis albofimbriata protein Mantis Fibroin 2b. SEQ ID NO:32 - Nucleotide sequence encoding honeybee silk protein Xenospiral (minus region encoding signal peptide).

SEQ ID NO: 33 - Nucleotide sequence encoding honeybee silk protein Xenospira2 (minus region encoding signal peptide).

SEQ ID NO:34 - Nucleotide sequence encoding honeybee silk protein Xenospira3 (minus region encoding signal peptide).

SEQ ID NO: 35 - Nucleotide sequence encoding honeybee silk protein Xenospira4 (minus region encoding signal peptide).

SEQ ID NO:36 - Nucleotide sequence encoding bumblebee silk protein BBF1 (minus region encoding signal peptide).

SEQ ID NO:37 - Nucleotide sequence encoding bumblebee silk protein BBF2 (minus region encoding signal peptide).

SEQ ID NO:38 - Nucleotide sequence encoding bumblebee silk protein BBF3 (minus region encoding signal peptide).

SEQ ID NO:39 - Nucleotide sequence encoding bumblebee silk protein BBF4 (minus region encoding signal peptide).

SEQ ID NO:40 - Nucleotide sequence encoding bulldog ant silk protein BAF1 (minus region encoding signal peptide).

SEQ ID NO:41 - Nucleotide sequence encoding bulldog ant silk protein BAF2 (minus region encoding signal peptide).

SEQ ID NO:42 - Nucleotide sequence encoding bulldog ant silk protein BAF3 (minus region encoding signal peptide).

SEQ ID NO:43 - Nucleotide sequence encoding bulldog ant silk protein BAF4 (minus region encoding signal peptide).

SEQ ID NO:44 - Nucleotide sequence encoding weaver ant silk protein GAF1 (minus region encoding signal peptide).

SEQ ID NO:45 - Nucleotide sequence encoding weaver ant silk protein GAF2 (minus region encoding signal peptide).

SEQ ID NO:46 - Nucleotide sequence encoding weaver ant silk protein GAF3 (minus region encoding signal peptide).

SEQ ID NO:47 - Nucleotide sequence encoding weaver ant silk protein GAF4 (minus region encoding signal peptide).

SEQ ID NO:48 - Nucleotide sequence encoding hornet silk protein Vssilk3 (minus region encoding signal peptide).

SEQ ID NO:49 - Nucleotide sequence encoding hornet silk protein Vssilk4 (minus region encoding signal peptide). SEQ ID NO:50 - Nucleotide sequence encoding hornet silk protein Vssilk2 (minus region encoding signal peptide).

SEQ ID NO:51 - Nucleotide sequence encoding hornet silk protein Vssilkl (minus region encoding signal peptide).

SEQ ID NO:52 - Nucleotide sequence encoding asiatic honeybee silk protein ABS1 (minus region encoding signal peptide).

SEQ ID NO:53 - Nucleotide sequence encoding asiatic honeybee silk protein ABS2 (minus region encoding signal peptide).

SEQ ID NO:54 - Nucleotide sequence encoding asiatic honeybee silk protein ABS3 (minus region encoding signal peptide).

SEQ ID NO:55 - Nucleotide sequence encoding asiatic honeybee silk protein ABS4 (minus region encoding signal peptide).

SEQ ID NO:56 - Nucleotide sequence encoding lacewing silk protein MalFl (minus region encoding signal peptide).

SEQ ID NO:57 - Nucleotide sequence encoding the mature form of Tenodera australasiae protein Mantis Fibroin 1.

SEQ ID NO:58 - Nucleotide sequence encoding the mature form of Tenodera australasiae protein Mantis Fibroin 2b.

SEQ ID NO: 59 - Nucleotide sequence encoding the mature form of Archimantis monstrosa protein Mantis Fibroin la.

SEQ ID NO:60 - Nucleotide sequence encoding the mature form of Archimantis monstrosa protein Mantis Fibroin 2.

SEQ ID NO:61 - Nucleotide sequence encoding the mature form of Pseudomantis albofimbriata protein Mantis Fibroin 1.

SEQ ID NO:62 - Nucleotide sequence encoding the mature form of Pseudomantis albofimbriata protein Mantis Fibroin 2b.

DETAILED DESCRIPTION OF THE INVENTION

General Techniques and Definitions

Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in silk protein processing, protein chemistry, cell culture, molecular genetics, and biochemistry).

Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in the present invention are standard procedures, well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al. (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbour Laboratory, (1988), and J.E. Coligan et al. (editors) Current Protocols in Immunology, John Wiley & Sons (including all updates until present).

The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.

As used herein, the term about, unless stated to the contrary, refers to +/- 20%, more preferably +/- 10%, even more preferably +/- 5%, of the designated value.

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

As used herein, the terms "silk protein" and "silk polypeptide" refer to a fibrous protein/polypeptide that can be used to produce materials such as silk fibre, silk film, silk sponges, silk particles and/or a fibrous silk protein complex. Typically, the silk proteins will be produced by recombinant expression. However, the silk proteins can be produced artificially such, for example, using a protein synthesizer. Silk proteins may have a sequence corresponding to a naturally occurring silk protein (for example honeybee silk proteins described herein) or be a man made variant thereof. Such variants not only include small substitutions, deletions and additions, but also encompass significant rearrangement of the native sequences where, for example, heptads are re-ordered so they bear no resemblance to the primary amino acid sequence of the native protein but because of the heptad structure are still functional silk proteins.

As used herein, a "silk fibre" refers to filaments comprising silk proteins which can be woven into various items such as textiles.

As used herein, the term "toughness" refers to the energy required to break the material. As used herein, the term "solvent stability" refers the ability of the material to stay insoluble in solvents such as water, SDS (for example 2% SDS), guanadium (for example 8M guanadium) or urea (for example 8M urea). As used herein, "stay insoluble in solvent" means that the material losses than less than 10% of its protein mass after 24 hours at room temperature in the solvent.

The term "signal peptide", "N-terminal signal sequence" and variations thereof refers to an amino terminal protein/pep tide preceding a secreted mature protein. The signal peptide is cleaved from and is therefore not present in the mature protein. Signal peptides have the function of directing and trans -locating secreted proteins across cell membranes. The signal peptide is also referred to as signal sequence, and are well known in the art.

Increasing Toughness and/or Solvent Stability of Material Comprising Coiled-Coil Silk Proteins

Dry heat treatment of amorphous or helical regenerated silkworm silk materials to above their T_g (~ 190°C) drives formation of thermally- induced β-sheet crystals (Magoshi et al., 1977). Similarly, in regenerated tussah silk heated to 230°C the random coil structure changes to β-sheet, although a-helix content remains fairly constant (Kweon et al., 2001). Regenerated collagen sponges and fibres are commonly stabilized by a form of heat curing involving heating the material under vacuum to temperatures of 100-120°C for several days (Yannas and Tobolsky, 1967). Heat curing of collagen causes degradation of the collagen, with protein fragmentation increasing with increased temperatures (Gorham et al., 1992). β-sheet structure in coiled coil silk materials has been induced by dry heating to 215°C which is well above the protein's glass transition temperature (Sutherland et al., 2011), however, increased mechanical strength was not observed through lack of cross-links which in hindsight was due to material not having been heated for a sufficient length of time. In contrast, the present inventors have found that exposing material comprising honeybee silk proteins, and/or related coiled coil silk proteins, to high levels of dry heat for a sufficient time promotes the formation of cross-links which confers increased the toughness and/or solvent stability to the heated material.

As the skilled person would appreciate, "dry heating" does not necessarily mean that no moisture be present. For instance, dry heating is often performed under normal room humidity conditions such as about 20% to about 80% humidity, or about 30% to about 50% humidity. The processes of the invention rely on heat treatment, the moisture content of the material before heating, and whether the heating step is performed under drying conditions (and the nature of the drying conditions). In light of the teachings herein, a suitable combination of these parameters can readily be determined using standard procedures. If there is any doubt, the benefits of the invention can readily be achieved by numerous means such as freeze-drying the material and heating to about 190°C for about 30 minutes, or by heating the material to about 100°C under a vacuum (such as that generated by standard laboratory equipment) for about 48 hours.

The material which is heated is in a solid state as too much water will have the effect of boiling the silk proteins. As used herein, "solid state" does not mean that there is absolutely no water in the material at, for example, room temperature or when frozen. In an embodiment, the starting material has a H₂0 content of about 1% to about 10%. In an embodiment, the starting material has a H₂0 content of less than about 5% or less than about 1%.

Performing the method under conditions which promote drying counters the above-mentioned boiling effect. Thus, when performed under conditions which promote drying the moisture content of the heated (for example heated to about 100°C to about 120°C) material can be higher than material with a low water content which is heated above 190°C. For example, the closer the vacuum (when used as a drying condition) is to a perfect vacuum the higher the H₂0 content can be.

In one embodiment, the heating is performed in the presence of a vacuum. Broadly, a vacuum is a region with a gaseous pressure much less than atmospheric pressure. The quality of a partial vacuum refers to how closely it approaches a perfect vacuum. Ultra-high vacuum chambers, common in chemistry, physics, and engineering, operate below one trillionth (10 ) of atmospheric pressure (100 nPa), and can reach around 100 particles/cm .

In an embodiment, the material is heated in a vacuum to about 100°C. Such a vacuum will probably not be a perfect vacuum. If the vacuum is not particularly strong, and/or a desiccant is not present, it may be necessary to increase the heat, for example to about 120°C to about 150°C. A specific combination of degree of vacuum (pressure) and temperature can readily be determined by the skilled person in view of the present teachings.

In another embodiment, the heating is performed in the presence of a desiccant (possibly also in the presence of a vaccum). Desiccants are well known to the skilled artisan and are commercially available and include, but are not limited to, silica gel, calcium sulfate, and calcium chloride. If the moisture content of the material which has been obtained is too high (for example the silk proteins are in solution), this can be reduced by drying the material using techniques such as, but not limited to, freeze-drying or precipitation (also known as coagulation).

Freeze-drying is also referred to in the art as, for example, lyophilization or cryodesiccation. Freeze-drying is achieved by freezing the material and then reducing the surrounding pressure to allow the frozen water in the material to sublimate directly from the solid phase to the gas phase. Examples of equipment that can be used to freeze-dry the material include a manifold freeze-dryer, a rotary freeze-dryer and a tray style freeze-dryer. This equipment typically comprises a vacuum pump to reduce the ambient gas pressure in a vessel containing the material and a condenser to remove the moisture by condensation on a surface cooled.

In an embodiment, the material or solution is frozen at about -20°C. In an embodiment, the frozen material or solution is freeze-dried for about 12 to about 48 hours. In a further embodiment, the frozen material or solution is freeze-dried for about 24 hours.

With regard to precipitation (coagulation), this term refers to converting the starting material (composition comprising silk proteins) from a fluid to a solid state. The material can be precipitated by a variety of techniques such as, but not limited to, the addition of an alcohol or a salt (salting out using, for example, using fluoride, sulfate, hydrogen phosphate, acetate, chloride, nitrate, bromide, chlorate, perchlorate, thiocyanate, ammonium, potassium, sodium, lithium, magnesium, calcium or guanidinium) to a solution comprising the silk proteins, or by reducing the pH of the solution to at least about 5.5, preferably at least about 4.5, or a combination of two or more thereof. In one embodiment, the silk proteins are precipitated in a solution comprising alcohol, the precipitate collected, air dried and used in step ii). Any suitable alcohol can be used, with preferred examples including methanol and ethanol.

The material can be dry heated using any suitable means known in the art. Examples include, but are not limited to, using an oven, a heat lamp or heat block. As the skilled person would appreciate, dry heat excludes processes which occur in high humidity such as autoclaving.

The present invention results in a substantial degree of cross-linking between individual silk protein molecules. The cross-linking appears to be a result of the presence of large number of amino acids in the silk proteins with the potential to form cross-links and the presence of these residues on the surface of the proteins, hence available to form cross-links, when in a coiled coil form. In an embodiment, the cross links are amide cross-links between one or more of glutamine, glutamic acid and aspartic acid residues, and/or ester cross-links between threonine and/or serine with glutamic acid and/or aspartic acid. In an embodiment, there are about two Lys-Glu isopeptide links on average between individual protein molecules.

To the inventor's surprise, the process of the invention results in improved properties of the material, at least an increased toughness and/or solvent stability. The extent of improvement depends on the nature of the material before heating. For example, when compared to an "untreated" sponge produced in accordance with the Examples the process essentially confers stability when immersed in water. Whilst there is little improvement in water solubility when compared to material previously treated with methanol, the process of the invention essentially confers stability in a solution comprising SDS (for example 2% SDS), urea (for example 8M urea) or guanadinium (for example 6M guanadinium), whereas methanol (for example 60% methanol) treated material is soluble in SDS, urea and guanadinium. In yet a further embodiment, the process of the invention increases toughness by at least about 20%, at least about 30%, at least about 40%, at least about 50% when compared to methanol (for example 60% methanol) treated material. In an embodiment, improved toughness and/or solvent stability is assessed when compared to untreated material where the silk proteins have been allowed to associate without additional treatments such as methanol treatment, water annealing or autoclaving.

Toughness can be measured using any suitable technique known in the art. In one instance, toughness is measured by determining the area under a standard stress- strain curve. In one embodiment, material produced using the method of the invention requires energy to break of at least about 120 MJ/m 3 , or least about 130 MJ/m 3 , or least about 140 MJ/m³, or least about 150 MJ/m³, or least about 160 MJ/m³. In a further embodiment, the method results in at least about a 1.5 fold increase, or at least about a 1.75 fold increase, or at least about a 2 fold increase, in toughness (energy to break) when compared to methanol (for example 60% methanol) treated material. Coiled-Coil Silk Proteins

The terms "polypeptide" and "protein" are generally used interchangeably and refer to a single polypeptide chain which may or may not be modified by addition of non-amino acid groups. The terms "proteins" and "polypeptides" as used herein also include variants, mutants, modifications, analogous and/or derivatives of the silk proteins described herein. In a preferred embodiment, a silk protein used in the invention is only comprised of naturally occurring amino acids. The % identity of a protein is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. The query sequence is at least 50 amino acids in length, and the GAP analysis aligns the two sequences over a region of at least 50 amino acids. More preferably, the query sequence is at least 100 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 100 amino acids. Even more preferably, the query sequence is at least 250 amino acids in length and the GAP analysis aligns the two sequences over a region of at least 250 amino acids. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

As used herein a "biologically active" fragment is a portion of a protein of the invention which maintains a defined activity of the full-length protein, namely the ability to be used to produce material described herein such as silk. Biologically active fragments can be any size as long as they maintain the defined activity.

The term "consisting essentially of", or variations thereof, means that the defined amino acid sequence may have a few, such as one, two, three or four, additional amino acids compared to that defined. For example, when absent from the defined sequence an N-terminal methionine may be added. The term "consists of", or variations thereof, means that the defined sequence does not have additional or less amino acids when compared to the defined sequence, particularly at the N- and C- termini.

With regard to a defined protein, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that the protein comprises an amino acid sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO. Amino acid sequence mutants of the naturally occurring silk proteins described herein can be prepared by introducing appropriate nucleotide changes into a nucleic acid encoding the silk protein, or by in vitro synthesis of the desired protein. Such mutants include, for example, deletions, insertions or substitutions of residues within the amino acid sequence. A combination of deletion, insertion and substitution can be made to arrive at the final construct, provided that the final protein product possesses the desired characteristics.

Mutant (altered) proteins can be prepared using any technique known in the art. For example, a polynucleotide of the invention can be subjected to in vitro mutagenesis. Such in vitro mutagenesis techniques include sub-cloning the polynucleotide into a suitable vector, transforming the vector into a "mutator" strain such as the E. coli XL- 1 red (Stratagene) and propagating the transformed bacteria for a suitable number of generations. In another example, the polynucleotides encoding the silk proteins are subjected to DNA shuffling techniques as broadly described by Harayama (1998). These DNA shuffling techniques may include one or more of the open reading frames defined herein (see SEQ ID NOs 32 to 62) possibly in addition to related open reading frames, such as silk genes from Hymenopteran or Neuroptean species other than the specific species mentioned herein. Products derived from mutated/altered DNA can readily be screened using techniques described herein to determine if they can be used as silk proteins.

In designing amino acid sequence mutants, the location of the mutation site and the nature of the mutation will depend on characteristic(s) to be modified. The sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conservative amino acid choices and then with more radical selections depending upon the results achieved, (2) deleting the target residue, or (3) inserting other residues adjacent to the located site.

Amino acid sequence deletions or insertions generally range from about 1 to 15 residues, more preferably about 1 to 10 residues and typically about 1 to 5 contiguous residues.

Substitution mutants have at least one amino acid residue in the protein molecule removed and a different residue inserted in its place. The sites of greatest interest for substitutional mutagenesis include sites identified as important for function. Other sites of interest are those in which particular residues obtained from various strains or species are identical. These positions may be important for biological activity. These sites, especially those falling within a sequence of at least three other identically conserved sites, are preferably substituted in a relatively conservative manner. Such conservative substitutions are shown in Table 1 under the heading "exemplary substitutions".

Table 1. Exem lar substitutions

Coiled-coil structures of silk proteins are characterized by heptad repeats represented by the consensus sequence (abcdefg )_n. In a preferred embodiment, the portion of the protein that has a coiled-coil structure comprises at least 10 copies of the heptad sequence abcdefg, and at least 25% of the amino acids at positions a and d are alanine residues. In a preferred embodiment, the protein that has a coiled-coil structure comprises at least 12 consecutive copies, more preferably at least 15 consecutive copies, and even more preferably at least 18 consecutive copies of the heptad. In further embodiments, the protein that has a coiled-coil structure can have up to at least 28 copies of the heptad. Typically, the copies of the heptad will be tandemly repeated. However, they do not necessarily have to be perfect tandem repeats, for example, as shown in Figures 5 and 6 of WO 2007/038837 a few amino acids may be found between two heptads, or a few truncated heptads may be found (see, for example, Xenospiral in Figure 5 of WO 2007/038837).

Guidance regarding amino acid substitutions which can be made to the silk proteins which have a coiled-coil structure is provided in Figures 5 and 6, as well as Tables 6 to 10, of WO 2007/038837. Where a predicted useful amino acid substitution based on the experimental data provided herein is in anyway in conflict with the exemplary substitutions provided in Table 1 of WO 2007/038837 it is preferred that a substitution based on the experimental data is used. Further guidance can be obtained by additional alignments of, for example, the proteins provided herein. Candidates for amino acid substiutions include substituting an amino acid with a different amino acid which is present at the corresponding position of a different coiled coil silk polypeptide.

Coiled-coil structures of the silk proteins preferably have a high content of alanine residues, particularly at amino acid positions a, d and e of the heptad. However, positions b, c, f and g also have a high frequency of alanine residues. In a preferred embodiment, at least 15% of the amino acids at one or more or all of positions a, d or e of the heptads are alanine residues, more preferably at least 25%, more preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%. In a further preferred embodiment, at least 25% of the amino acids at both positions a and d of the heptads are alanine residues, more preferably at least 30%, more preferably at least 40%, and even more preferably at least 50%. Furthermore, it is preferred that at least 15% of the amino acids at positions b, c, / and g of the heptads are alanine residues, more preferably at least 20%, and even more preferably at least 25%.

Typically, the heptads will not comprise any proline or histidine residues. Furthermore, the heptads will comprise few (1 or 2), if any, phenylalanine, methionine, tyrosine, cysteine, glycine or tryptophan residues. Apart from alanine, common (for example greater than 5%, more preferably greater than 10%) amino acids in the heptads include leucine (particularly at positions b and d), serine (particularly at positions b, e and f), glutamic acid (particularly at positions c, e and f), lysine (particularly at positions b, c, d, / and g) as well as arginine at position g.

In a preferred embodiment, the heptads are determined by using the pattern recognition programs described in one or more of Delorenzi and Speed (2002), Berger et al. (1995), Wolf et al. (1997), or Lupas et al. (1997).

As the skilled addressee will appreciate, the entire silk protein does not necessarily have a coiled-coil structure. In an embodiment, about 45% to about 90%, more preferably about 55% to about 70%, and even more preferably about 60% to about 66%, of the silk protein has a coiled-coil structure.

In an embodiment, the portion of the silk proteins which comprises a coiled-coil structure comprises at least 10 copies of the heptad sequence abcdefg, and wherein at least 25% of the amino acids at positions a and d are alanine residues.

In an embodiment, the silk proteins comprise a sequence selected from:

a) an amino acid sequence as provided in any one of SEQ ID NOs 1 to 31, b) an amino acid sequence which is at least 30% identical to any one or more of

SEQ ID NOs 1 to 31, and

c) a biologically active fragment of a) or b).

In an embodiment, the material comprises a single type of silk protein. For example, each of the proteins in the material consist of an amino acid sequence provided as SEQ ID NO:3.

In an alternate embodiment, the material comprises two, three, four or more different types of silk proteins. For example, in one embodiment, the silk proteins comprise a first silk protein which comprises at least one of the following

a) an amino acid sequence as provided in any one of SEQ ID NOs 1, 5, 9, 13, 17 or 21 ;

b) an amino acid sequence which is at least 30% identical to any one or more of SEQ ID NOs 1, 5, 9, 13, 17 or 21 ; and

c) a biologically active fragment of a) or b),

a second silk protein which comprises at least one of the following

d) an amino acid sequence as provided in any one of SEQ ID NOs 2, 6, 10, 14,

18 or 22;

e) an amino acid sequence which is at least 30% identical to any one or more of SEQ ID NOs 2, 6, 10, 14, 18 or 22; and

f) a biologically active fragment of d) or e),

a third silk protein which comprises at least one of the following g) an amino acid sequence as provided in any one of SEQ ID NOs 3, 7, 11, 15,

19 or 23;

h) an amino acid sequence which is at least 30% identical to any one or more of SEQ ID NOs 3, 7, 11, 15, 19 or 23; and

i) a biologically active fragment of g) or h), and

a fourth silk protein which comprises at least one of the following

j) an amino acid sequence as provided in any one of SEQ ID NOs 4, 8, 12, 16,

20 or 24;

k) an amino acid sequence which is at least 30% identical to any one or more of SEQ ID NOs 4, 8, 12, 16, 20 or 24; and

1) a biologically active fragment of j) or k).

In a further embodiment, the material comprises approximate equimolar amounts of the first silk protein, the second silk protein, the third silk protein and the fourth silk protein.

In another embodiment, the silk proteins comprise a first silk protein which comprises at least one of the following

a) an amino acid sequence as provided in any one of SEQ ID NOs 26, 28 or 30; b) an amino acid sequence which is at least 30% identical to any one or more of SEQ ID NOs 26, 28 or 30; and

c) a biologically active fragment of a) or b), and

a second silk protein which comprises at least one of the following

d) an amino acid sequence as provided in any one of SEQ ID NOs 27, 29 or 31; e) an amino acid sequence which is at least 30% identical to any one or more of SEQ ID NOs 27, 29 or 31; and

f) a biologically active fragment of d) or e).

In a further embodiment, the material comprises approximate equimolar amounts of the first silk protein and the second silk protein.

Proteins (and polynucleotides) useful for the methods of the invention can be purified (isolated) from a wide variety of Hymenopteran and Neuropteran species. Examples of Hymenopterans include, but are not limited to, any species of the Suborder Apocrita (bees, ants and wasps), which include the following Families of insects; Chrysididae (cuckoo wasps), Formicidae (ants), Mutillidae (velvet ants), Pompilidae (spider wasps), Scoliidae, Vespidae (paper wasps, potter wasps, hornets), Agaonidae (fig wasps), Chalcididae (chalcidids), Eucharitidae (eucharitids), Eupelmidae (eupelmids), Pteromalidae (pteromalids), Evaniidae (ensign wasps), Braconidae, Ichneumonidae (ichneumons), Megachilidae, Apidae, Colletidae, Halictidae, and Melittidae (oil collecting bees). Examples of Neuropterans include species from the following insect Families: Mantispidae (see Walker et al., 2012), Chrysopidae (lacewings), Myrmeleontidae (antlions), and Ascalaphidae (owlflies).

Also included within the scope of the invention are proteins which are differentially modified during or after synthesis, e.g., by biotinylation, benzylation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. These modifications may serve to increase the stability and/or bioactivity of the protein.

Production of the Starting Material

Typically, the starting material (the material used in step ii) of the invention) is obtained from recombinant cells expressing the silk proteins. The starting material can be in any suitable form such as but not limited to, a sponge, particle, fiber or film. In an embodiment, the silk proteins constitute at least about 5%, at least about 10%, at least about 25%, at least about 50%, at least about 75%, at least about 90%, at least about 95% of the protein in the material, more preferably of the material per se. Methods for producing such material include those described in WO 2007/038837 and WO 2011/022771.

In one embodiment, the method involves removal of native cell proteins from homogenized cells/tissues/plants etc. by lowering pH and heating, followed by ammonium sulfate fractionation. Briefly, total soluble proteins are extracted by homogenizing cells/tissues/plants. Native proteins are removed by precipitation at pH 4.7 and then at 60°C. The resulting supernatant is then fractionated with ammonium sulfate at 40% saturation. The resulting protein will be of the order of 95% pure. Additional purification may be achieved with conventional gel or affinity chromatography.

In another example, cell lysates are treated with high concentrations of acid e.g. HC1 or propionic acid to reduce pH to -1-2 for 1 hour or more which will solubilise the silk proteins but precipitate other proteins.

In a further example, the silk proteins are expressed in inclusion bodies in a bacterial cell such as E. coli. The cells are lysed to liberate silk proteins produced and contained within the cells. This step can be performed by any means known in the art. For example, the cell suspension is typically centrifuged to pellet the cells and the cells resuspended into a more concentrated solution ready for lysis. Cells can be lysed, for example, by passages through a French Press, homogenized using a Polytron (Brinkman Instruments) or sonicated on ice. Alternate methods of lysing cells, such as bacterial cells, are well known to those of skill in the art (see, e.g., Sambrook et al., supra). Various kits are available for cell lysis and are well known in the art, for example the Bugbuster kit (Novagen) and the ProteaPrep kit (Protea Biosciences, Inc.).

Several protocols are suitable for purification of protein inclusion bodies. For example, purification of inclusion bodies typically involves the extraction, separation and/or purification of inclusion bodies by disruption of bacterial cells by the methods discussed above. In an embodiment, the cells are lysed, the cell membranes solubilised, and the insoluble fraction comprising the inclusion bodies is isolated for further processing.

The process of preparing the material to be heat treated may comprise producing silk dope using a surfactant (WO 2011/022771). In an embodiment, the surfactant is an anionic surfactant. Examples of anionic surfactants useful for the invention include, but are not limited to, sodium dodecyl sulfate (SDS), ammonium lauryl sulfate and other alkyl sulfate salts, sodium 1-octanesulfonate monohydrate, sodium lauroyl sarcosinate, sodium lauryl ether sulfate (SLES), sodium taurodeoxycholate hydrate, and alkyl benzene sulfonate; or a combination of two or more thereof. In a preferred embodiment, the anionic surfactant is SDS.

Any concentration of the surfactant can be used which increases the solubility of the silk proteins can be used. For example, at least about 0.1% v/v of the surfactant is used. In an embodiment, about 0.1% to about 10% v/v, more preferably, about 0.5% to about 2% v/v or about 0.5% to about 5% v/v, of the surfactant is used.

A further step may comprise reducing the amount of surfactant in solution by adding a compound which precipitates the surfactant to assist in the correct folding of the silk proteins. Any compound may be used which associates which, and reduces the solubility of, the surfactant. Examples include, but are not limited to, a salt or a carbohydrate such as a-cyclodextrin; or a combination of two or more thereof. Preferably, the salt is a potassium salt or a sodium salt. Preferably, the potassium salt is potassium chloride and the sodium salt is sodium acetate. Any concentration of the compound can be used which results in a reduction in the amount of surfactant in solution. For example, the compound is added to a final concentration of about 1 mM to about 1 M, more preferably about 40 mM to about 100 mM, or about 40 mM to 400 mM. A further step comprises separating the solution from the precipitate formed following the addition of the compound. This can be achieved by any method known in the art such as using centrifugation, for example at 16000g for 5 minutes, and removing the supernatant (solution) comprising (which is) the silk dope. Preferably, after this step the silk proteins constitute at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%, more preferably at least 97%, more preferably at least 99%, and even more preferably 100% of the protein in solution.

When secreted from a cell, the silk proteins may be recovered from the supernatant. Again, this can be achieved by any method known in the art. In one embodiment, this is achieved by contacting the supernatant with an agent which precipitates the silk proteins such as, but not limited to, ammonium sulfate, trichloroacetic acid, perchloric acid and acetone, or commercial precipitant cocktails such as PlusOne (Amersham Biosciences), or Perfect-Focus (Geno Technology Inc.).

When desired, the concentration of silk proteins in a solution, such as silk dope, can be increased. Again, the can be achieved by any method known in the art for increasing the concentration of a protein an aqueous solution. In a particularly useful embodiment, the silk dope (for example) is concentrated by dialysing against a dehydrating solution such as a solution comprising a hygroscopic polymer. Examples suitable hygroscopic polymers include, but are not limited to, polyethylene glycol (PEG), amylase, and sericin, or a combination of two or more thereof. PEG molecules are available in a range of molecular sizes and the selection of the PEG will be determined by the membrane chosen for dialysis and the rate of concentration required. Preferably, the PEG is of a molecular weight of about 8,000 to about 10,000 g/mol and has a concentration of about 25% to about 50%. In another embodiment, the concentrating step results in a solution comprising about 0.2% to 0.3 % SDS. In a further embodiment, the concentrating step results in a solution about 70% to about 90%, or about 80%, dry weigth of silk proteins.

Fibrillar aggregates will form from solutions by spontaneous self-assembly of coiled coil silk proteins when the protein concentration exceeds a critical value. The aggregates may be gathered and mechanically spun into macroscopic fibers according to the method of O'Brien et al. (I. O'Brien et al., "Design, Synthesis and Fabrication of Novel Self-Assembling Fibrillar Proteins", in Silk Polymers: Materials Science and Biotechnology, pp. 104-117, Kaplan, Adams, Farmer and Viney, eds., c. 1994 by American Chemical Society, Washington, D.C.).

By nature of the inherent coiled coil secondary structure silk proteins useful for the invention typically spontaneously form the coiled coil secondary structure upon dehydration.

Fibers may be spun from solutions having properties characteristic of a liquid crystal phase. The fiber concentration at which phase transition can occur is dependent on the composition of a protein or combination of proteins present in the solution. Phase transition, however, can be detected by monitoring the clarity and birefringence of the solution. Onset of a liquid crystal phase can be detected when the solution acquires a translucent appearance and registers birefringence when viewed through crossed polarizing filters.

In one fiber-forming technique, fibers can first be extruded from the protein solution through an orifice into methanol, until a length sufficient to be picked up by a mechanical means is produced. Then a fiber can be pulled by such mechanical means through a methanol solution, collected, and dried. Methods for drawing fibers are considered well-known in the art.

Further examples of methods which may be used for producing the starting material comprising coiled coil silk proteins are described in US 2004/0170827 and US 2005/0054830. Polynucleotides

The term "polynucleotide" is used interchangeably herein with the term "nucleic acid".

When it is preferred that as little as possible of the silk proteins is secreted from a recombinant cell, the encoded silk proteins do not comprise an N-terminal signal sequence. Examples of polynucleotides encoding such silk proteins include those comprising, more preferably consisting essentially of, even more preferably consisting of, a sequence selected from:

a) a nucleotide sequence as provided in any one of SEQ ID NOs 32 to 62, b) a nucleotide sequence which is at least 30% identical to any one or more of SEQ ID NOs 32 to 62,

c) a biologically active fragment encoding portion of a) or b).

Other embodiments of the invention rely on the expression of silk proteins with an N-terminal signal sequence, and/or the co-production (in the same or different cells) of, for example, one or more or all a first silk protein, second silk protein, third silk protein and fourth silk protein as defined herein. Based on the sequence information provided in the Sequence Listing, the skilled person could readily identifying representative polynucleotides for expression for each embodiment of the invention. Examples of suitable signal segments include, but are not limited to, tissue plasminogen activator (t-PA), interferon, interleukin, growth hormone, viral envelope glycoprotein signal segments, Nicotiana nectarin signal peptide (US 5,939,288), tobacco extensin signal, the soy oleosin oil body binding protein signal, Arabidopsis thaliana vacuolar basic chitinase signal peptide, as well as native signal sequences of the silk polypeptides defined herein and described in WO 2007/038837.

The % identity of a polynucleotide is determined by GAP (Needleman and Wunsch, 1970) analysis (GCG program) with a gap creation penalty=5, and a gap extension penalty=0.3. Unless stated otherwise, the query sequence is at least 45 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 45 nucleotides. Preferably, the query sequence is at least 150 nucleotides in length, and the GAP analysis aligns the two sequences over a region of at least 150 nucleotides. More preferably, the query sequence is at least 300 nucleotides in length and the GAP analysis aligns the two sequences over a region of at least 300 nucleotides. Even more preferably, the GAP analysis aligns the two sequences over their entire length.

With regard to the defined polynucleotides, it will be appreciated that % identity figures higher than those provided above will encompass preferred embodiments. Thus, where applicable, in light of the minimum % identity figures, it is preferred that a polynucleotide of the invention comprises a sequence which is at least 40%, more preferably at least 45%, more preferably at least 50%, more preferably at least 55%, more preferably at least 60%, more preferably at least 65%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, more preferably at least 99%, more preferably at least 99.1%, more preferably at least 99.2%, more preferably at least 99.3%, more preferably at least 99.4%, more preferably at least 99.5%, more preferably at least 99.6%, more preferably at least 99.7%, more preferably at least 99.8%, and even more preferably at least 99.9% identical to the relevant nominated SEQ ID NO.

Polynucleotides for use in the methods of the present invention may possess, when compared to naturally occurring molecules, one or more mutations which are deletions, insertions, or substitutions of nucleotide residues. Mutants can be either naturally occurring (that is to say, isolated from a natural source) or synthetic (for example, by performing site-directed mutagenesis on the nucleic acid).

Polynucleotides for use in the invention can also hybridize to a silk protein encoding nucleotide sequence as provided herein, such as one or more of SEQ ID NOs 32 to 62, under stringent conditions. The term "stringent hybridization conditions" and the like as used herein refers to parameters with which the art is familiar, including the variation of the hybridization temperature with length of an oligonucleotide. Nucleic acid hybridization parameters may be found in references which compile such methods, Sambrook, et al. (supra), and Ausubel, et al. (supra). For example, stringent hybridization conditions, as used herein, can refer to hybridization at 65 °C in hybridization buffer (3.5xSSC, 0.02% Ficoll, 0.02% polyvinyl pyrrolidone, 0.02% Bovine Serum Albumin (BSA), 2.5 mM NaH₂P0₄ (pH7), 0.5% SDS, 2 mM EDTA), followed by one or more washes in 0.2.xSSC, 0.01% BSA at 50°C.

Nucleic Acid Constructs

Starting material such as silk sponges are typically obtained from culturing cells expressing the silk proteins. These cells will typically comprise a nucleic acid construct(s) encoding the silk protein(s). The construct may be integrated into the genome of the cell, or be extrachromosal such as be a recombinant vector. Such a vector contains heterologous polynucleotide sequences, that is polynucleotide sequences that are not naturally found adjacent to the polynucleotide molecule encoding the silk protein, and that preferably are derived from a species other than the species from which the polynucleotide molecule(s) are derived. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a transposon (such as described in US 5,792,294), a virus or a plasmid.

One type of recombinant vector comprises a polynucleotide molecule encoding the silk protein operatively linked to an expression vector. The phrase operatively linked refers to insertion of a polynucleotide molecule into an expression vector in a manner such that the molecule is able to be expressed when transformed into a host cell. As used herein, an expression vector is a DNA or RNA vector that is capable of transforming a host cell and of effecting expression of a specified polynucleotide molecule. Preferably, the expression vector is also capable of replicating within the host cell. Expression vectors can be either prokaryotic or eukaryotic, and are typically viruses or plasmids. Expression vectors include any vectors that function (i.e., direct gene expression) in recombinant cells of the present invention, including in bacterial, fungal, endoparasite, arthropod, insect, animal, and plant cells. Particularly preferred expression vectors can direct gene expression in bacterial cells. Vectors can also be used to produce the protein in a cell-free expression system, such systems are well known in the art.

In particular, the nucleic acid construct contains regulatory sequences such as transcription control sequences, translation control sequences, origins of replication, and other regulatory sequences that are compatible with the recombinant cell and that control the expression of the polynucleotide molecules. Transcription control sequences are sequences which control the initiation, elongation, and termination of transcription. Particularly important transcription control sequences are those which control transcription initiation, such as promoter, enhancer, operator and repressor sequences. Suitable transcription control sequences include any transcription control sequence that can function in at least one of the recombinant cells of the present invention. A variety of such transcription control sequences are known to those skilled in the art. Preferred transcription control sequences include those which function in bacterial, yeast, arthropod, plant or mammalian cells, such as, but not limited to, tac, lac, trp, trc, oxy-pro, omp/lpp, rrnB, bacteriophage lambda, bacteriophage T7, T71ac, bacteriophage T3, bacteriophage SP6, bacteriophage SP01, metallothionein, alpha- mating factor, Pichia alcohol oxidase, alphavirus subgenomic promoters (such as Sindbis virus subgenomic promoters), antibiotic resistance gene, baculovirus, Heliothis zea insect virus, vaccinia virus, herpesvirus, raccoon poxvirus, other poxvirus, adenovirus, cytomegalovirus (such as intermediate early promoters), simian virus 40, retrovirus, actin, retroviral long terminal repeat, Rous sarcoma virus, heat shock, phosphate and nitrate transcription control sequences as well as other sequences capable of controlling gene expression in prokaryotic or eukaryotic cells. Cells

Transformation of a polynucleotide molecule, such as DNA construct defined herein, into a cell can be accomplished by any method by which a polynucleotide molecule can be inserted into the cell. Transformation techniques include, but are not limited to, transfection, electroporation, microinjection, lipofection, adsorption, and protoplast fusion. A recombinant cell may remain unicellular or may grow into a tissue, organ or a multicellular organism. Transformed polynucleotide molecules can remain extrachromosomal or can integrate into one or more sites within a chromosome of the transformed (i.e., recombinant) cell in such a manner that their ability to be expressed is retained.

Suitable host cells to transform include any cell that can be transformed with a polynucleotide encoding a silk polypeptide as defined herein. Host cells either can be endogenously (i.e., naturally) capable of producing the silk polypeptides or can be capable of producing such polypeptides after being transformed with at least one polynucleotide molecule as defined herein. Host cells can be any cell capable of producing at least one silk protein as defined herein, and include bacterial, fungal (including yeast), parasite, arthropod, animal and plant cells. Examples of host cells include Salmonella, Escherichia, Bacillus, Listeria, Saccharomyces, Spodoptera, Mycobacteria, Trichoplusia, BHK (baby hamster kidney) cells, MDCK cells, CRFK cells, CV-1 cells, COS (e.g., COS-7) cells, and Vero cells. Further examples of host cells are E. coli, including E. coli K-12 derivatives; Salmonella typhi; Salmonella typhimurium, including attenuated strains; Spodoptera frugiperda; Trichoplusia ni; and non-tumorigenic mouse myoblast G8 cells (e.g., ATCC CRL 1246). Additional appropriate mammalian cell hosts include other kidney cell lines, other fibroblast cell lines (e.g., human, murine or chicken embryo fibroblast cell lines), myeloma cell lines, Chinese hamster ovary cells, mouse NIH/3T3 cells, LMTK cells and/or HeLa cells. Particularly preferred host cells are bacterial cells.

The skilled person can readily determine suitable culture conditions such as media, temperature and time for a particular cell type. For example, in an embodiment the cells are Escherichia coli cultered at about 30°C to about 37°C for a period of about 24h to about 48h.

Recombinant DNA technologies can be used to improve expression of a transformed polynucleotide molecule by manipulating, for example, the number of copies of the polynucleotide molecule within a host cell, the efficiency with which those polynucleotide molecules are transcribed, the efficiency with which the resultant transcripts are translated, and the efficiency of post-translational modifications. Recombinant techniques useful for increasing the expression of polynucleotide molecules include, but are not limited to, operatively linking polynucleotide molecules to high-copy number plasmids, integration of the polynucleotide molecule into one or more host cell chromosomes, addition of vector stability sequences to plasmids, substitutions or modifications of transcription control signals (e.g., promoters, operators, enhancers), substitutions or modifications of translational control signals (e.g., ribosome binding sites, Shine-Dalgarno sequences), modification of polynucleotide molecules to correspond to the codon usage of the host cell, and the deletion of sequences that destabilize transcripts. Uses

The material comprising coiled coil silk proteins produced using the methods of the invention can be used for a broad and diverse array of medical, military, industrial and commercial applications. For example, silk fibres can be used in the manufacture of medical devices such as sutures, skin grafts, cellular growth matrices, replacement ligaments, and surgical mesh, and in a wide range of industrial and commercial products, such as, for example, cable, rope, netting, fishing line, clothing fabric, bullet- proof vest lining, container fabric, backpacks, knapsacks, bag or purse straps, adhesive binding material, non-adhesive binding material, strapping material, tent fabric, tarpaulins, pool covers, vehicle covers, fencing material, sealant, construction material, weatherproofing material, flexible partition material, sports equipment; and, in fact, in nearly any use of fibre or fabric for which high tensile strength and elasticity are desired characteristics. The silk also have applications for use in the production of compositions for personal care products such as cosmetics, skin care, hair care and hair colouring; and in coating of particles, such as pigments.

The silks may be used in their native form or they may be modified to form derivatives, which provide a more beneficial effect. For example, the silks may be modified by conjugation to a polymer to reduce allergenicity as described in US 5,981,718 and US 5,856,451. Suitable modifying polymers include, but are not limited to, polyalkylene oxides, polyvinyl alcohol, poly-carboxylates, poly(vinylpyrolidone), and dextrans. In another example, the silks may be modified by selective digestion and splicing of other protein modifiers. For example, the silk proteins may be cleaved into smaller peptide units by treatment with acid at an elevated temperature of about 60°C. The useful acids include, but are not limited to, dilute hydrochloric, sulfuric or phosphoric acids. Alternatively, digestion of the silk proteins may be done by treatment with a base, such as sodium hydroxide, or enzymatic digestion using a suitable protease may be used.

The proteins may be further modified to provide performance characteristics that are beneficial in specific applications for personal care products. The modification of proteins for use in personal care products is well known in the art. For example, commonly used methods are described in US 6,303,752, US 6,284,246, and US 6,358,501. Examples of modifications include, but are not limited to, ethoxylation to promote water-oil emulsion enhancement, siloxylation to provide lipophilic compatibility, and esterification to aid in compatibility with soap and detergent compositions. Additionally, the silk proteins may be derivatized with functional groups including, but not limited to, amines, oxiranes, cyanates, carboxylic acid esters, silicone copolyols, siloxane esters, quaternized amine aliphatics, urethanes, polyacrylamides, dicarboxylic acid esters, and halogenated esters. The silk proteins may also be derivatized by reaction with diimines and by the formation of metal salts.

Consistent with the above definitions of "polypeptide" (and "protein"), such derivatized and/or modified molecules are also referred to herein broadly as "polypeptides" and "proteins". The silk fibre can be spun together and/or bundled or braided with other fibre types. Examples include, but are not limited to, polymeric fibres (e.g., polypropylene, nylon, polyester), fibres and silks of other plant and animal sources (e.g., cotton, wool, Bombyx mori or spider silk), and glass fibres. A preferred embodiment is silk fibre braided with 10% polypropylene fibre. The present invention contemplates that the production of such combinations of fibres can be readily practiced to enhance any desired characteristics, e.g., appearance, softness, weight, durability, water-repellant properties, improved cost-of-manufacture, that may be generally sought in the manufacture and production of fibres for medical, industrial, or commercial applications.

Personal Care Products

Cosmetic and skin care compositions may be anhydrous compositions comprising an effective amount of silk in a cosmetically acceptable medium. The uses of these compositions include, but are not limited to, skin care, skin cleansing, makeup, and anti- wrinkle products. An effective amount of a silk for cosmetic and skin care compositions is herein defined as a proportion of from about 10^"4 to about 30% by weight, but preferably from about 10^" to 15% by weight, relative to the total weight of the composition. This proportion may vary as a function of the type of cosmetic or skin care composition. Suitable compositions for a cosmetically acceptable medium are described in US 6,280,747. For example, the cosmetically acceptable medium may contain a fatty substance in a proportion generally of from about 10 to about 90% by weight relative to the total weight of the composition, where the fatty phase containing at least one liquid, solid or semi-solid fatty substance. The fatty substance includes, but is not limited to, oils, waxes, gums, and so-called pasty fatty substances. Alternatively, the compositions may be in the form of a stable dispersion such as a water-in-oil or oil- in-water emulsion. Additionally, the compositions may contain one or more conventional cosmetic or dermatological additives or adjuvants, including but not limited to, antioxidants, preserving agents, fillers, surfactants, UVA and/or UVB sunscreens, fragrances, thickeners, wetting agents and anionic, nonionic or amphoteric polymers, and dyes or pigments.

Emulsified cosmetics and quasi drugs which are producible with the use of emulsified materials comprising silk produced by a method of the invention, for example, cleansing cosmetics (beauty soap, facial wash, shampoo, rinse, and the like), hair care products (hair dye, hair cosmetics, and the like), basic cosmetics (general cream, emulsion, shaving cream, conditioner, cologne, shaving lotion, cosmetic oil, facial mask, and the like), make-up cosmetics (foundation, eyebrow pencil, eye cream, eye shadow, mascara, and the like), aromatic cosmetics (perfume and the like), tanning and sunscreen cosmetics (tanning and sunscreen cream, tanning and sunscreen lotion, tanning and sunscreen oil, and the like), nail cosmetics (nail cream and the like), eyeliner cosmetics (eyeliner and the like), lip cosmetics (lipstick, lip cream, and the like), oral care products (tooth paste and the like) bath cosmetics (bath products and the like), and the like.

The cosmetic composition may also be in the form of products for nail care, such as a nail varnish. Nail varnishes are herein defined as compositions for the treatment and colouring of nails, comprising an effective amount of silk in a cosmetically acceptable medium. An effective amount of a silk for use in a nail varnish composition is herein defined as a proportion of from about 10^~4 to about 30% by weight relative to the total weight of the varnish. Components of a cosmetically acceptable medium for nail varnishes are described in US 6,280,747. The nail varnish typically contains a solvent and a film forming substance, such as cellulose derivatives, polyvinyl derivatives, acrylic polymers or copolymers, vinyl copolymers and polyester polymers. The composition may also contain an organic or inorganic pigment.

Hair care compositions are herein defined as compositions for the treatment of hair, including but not limited to shampoos, conditioners, lotions, aerosols, gels, and mousses, comprising an effective amount of silk in a cosmetically acceptable medium. An effective amount of a silk for use in a hair care composition is herein defined as a proportion of from about 10^" to about 90% by weight relative to the total weight of the composition. Components of a cosmetically acceptable medium for hair care compositions are described in US 2004/0170590, US 6,280,747, US 6,139,851, and US 6,013,250. For example, these hair care compositions can be aqueous, alcoholic or aqueous-alcoholic solutions, the alcohol preferably being ethanol or isopropanol, in a proportion of from about 1 to about 75% by weight relative to the total weight, for the aqueous-alcoholic solutions. Additionally, the hair care compositions may contain one or more conventional cosmetic or dermatological additives or adjuvants, as given above.

Hair colouring compositions are herein defined as compositions for the colouring, dyeing, or bleaching of hair, comprising an effective amount of silk in a cosmetically acceptable medium. An effective amount of a silk for use in a hair colouring composition is herein defined as a proportion of from about 10^"4 to about 60% by weight relative to the total weight of the composition. Components of a cosmetically acceptable medium for hair colouring compositions are described in US 2004/0170590, US 6,398,821 and US 6,129,770. For example, hair colouring compositions generally contain a mixture of inorganic peroxygen-based dye oxidizing agent and an oxidizable coloring agent. The peroxygen-based dye oxidizing agent is most commonly hydrogen peroxide. The oxidative hair coloring agents are formed by oxidative coupling of primary intermediates (for example p-phenylenediamines, p- aminophenols, p-diaminopyridines, hydroxyindoles, aminoindoles, aminothymidines, or cyanophenols) with secondary intermediates (for example phenols, resorcinols, m- aminophenols, m-phenylenediamines, naphthols, pyrazolones, hydroxyindoles, catechols or pyrazoles). Additionally, hair colouring compositions may contain oxidizing acids, sequestrants, stabilizers, thickeners, buffers carriers, surfactants, solvents, antioxidants, polymers, non-oxidative dyes and conditioners.

The silks can also be used to coat pigments and cosmetic particles in order to improve dispersibility of the particles for use in cosmetics and coating compositions. Cosmetic particles are herein defined as particulate materials such as pigments or inert particles that are used in cosmetic compositions. Suitable pigments and cosmetic particles, include, but are not limited to, inorganic color pigments, organic pigments, and inert particles. The inorganic color pigments include, but are not limited to, titanium dioxide, zinc oxide, and oxides of iron, magnesium, cobalt, and aluminium. Organic pigments include, but are not limited to, D&C Red No. 36, D&C Orange No. 17, the calcium lakes of D&C Red Nos. 7, 11, 31 and 34, the barium lake of D&C Red No. 12, the strontium lake D&C Red No. 13, the aluminium lake of FD&C Yellow No. 5 and carbon black particles. Inert particles include, but are not limited to, calcium carbonate, aluminium silicate, calcium silicate, magnesium silicate, mica, talc, barium sulfate, calcium sulfate, powdered Nylon™, perfluorinated alkanes, and other inert plastics.

The silks may also be used in dental floss (see, for example, US 2005/0161058). The floss may be monofilament yarn or multifilament yarn, and the fibres may or may not be twisted. The dental floss may be packaged as individual pieces or in a roll with a cutter for cutting pieces to any desired length. The dental floss may be provided in a variety of shapes other than filaments, such as but not limited to, strips and sheets and the like. The floss may be coated with different materials, such as but not limited to, wax, polytetrafluoroethylene monofilament yarn for floss.

The silks may also be used in soap (see, for example, US 2005/0130857). Pigment and Cosmetic Particle Coating

The effective amount of a silk for use in pigment and cosmetic particle coating is herein defined as a proportion of from about 10^~4 to about 50%, but preferably from about 0.25 to about 15% by weight relative to the dry weight of particle. The optimum amount of the silk to be used depends on the type of pigment or cosmetic particle being coated. For example, the amount of silk used with inorganic color pigments is preferably between about 0.01% and 20% by weight. In the case of organic pigments, the preferred amount of silk is between about 1% to about 15% by weight, while for inert particles, the preferred amount is between about 0.25% to about 3% by weight. Methods for the preparation of coated pigments and particles are described in US 5,643,672. These methods include: adding an aqueous solution of the silk to the particles while tumbling or mixing, forming a slurry of the silk and the particles and drying, spray drying a solution of the silk onto the particles or lyophilizing a slurry of the silk and the particles. These coated pigments and cosmetic particles may be used in cosmetic formulations, paints, inks and the like.

Biomedical

The silks may be used as a coating on a bandage to promote wound healing. For this application, the bandage material is coated with an effective amount of the silk. For the purpose of a wound-healing bandage, an effective amount of silk is herein defined as a proportion of from about 10^"4 to about 30% by weight relative to the weight of the bandage material. The material to be coated may be any soft, biologically inert, porous cloth or fibre. Examples include, but are not limited to, cotton, silk, rayon, acetate, acrylic, polyethylene, polyester, and combinations thereof. The coating of the cloth or fibre may be accomplished by a number of methods known in the art. For example, the material to be coated may be dipped into an aqueous solution containing the silk. Alternatively, the solution containing the silk may be sprayed onto the surface of the material to be coated using a spray gun. Additionally, the solution containing the silk may be coated onto the surface using a roller coat printing process. The wound bandage may include other additives including, but not limited to, disinfectants such as iodine, potassium iodide, povidon iodine, acrinol, hydrogen peroxide, benzalkonium chloride, and chlorohexidine; cure accelerating agents such as allantoin, dibucaine hydrochloride, and chlorophenylamine malate; vasoconstrictor agents such as naphazoline hydrochloride; astringent agents such as zinc oxide; and crust generating agents such as boric acid. The silk may also be used in the form of a film as a wound dressing material. The use of silk, in the form of an amorphous film, as a wound dressing material is described in US 6,175,053. The amorphous film comprises a dense and nonporous film of a crystallinity below 10% which contains an effective amount of silk. For a film for wound care, an effective amount of silk is herein defined as between about 1 to 99% by weight. The film may also contain other components including but not limited to other proteins such as sericin, and disinfectants, cure accelerating agents, vasoconstrictor agents, astringent agents, and crust generating agents, as described above. Other proteins such as sericin may comprise 1 to 99% by weight of the composition. The amount of the other ingredients listed is preferably below a total of about 30% by weight, more preferably between about 0.5 to 20% by weight of the composition. The wound dressing film may be prepared by dissolving the above mentioned materials in an aqueous solution, removing insolubles by filtration or centrifugation, and casting the solution on a smooth solid surface such as an acrylic plate, followed by drying.

The silk may also be used to produce sutures (see, for example, US

2005/0055051). Such sutures can feature a braided jacket made of ultrahigh molecular weight fibres and silk fibres. The polyethylene provides strength. Polyester fibres may be woven with the high molecular weight polyethylene to provide improved tie down properties. The silk may be provided in a contrasting color to provide a trace for improved suture recognition and identification. Silk also is more tissue compliant than other fibres, allowing the ends to be cut close to the knot without concern for deleterious interaction between the ends of the suture and surrounding tissue. Handling properties of the high strength suture also can be enhanced using various materials to coat the suture. The suture advantageously has the strength of Ethibond No. 5 suture, yet has the diameter, feel and tie-ability of No. 2 suture. As a result, the suture is ideal for most orthopedic procedures such as rotator cuff repair, Achilles tendon repair, patellar tendon repair, ACL/PCL reconstruction, hip and shoulder reconstruction procedures, and replacement for suture used in or with suture anchors. The suture can be uncoated, or coated with wax (beeswax, petroleum wax, polyethylene wax, or others), silicone (Dow Corning silicone fluid 202A or others), silicone rubbers, PBA (polybutylate acid), ethyl cellulose (Filodel) or other coatings, to improve lubricity of the braid, knot security, or abrasion resistance, for example.

The silk may also be used to produce stents (see, for example, US 2004/0199241). For example, a stent graft is provided that includes an endoluminal stent and a graft, wherein the stent graft includes silk. The silk induces a response in a host who receives the stent graft, where the response can lead to enhanced adhesion between the silk stent graft and the host's tissue that is adjacent to the silk of the silk stent graft. The silk may be attached to the graft by any of various means, e.g., by interweaving the silk into the graft or by adhering the silk to the graft (e.g., by means of an adhesive or by means of suture). The silk may be in the form of a thread, a braid, a sheet, powder, etc. As for the location of the silk on the stent graft, the silk may be attached only the exterior of the stent, and/or the silk may be attached to distal regions of the stent graft, in order to assist in securing those distal regions to neighbouring tissue in the host. A wide variety of stent grafts may be utilized within the context of the present invention, depending on the site and nature of treatment desired. Stent grafts may be, for example, bifurcated or tube grafts, cylindrical or tapered, self-expandable or balloon-expandable, unibody or, modular, etc.

In addition to silk, the stent graft may contain a coating on some or all of the silk, where the coating degrades upon insertion of the stent graft into a host, the coating thereby delaying contact between the silk and the host. Suitable coatings include, without limitation, gelatin, degradable polyesters (e.g., PLGA, PLA, MePEG-PLGA, PLGA-PEG-PLGA, and copolymers and blends thereof), cellulose and cellulose derivatives (e.g., hydroxypropyl cellulose), polysaccharides (e.g., hyaluronic acid, dextran, dextran sulfate, chitosan), lipids, fatty acids, sugar esters, nucleic acid esters, polyanhydrides, polyorthoesters and polyvinylalcohol (PVA). The silk-containing stent grafts may contain a biologically active agent (drug), where the agent is released from the stent graft and then induces an enhanced cellular response (e.g., cellular or extracellular matrix deposition) and/or fibrotic response in a host into which the stent graft has been inserted.

The silk may also be used to produce a matrix for producing ligaments and tendons ex vivo (see, for example, US 2005/0089552). A silk-fibre-based matrix can be seeded with pluripotent cells, such as bone marrow stromal cells (BMSCs). The bioengineered ligament or tendon is advantageously characterized by a cellular orientation and/or matrix crimp pattern in the direction of applied mechanical forces, and also by the production of ligament and tendon specific markers including collagen type I, collagen type III, and fibronectin proteins along the axis of mechanical load produced by the mechanical forces or stimulation, if such forces are applied. In a preferred embodiment, the ligament or tendon is characterized by the presence of fibre bundles which are arranged into a helical organization. Some examples of ligaments or tendons that can be produced include anterior cruciate ligament, posterior cruciate ligament, rotator cuff tendons, medial collateral ligament of the elbow and knee, flexor tendons of the hand, lateral ligaments of the ankle and tendons and ligaments of the jaw or temporomandibular joint. Other tissues that may be produced by methods of the present invention include cartilage (both articular and meniscal), bone, muscle, skin and blood vessels.

The silk may also be used to produce hydrogels (see, for example, US 2005/0266992). Silk fibroin hydrogels can be characterized by an open pore structure which allows their use as tissue engineering scaffolds, substrate for cell culture, wound and burn dressing, soft tissue substitutes, bone filler, and as well as support for pharmaceutical or biologically active compounds.

The silk may also be used to produce dermatological compositions (see, for example, US 2005/0019297). Furthermore, the may also be used to produce sustained release compositions (see, for example, US 2004/0005363).

Textiles

The silk may also be used to produce a coating for the surface of fibres for subsequent use in textiles. This provides a monolayer of the protein film on the fibre, resulting in a smooth finish. US 6,416,558 and US 5,232,611 describe the addition of a finishing coat to fibres. The methods described in these disclosures provide examples of the versatility of finishing the fibre to provide a good feel and a smooth surface. For this application, the fibre is coated with an effective amount of the silk. For the purpose of fibre coating for use in textiles, an effective amount of silk is herein defined as a proportion of from about 1 to about 99% by weight relative to the weight of the fibre material. The fibre materials include, but are not limited to textile fibres of cotton, polyesters such as rayon and Lycra™, nylon, wool, and other natural fibres including native silk. Compositions suitable for applying the silk onto the fibre may include co-solvents such as ethanol, isopropanol, hexafluoranols, isothiocyanouranates, and other polar solvents that can be mixed with water to form solutions or microemulsions. The silk containing solution may be sprayed onto the fibre or the fibre may be dipped into the solution. While not necessary, flash drying of the coated material is preferred. An alternative protocol is to apply the silk composition onto woven fibres. An ideal embodiment of this application is the use of silks to coat stretchable weaves such as used for stockings.

Composite Materials

Silk fibres can be added to polyurethane, other resins or thermoplastic fillers to prepare panel boards and other construction material or as moulded furniture and benchtops that replace wood and particle board. The composites can be also be used in building and automotive construction especially rooftops and door panels. The silk fibres re-enforce the resin making the material much stronger and allowing lighterweight construction which is of equal or superior strength to other particle boards and composite materials. Silk fibres may be isolated and added to a synthetic composite-forming resin or be used in combination with plant-derived proteins, starch and oils to produce a biologically-based composite materials. Processes for the production of such materials are described in JP 2004284246, US 2005175825, US 4,515,737, JP 47020312 and WO 2005/017004. Paper Additives

The fibre properties of the silk can add strength and quality texture to paper making. Silk papers are made by mottling silk threads in cotton pulp to prepare extra smooth handmade papers is used for gift wrapping, notebook covers, carry bags. Processes for production of paper products from silk are generally described in JP 2000139755.

Advanced Materials

Silks produced using the methods of the invention have considerable toughness and stands out among other silks in maintaining these properties when wet (Hepburn et al., 1979).

Areas of substantial growth in the clothing textile industry are the technical and intelligent textiles. There is a rising demand for healthy, high value functional, environmentally friendly and personalized textile products. Fibres, such as those of the invention, that do not change properties when wet and in particular maintain their strength and extensibility are useful for functional clothing for sports and leisure wear as well as work wear and protective clothing.

Developments in the weapons and surveillance technologies are prompting innovations in individual protection equipments and battle-field related systems and structures. Besides conventional requirements such as material durability to prolonged exposure, heavy wear and protection from external environment, silk textiles produced from silk can be processed to resist ballistic projectiles, fire and chemicals. Processes for the production of such materials are described in WO 2005/045122 and US 2005268443. EXAMPLES

EXAMPLE 1 - Materials and Methods

Generation of protein sponges for increasing mechanical strength

A honeybee silk protein (AmelF3; NCBI accession no: NP_001129680) was expressed into Escherichia coli inclusion bodies and purified as previously described (Weisman et al., 2010). Briefly, purified inclusion bodies were solubilised in 3% SDS solution then treated with 300 mM KC1. The potassium serves to precipitate the dodecyl sulfate of SDS which can be removed by centrifugation, thereby reducing SDS levels to around 0.3% (weight/volume) and producing high-purity AmelF3 solution (Weisman et al., 2010). The AmelF3 solution was dialysed against 15% polyethylene glycol (PEG), 0.25% SDS to remove salt and generate a solution containing 3.6% protein, 0.3% SDS. The solution also contained 0.1% salt comprising residual Na⁺ derived from the SDS after precipitation of the dodecyl sulfate and K⁺ with CI^" counter ions. SDS concentration was determined according to Rusconi er al. (2001), salt concentration was determined by calculation of dilution during dialysis, and total solute weight was determined by weighing aliquots of dried solution. The 4 dry wt% solution was poured into silicone rubber moulds (14 x 5 x 6 mm; RL060, ProSciTech, QLD), frozen at -20 °C overnight, and placed in a freeze-dryer (FD355DMP, FTS Systems) for 24 hours to generate sponges typically of 12.6 x 4.5 x 5.4 mm. The silk sponges were stored in sealed plastic bags at room temperature until use.

For comparison, stabilized collagen sponges for comparison were made from ovine skin collagen. Minced ovine skin was digested with 1 mg ml^"1 pepsin at 4 °C for 24 h in lOOmM acetic acid, adjusted to pH 2.5 with HC1. The hydrolysate was purified using two 0.7 M NaCl precipitation steps (Miller and Rhodes, 1982). SDS-PAGE showed that the collagen was predominantly type I, but a small quantity of type III collagen, <5%, was also present. PEG 4000 was used to precipitate collagen as fibrous-like aggregates (Ramshaw et al, 1984) as previously described (US 4,980,403). Purified collagen was dissolved at 2 mg ml^"1 in 20 mM acetic acid then adjusted to pH 7.0 in 40 mM sodium phosphate buffer. PEG (40% w/v) was added slowly with stirring to a final concentration of 4% and the solution was left at 4°C for 16 h. Precipitate was collected as a loose pellet by centrifugation at 1000 x g for 15 min then resuspended in 20 mM sodium phosphate buffer, pH 7.0, and the precipitate recollected as above after 1 h. The precipitate was resuspended in 10 mM sodium phosphate buffer, pH 7.0, and the precipitate recollected after 1 h at 1000 x g for 30 min. The collagen paste was transferred into silicone rubber moulds (RL060, ProSciTech, QLD) and freeze-dried. Sponges were suspended in dry ethanol and air in pores removed by evacuation prior to cross-linking with a 10: 1 w/w excess of l-ethyl-3-[3- dimethylaminopropyl]carbodiimide hydrochloride for 16 h. The sponges were then washed extensively in ethanol and air dried. Treatment of honeybee silk sponges and water stability testing

Honeybee silk sponges were treated to decrease water sensitivity by either heating in air to temperatures of 160 - 190°C for times ranging between 10 - 120 min, or by immersion for 48 h in aqueous methanol solutions with methanol concentrations ranging from 0 - 100%. In order to test water stability sponges were cut into 4 pieces and each sample, under ambient laboratory conditions, was weighed on a microbalance prior to heat or methanol treatment. After heat or methanol treatment sponge samples were transferred through a dilution series of 40, and 20 methanol solutions into 1 mL water and incubated at room temperature on an orbital shaker for time periods ranging from 1 to 32 days, dried at 60°C for 2 h and then weighed to determine mass loss. Mass loss is reported as the percentage remaining of the initial mass after heat or methanol treatment. Initial mass loss and rate of mass loss were determined from the y-intercept and slope of the linear regression through the mass loss vs. time data points, respectively. Four samples per treatment were assessed and data collected at day 0, 1, 2, 4, 8, 16 and 32 days. SDS levels in the methanol solutions or water after sponges were removed were calculated according to Rusconi et al. (2001), protein concentration was determined using the QuantiPro BCA assay kit (Sigma; St Louis, MO).

Compression testing

The mechanical properties of honeybee silk sponges and stabilized collagen sponges were measured using compression tests on an Instron 5500R (Instron, USA) fitted with a 2.5N static load cell. 'Methanol-treated' samples were treated in 60 - 100 % methanol in water for 48 h and then transferred through a dilution series of 80, 60, 40 and 20% methanol solutions each for 10 min and then soaked for 24 h in phosphate buffered saline (PBS) prior to testing. Heat-treated samples and collagen sponges were soaked for 5 min in PBS with gentle squeezing to ensure saturation prior to testing. Sponge samples were compressed to 190-200 mN at 2 mm/min then decompressed at 2 mm/min. The compression tests were repeated three times on each sample with 1 min relaxation times between tests. The cross- sectional area of the samples, measured on a light microscope to be around 57 mm , was used to convert force values to stress values. Sample thickness was determined during the compression test, as the distance between the base plate and the point at which the compression plate contacts the sample (F = lmN).

Raman spectroscopy

Raman spectra were obtained from dried honeybee silk sponges at a resolution of 4 cm^"1 using a Bruker RFS-100 FT-Raman spectrometer (Karlsruhe, Germany) equipped with an Adlas Nd:YAG laser operating at 1.064 μιη and 500 mW and with a liquid nitrogen cooled Germanium diode detector. Spectra were collected using 180° backscatter geometry with the samples held in a compression cell described elsewhere (Church et al., 1994). Data acquisition over 512 scans was performed using Bruker OPUS software (version 3.1). Four spectra were obtained from different areas of each sample and co-averaged to produce a final spectrum for analysis. All spectra obtained from a given sample were found to be in excellent agreement. Data manipulation was carried out using Grams AI v8.0 software (Thermo Electron Corp., USA). All spectra were normalized on the C-H bending mode at 1449 cm^"1 as this mode is insensitive to protein conformation. The structural changes of the honeybee silk sponges after the different treatments was also assessed by the quantitation of the overlap area between the second derivatives of the amide I bands (Kendrick et al., 1996). Briefly, the method, which was implemented using Matlab R2010a (Math Works, USA), involves the normalization of the spectral region of interest followed by the calculation of their 2^nd derivatives. The two 2^nd derivative spectra to be compared are passed through a logical filter which takes the lowest intensity value of the pair and creates an array that constitutes a spectrum representing the overlap area. Amino acid analysis

Untreated and heat-treated honeybee silk sponge samples were sent to the Australian Proteome Analysis Facility for quantitative amino acid analysis via acid hydrolysis. EXAMPLE 2 - Water stability of treated sponges

Untreated recombinant honeybee silk sponges swell and rapidly dissolve in water and therefore water solubility can be used as an indicator of efficiency of post fabrication treatments. Sponges that were treated either by heating dry material for various times or by extended immersion in aqueous methanol solutions were incubated in water for up to 32 days to test their water solubility (Table 2). Both methanol treatment in solutions containing > 50% methanol and heat treatment at a temperature of 190°C impart a high degree of water stability to the sponges. The sponges treated in 100% methanol swelled extensively when transferred to water, producing a very weak material that could not be lifted out of the solution intact. These sponges could be weighed after they were pelleted by centrifugation. The sponges were too weak to undergo mechanical testing. Samples treated with aqueous methanol solutions containing <50% methanol dissolved in the treatment solutions.

Table 2. Stability of honeybee silk sponges in water over 32 days.

Treatment to cross-link Initial mass Average further mass Degree of

proteins loss (%) loss per day (%) swelling

Untreated 100 - -

48 h in 60% MeOH 18 (1 ) 0.0 (ns) low

48 h in 80% MeOH 23 (1 ) 0.0 (ns) med

48 h in 100% MeOH 21 (4) 1 .0 (0.2) high

10 min @ 1 60°C 100 - -

10 min @ 1 80°C 100 - -

10 min @ 1 90°C 18 (1 ) 0.3 (0.1 ) med

30 min @ 1 90°C 17 (1 ) 0.0 (ns) low

60 min @ 1 90°C 18 (1 ) 0.0 (ns) low

120 min @ 190 Ό 19 (1 ) 0.0 (ns) low

Standard errors are given in brackets, ns = not significant at the 95% confidence limit.

Samples that had been heat treated at temperatures <190°C (160°C, 180°C) were significantly less water-stable than those treated for equivalent times at 190°C, the samples treated for 10 min dissolving. Protein was continually leached from samples treated with 100% methanol or heated for 10 minutes at 190°C at a respective rate of around 1% and 0.3% per day, whereas the samples heated for longer time periods were effectively impervious to water after their initial mass loss (Table 2).

When a sponge was subjected to its initial wet exposure, either as part of its treatment and/or during water stability testing, a mass loss of 18-23% occurred during the treatment or within the first day of incubation in water (Table 2). This loss was primarily due to leaching of SDS from the scaffolds. The detergent was detected in the methanol solutions or water at levels equivalent to what was originally in the scaffold (data not shown). The remaining mass lost is likely to be made up of leaching of salt and low molecular weight peptides present as contaminants or degradation products, and bound water loss during the dry heat treatment process (4-6%) or resulting from molecular rearrangement. EXAMPLE 3 - Mechanical properties of recombinant materials

Sponges

Honeybee silk sponges treated by dry heating to 190°C for 1 hr or by immersion in aqueous methanol solutions were subjected to compression tests in PBS to assess their wet mechanical properties. For comparison, chemically cross-linked collagen sponges were also tested. Representative compression curves are shown in Figure 1 and summarized results are shown in Table 3. Since the scaffolds have been compressed to a constant force, the compressive strain at this load is inversely related to the stiffness of the samples. The best measure of stiffness however is the stress at a given strain. Clearly, the honeybee silk scaffolds are softer than the collagen scaffolds requiring less stress to compress the scaffold to 25% of its initial thickness (ie. at 25% strain, Table 2). There is no significant difference between the honeybee samples treated in 60% or 80% methanol, both of which are softer than the samples heated at 190 ° C for greater than 10 min (compare stress at 25% and 50% strain). The energy required to compress the scaffolds is influenced by both the stiffness and shape of the curve and so is best used only as an indicator of the recovery of the samples between successive compressions (Table 3 and Figure 4). The samples that were heat-treated show an initial J-shaped compression curve after 10 min heating (Figure 2). At longer heat treatment times a small yield point is evident (Figure 2) and the sample stiffness (stress at 50% strain) and energy required for compression increase logarithmically with time of treatment (Figure 3 and Table 3).

Resilience is measured as the ratio of the energy required to deform a sample, to the energy recovered when the load is removed. In other words, it is a measure of a material's ability to recover from deformation. Resilience values of the stabilized honeybee silks vary between 40 and 50%, substantially lower than the 80% shown by collagen. It should be noted, however, that the collagen sample, being much stiffer, was only compressed to about 30% compared to 70-90% for the honey bee silk samples. As a general rule, resilience decreases linearly with increasing energy of deformation (Huson et al., 2006), therefore comparing samples which require vastly different Table 3. Average values of the mechanical properties of scaffolds subjected to cyclic compression testing in PBS. All values are for the first cycle except the sample marked by an asterisk (*). These values are the first cycle of the 3 rd compression experiment after 32 days in PBS. Values in brackets are standard deviations.

Sample Treatment to Max Strain at Stress at Stress at Energy to Resilience

cross-link proteins Load max load 15% strain 50% strain max load

(%)

(N) (%) (kPa) (kPa) (J/m³)

Collagen carbodiimide 0.7 (0) 84 (0) 1 .77 (0.13) 5.10 (0.39) 397 (23) 77 (2)

Collagen carbodiimide 0.2 (0) 37 (1 1 ) 1 .97 - 69 81 (5)

AmelF3 48h in 60%

0.2 93 (3) 0.08 (0.04) 0.38 (0.1 1 ) 130 41 (2)

MeOH

AmelF3 48h in 80%

0.2 90 (2) 0.1 1 (0.04) 0.44 (0.04) 150 46 (2)

MeOH

AmelF3 10 min @ 190°C 0.2 83 (1 ) 0.10 (0.02) 0.29 (0.07) 96 45 (2)

AmelF3 30 min @ 190°C 0.2 76 (2) 0.29 (0.03) 0.89 (0.21 ) 167 53 (1 )

AmelF3 60 min @ 190°C 0.2 72 (6) 0.26 (0.1 1 ) 1 .20 (0.49) 187 48 (4)

AmelF3 120 min @ 190°C 0.2 65 (8) 0.50 (0.43) 1 .82 (0.86) 205 38 (4)

AmelF3 ^* 120 min @ 190°C 0.2 84 (7) 0.10 (0.03) 0.97 (0.40) 208 55 (2)

All values are for the first cycle except the sample marked by an asterisk (*). These values are the first cycle of the 3¹ compression experiment after 32 days in PBS. Values in brackets are standard deviations.

energies of deformation can be misleading. When the collagen scaffold was compressed to 70% its resilience dropped, a value similar to that of the honeybee silk scaffolds. It is important to note too, that the values in Table 3 are a measure of the instant recovery during the cyclic test and that given more time viscoelastic materials will recover further. Thus, although the honeybee silk scaffolds only recover about 50% of the deformation energy during the cyclic test, if they are allowed one minute relaxation time then they show only a 10% decrease in energy to compress (Figure 4).

If a sponge is left in solution for 24 hours after a series of compressions its mechanical properties recover fully to those seen in the initial compression. Table 3 includes data for the seventh compression of a 120 min heat-treated sample compressed seven times over a period of 32 days in PBS. Clearly its properties have been well maintained even after multiple compressions and a lengthy immersion in PBS.

Fibres

Fibres were produced by extruding into a methanol coagulation bath, drying and then drawing to approximately 3 times their original length in a methanol bath. The average strength of the fibres was 185 MPa (range 178-195 MPa) with 21% extension at break (range 15-31%) and around 80 MJ.m-3 energy to break (toughness). Heat treatment (190°C for 60 min) on dried fibres substantially improved the mechanical performance of the fibres with the average strength of the heated fibres being 330 MPa (range 273 - 456 MPa) with 63% extension at break (range 34 - 84%) and substantially improved toughness (energy to break: average 165 MJ.m-3) (Table 4). The heated fibre came off the same roll as the control fibre. All tested fibres were ~25-30um in diameter.

Table 4. Summary toughess data for six heat treated fibres.

Fibre Diameter Modulus Yield Energy to Stress to Elongation

Stress break break at break

(micron) (Gpa) (MPa) (MJ/m^A3) (Mpa) (%)

1 32 9.3 298 157 287 62

2 25 10.2 353 239 457 68

3 30 6.7 249 194 316 80

4 29 6.7 263 111 274 46

5 28 8.8 320 215 357 72

6 30 7.8 281 71 255 30

Mean 29 8.3 294 165 324 60

Max 32 10.2 353 239 457 80

Min 25 6.7 249 71 255 30 EXAMPLE 4 - Protein structure changes in treated sponges

Raman spectra of aqueous methanol-treated and heat-treated silk sponges are shown in Figure 5. Spectra obtained from sponges that had been soaked in water for 24 h after aqueous methanol or heat treatment then dried and analyzed were effectively identical to the spectra collected from dry sponges immediately after treatment, indicating that post-treatment water immersion did not affect the protein secondary structure. The shown spectra are from sponges immediately after treatment.

Examination of the amide I region of the spectra for aqueous methanol-treated sponges indicates a distinct change in protein conformation compared to untreated sponge (Figure 5 A) has taken place. There is an increase in the peak at 1666 cm^"1, attributed to beta-sheet, and a corresponding decrease in the peak at 1655 cm^"1, attributed to coiled coil. As it is not possible to resolve β-sheet and disordered protein structure in the amide I region (Frushour and Koenig, 1975) there is a possibility that both are actually present. In the amide III region (not shown) where the β-sheet and disordered proteins are better resolved (Frushour and Koenig, 1975), there is a slight increase in a feature at 1258 cm^"1 confirming that disordered protein is being formed, particularly at the lower methanol concentrations. There is a trend towards decreasing structural change with increasing methanol concentration (Figure 5 A, inset), so that the sample treated with 60% methanol has the highest β-sheet content (corresponding with the lowest level of water swelling from Table 2), and the sample treated with 100% methanol has lowest β-sheet content (corresponding with highest level water swelling and some water solubility from Table 2).

The structural change upon methanol / water treatment was further investigated using the band overlap approach developed Kendrick et al. (1996) for the analysis of amide I bands in the infrared. Consistent with the above analysis, the greatest conformational change (26.4%) was observed for the 60% methanol treated sponge while the least (11.2%) was found for the 100% methanol treated sponge. The % conformational change was found to be a linear function of methanol concentration with an R of 0.9838. The changes consisted of a major loss in coiled coil structure (1653 cm-1) off-set by a major gain in beta sheet structure (1669 cm-1). There is also a minor loss in β-sheet structure observed at 1688 cm-1. These results are presented as Table 5 and Figure 6.

Heat-treated sponges appear to undergo a slight structural transition in the direction of β-sheet (Figure 5B). In the series of samples treated at 190°C, the effect is smallest in the sample treated for 10 min then approximately constant for treatments of 30 min or longer (Figure 5B, insert). In all heat-treated sponges the magnitude of the apparent structural transition is less than that seen in samples treated with 100% methanol, however the heat-treated samples have much higher water stability than 100% methanol samples (Table 2). This suggests that the heat treated samples are undergoing stabilizing changes other than or in addition to protein structural transitions. The amide I regions observed for samples that were heat treated at lower temperatures, 160 and 180°C for 60 min, were found to be very similar to that obtained from the untreated sponge. These sponges were all found to dissolve immediately upon exposure to water (Table 2).

Table 5. Percenta ge conformational change as a function of methanol/water concentration.

Treatment Conformational Description

Change (%)

60% MeOH 48 h 26 Major loss of coiled coil structure (1653 cm^"1) 70% MeOH 48 h 24 off-set by a major gain in β-sheet structure 80% MeOH 48 h 19 (1669 cm^"1). There is also a minor loss in β- 90% MeOH 48 h 14 sheet structure observed at 1688 cm^"1

100% MeOH 48 h 11

160°C 60 min 6

180°C 60 min 6 Loss of coiled coil structure (1653 cm^"1)

190°C 10 min 5 coupled with gain in β-sheet structure (1688 cm^"1)

190°C 30 min 10 Greater loss of coiled coilstructure (1653 cm^"1) 190°C 60 min 12 coupled with gains in β-sheet structure at 1669 and 1688 cm^"1

190°C 120 min 10 No significant further loss of coiled coil

structure (1653 cm^"1) but no apparent gain in β- sheet structure at 1669 cm^"1 The Raman amide I bands observed for all of the thermally treated sponges are very similar (Figure 5B). No significant differences were observed in the amide III region. Amide I Band overlap analysis results are presented in Table 5 and Figure 7. The conformational similarity of the sponges treated at 160 and 180°C for 60 min and 190°C for 10 min is confirmed with all undergoing changes of the order of 5.5% upon treatment. These changes can be associated with the loss of coiled coil structure coupled with gains in β-sheet structure. Sponges treated at 190°C for more than 30 min also have very similar conformations to each other, having undergone conformational change of the order of 10%. These sponges exhibit greater loss of alpha-helical structure along with complementary gains in β-sheet structure compared to those treated at lower temperature or for less time.

EXAMPLE 5 - Cross-linking in heat-treated sponges

As described above, no significant secondary structural changes are seen in the heat treated materials, yet significant changes in their mechanical properties are observed. These results strongly indicate covalent cross-linking mechanisms instead of the well described β-sheet cross-linking mechanism found in silks. Amino acid analysis was performed on control and heat treated (60 min at 190°C) sponges and results are shown in Figure 8. Significantly, heat treatment caused losses in lysine (21.5 mg/g sponge, ~6 lysine residues per protein) and serine 8.9 mg/g sponge, ~4 serine residues per protein). Serine and lysine are known to form lysinoalanine cross-links in other proteins subjected to heat treatment through dehydration of the serine to form a dehydroalanine electrophile which can then undergo nucleophilic attack by the lysine's primary amine (Friedman et al., 1999). The analogous reaction with threonine, which also exhibits a significant reduction, is known to occur, forming methyl-lysinoalanine. Both lysinoalanine and methyl lysinoalanine would be difficult to detect as the strongest Raman bands, those associated with the symmetric C-N-C stretching vibrations, are expected in the 850 to 900 cm^"1 region (Dollish et al., 1974) which is already rich (Frushour and Koenig, 1975) in protein skeletal vibrations. No significant spectral changes were observed in this region as a function of thermal treatment.

Heat treatment will also generate isopeptide bonds or esters between amine- functional, or potentially amine-functional sidechains (Lys, Arg, His, Asn, Gin), and acidic residues such as Asp and Glu in close proximity (Mohammed et al., 2000). Isopeptide and ester bonds cannot be inferred from the amino acid analysis as acid hydrolysis will hydrolyse all amide and ester bonds, returning the original amine, hydroxyl and carboxylate functions, except in the case where Arg could be thermally converted to a primary amine and form isopeptide bonds. In this event, hydrolysis would, of course, only return the primary amine. Indeed, there does appear to be a slight reduction in Arg content upon heat treatment at 190°C for 60 min which may be indicative of this manner of crosslinking having occurred. Isopeptide bonds are difficult to observe directly in the Raman spectra owing to their relatively low abundance compared to the backbone amides which will absorb in the same region (Frushour and Koenig, 1975). While the carbonyl stretching vibrations of ester groups are expected on the high frequency side of the amide I band, its weak intensity in the Raman compared to that in the infrared Dollish et al., 1974) making it hard to detect at low concentrations.

The nature and amount of cross-linking was confirmed by protease disgestion of the proteins followed separartion of the resulting peptides by liquid chromatography, and analysis of the mass of the peptides using mass spectrometry and/or sequencing using tandem mass spectrometry (MS/MS).

Amino acid analysis

Quantitative amino acid analysis via acid hydrolysis was carried out on untreated and heat treated honeybee silk sponge samples. Digestions were carried out in triplicate, analyzed in duplicate and the results reported as an average. Deficits of amino acids in the treated samples relative to untreated samples were taken to indicate residues that had been modified by heat- treatment. Modifications such as dehydration reactions producing isopeptide bonds would be reversed by acid hydrolysis.

In order to identify residues involved in such modifications, amino acid analyses were also performed following enzymatic digestions of silk sponge samples. Enzymatic digestions were performed in duplicate on samples of heat-treated and non- heat-treated honeybee silk sponges that were first washed with 70% methanol. Methanol would not be expected to induce any covalent modification but removes detergent that might interfere with enzymatic digestion. Approximately 3 mg of sample was accurately weighed and resuspended in 100 mM HEPES buffer pH 7.5. The samples underwent a 24 hour enzymatic hydrolysis at 37 °C with Pronase E, Leucine Aminopeptidase M, and Prolidase in a final volume of 500 μL· Samples were heated at 100°C for 10 minutes before undergoing a further 24 hour enzymatic hydrolysis at 37°C with carboxypeptidase.

Levels of amino acids after both treatments were analyzed using the Waters AccQTag Ultra chemistry on a Waters ACQUITY UPLC system. The non-standard amino acid s-(y-glutamyl)-lysine was quantified by reference to a standard (Sigma) and eluted with a retention time similar to cysteine, an amino acid that is conveniently absent from AmelF3.

The enzymatically hydrolysed heat treated material showed significant losses in lysine, asparagine, aspartic acid and glutamic acid in comparison to the control sponge (Figure 14, panel A). The concentrations of histidine and tyrosine were not found to decrease. These differences as well as that observed for serine are represented as mole percentages in Figure 14, panel B. These results are consistent with the formation of isopeptide linkages between lysine/asparagine and aspartic acid/glutamic acid as well as the formation of lysinoalanine linkages between lysine and serine and ester linkages between aspartic acid/glutamic acid and serine.

Liquid chromatography-tandem mass spectrometry

Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to test for the presence of s-(y-glutamyl)-lysine, a dipeptide that would remain following enzymatic digestion of honeybee silk sponges containing Glu-Lys isopeptide crosslinks. Heat-treated and non-heat-treated material was digested as above, acidified with formic acid (1% v/v final) and 1 μΐ was separated on an Agilent 1200 capillary LC system coupled by electrospray ionization to an Agilent XCT ion trap mass spectrometer. The column (Agilent Zorbax 300SB-C18, 3.5 μιη, 50x0.3 mm) was eluted isocratically with 3% acetonitrile/0.1% formic acid at 5 μ!7ηιίη for 5 minutes before washing with 90% acetonitrile/0.1% formic acid and re-equilibration with 3% acetonitrile/0.1% formic acid. The ion trap was tuned to retain only ions of 276 m/z, corresponding to the MH⁺ ion from s-(y-glutamyl)-lysine. Fragmentation energy was adjusted to produce two dominant ions at 131 and 148 m/z from a standard solution of s-(y-glutamyl)-lysine (Sigma). The standard eluted at 1-2 minutes. Mass spectra from the same retention time were averaged for digested honeybee silk sponges for comparison with the spectrum from the iso-peptide standard.

Enzymatic hydrolysis of material containing Lys-Glu isopeptide bonds will liberate s-(y-glutamyl)-lysine. Targeted LC-MS/MS directly demonstrated the presence of s-(y-glutamyl)- lysine in enzymatic digests of heat-treated honeybee silk sponges but not in non-heat-treated sponges (Figure 15). Following calibration with a s-(y-glutamyl)- lysine standard, the amino acid analysis traces were re-examined. A peak corresponding to s-(y-glutamyl)- lysine was present in the heat-treated sponge at about 4.4 mg/g sponge corresponding to approximately one s-(y-glutamyl)-lysine moiety per protein molecule or two Lys-Glu isopeptide links on average between protein molecules. Heat treatment is also expected to result in analogous isopeptide bonds between lysine and aspartate residues.

EXAMPLE 6 - Effect of vacuum

Scaffolds

Scaffold pieces were treated by heating under vacuum then placed in water. The extent of swelling is proxy for the extent of cross-linking. Figure 9 shows the results 70 hours after water was added but the result looks the same at 1 hour or 2 weeks. 90°C/lh dissolved almost instantly. 120°C/48h has swollen less than the usual treatment of 190°C/lh/no vacuum and is not discoloured, but 120°C/48h without vacuum was very weak. Treatment with 90% methanol for 1 hour to induce beta sheet formation prior to the vacuum treatment produced stronger scaffolds. Fibres

Fibres were produced from 9-10 % silk protein, 0.25-0.30% SDS, 15 mM salt solutions as described in Example 7. The fibres were untreated, treated for 1 hr at 180°C or for various lengths of time (as indicated) under vacuum (30 in.Hg which is equivalent to 1 bar or 1 atm or lOOkPa).

The stability of the untreated and treated fibres in SDS (Figure 16A), guanidinium (Figure 16B) and the stress to break (Figure 16C) was compared. The untreated fibres swelled and dissolved in detergent and guanidinium within one hour. Swelling and dissolution was also observed in fibres treated for 8 hrs at 120°C under vacuum. However as the treatment time at 120°C under vacuum increased the fibres became more stable so that after 7 days treatment they were essentially equivalent to fibres treated at 180°C without vacuum.

EXAMPLE 7 - Production of silk fibres

A clear, viscous protein solution (350 g) containing 9.3 % silk protein, 0.28% SDS, <15 mM salt was prepared for the development of continuous fibre production. Initially, a ΙΟΟμιη capillary tube used for injection kept blocking and therefore a 30G needle (30G = ~140μιη diameter) was used for injection. A graphic of the set up of the system is shown in Figure 10. Initially, over 20 m of continuously drawn fibre was collected without breakages over 7 min extrusion time. In subsequent trials up to 44 m of fibre was produced as a single thread.

The average strength of continuously produced fibres (drawn ~3x) was measured at 185 MPa (range 178-195 MPa) with 21% extension at break (range 15- 31%). Heat treatment (190°C for 60 min) substantially improved the mechanical performance of the fibres with the average strength of the heated fibres (drawn ~3x) being 330 MPa (range 273 - 456 MPa) with 63% extension at break (range 34 - 84%). The heated fibre came off the same roll as the control fibre. All tested fibres were -25- 30um in diameter.

Samples of 1 m of continuously produced fibre heat treated (190°C for 60 min), were tested in a small scale knitting machine to determine if they were amenable to the knitting process (Figure 11). Results indicate that the heat treated fibre is suitable for knitting (Figure 12). Dried fibres were flexible and did not crack when knotted (Figure 13).

EXAMPLE 8 - Solvent stability of heat treated materials

Freeze dried sponges comprising silk proteins were treated for 60 min at 190°C, cut into 3 pieces and weighed accurately on a microbalance. Each piece was placed into 2 mL of either 2 M Urea, 4 M Urea, 8 M Urea, 1% SDS, 2% SDS, 4% SDS, 2 M guanadinium, 4 M guanadinium, 6 M guanadinium and incubated for 24 hrs on a shaking platform. After incubation, the samples were washed extensively, dried and weighed. No mass loss was observed in any of the samples.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

The present application claims priority from US 61/600,451 filed 17 February 2012, the entire contents of which are incorporated herein by reference.

All publications discussed and/or referenced herein are incorporated herein in their entirety.

Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

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Claims

1. A process for forming cross-links between coiled coil silk proteins in a material comprising the silk proteins, the process comprising

i) obtaining the material in a solid state, and

2. The process of claim 1, wherein following the process the material has an increased toughness and/or solvent stability when compared to the material before step ii).

3. The process of claim 1 or claim 2, wherein step ii) is conducted under conditions which promote drying of the material.

4. The process of claim 3, wherein step ii) is conducted in a vacuum and/or in the presence of a desiccant.

5. The process of claim 3 or claim 4, wherein step ii) is performed for at least about 10 hours.

6. The process of claim 5, wherein step ii) is performed for about 10 hours to about 100 hours.

7. The process according to any one of claims 3 to 6, wherein the temperature used in step ii) is between about 100°C and about 150°C.

8. The process of claim 7, wherein the temperature used in step ii) is between about 100°C and about 120°C.

9. The process of claim 1 or claim 2, wherein the temperature used in step ii) is at least about 190°C.

10. The process of claim 9, wherein the temperature used in step ii) is between about 190°C and about 220°C.

11. The process of claim 10, wherein the temperature used in step ii) is between about 190°C and about 200°C.

12. The process according to any one of claims 9 to 11, wherein step ii) is performed for at least about 20 minutes.

13. The process of claim 12, wherein step ii) is performed for about 20 minutes to about 120 minutes.

14. The process of claim 12, wherein step ii) is performed for about 30 minutes to about 60 minutes.

15. The process according to any one of claims 1 to 14 which further comprises reducing the concentration of water in a material or solution comprising the silk proteins before step ii).

16. The process of claim 15, wherein the material or solution is frozen and then freeze-dried.

17. The process of claim 16, wherein the material or solution is frozen at about - 20°C.

18. The process of claim 16 or claim 17, wherein the frozen material or solution is freeze-dried for about 12 to about 48 hours.

19. The process of claim 18, wherein the frozen material or solution is freeze-dried for about 24 hours.

20. The process of claim 15, wherein the silk proteins are precipitated.

21. The process of claim 20, wherein the silk proteins are is precipitated by the addition of an alcohol or a salt to a solution comprising the silk proteins, or by reducing the pH of the solution to at least about 5.5, preferably at least about 4.5, or a combination of two or more thereof.

22. The process of claim 21, wherein the silk proteins are precipitated in a solution comprising alcohol, the precipitate collected, air dried and used in step ii).

23. The process of claim 21 or claim 22, wherein the alcohol is methanol or ethanol.

24. The process according to any one of claims 1 to 23, wherein before step ii) the material has a H₂0 content of about 1% to about 10%.

25. The process of claim 24, wherein before step ii) the material has a H₂0 content of less than about 5%.

26. The process according to any one of claims 1 to 25 which further comprises treating the material with a solution comprising at least about 60% alcohol before or after step ii).

27. The process of claim 26, wherein the solution comprises about 60% to about 80% alcohol.

28. The process of claim 26 or claim 27, wherein the alcohol is methanol or ethanol.

29. The according to any one of claim 26 to 28, wherein the material is treated for about 24 to about 48 hours.

30. The process according to any one of claim 1 to 29, wherein following step ii) silk proteins in the material have a coiled coil structure.

31. The process according to any one of claims 1 to 30, wherein the material in step i) is in the form of a sponge, particle, fiber or film.

32. The process according to any one of claims 1 to 31, wherein following the process the material has at least a 75% reduction in water solubility solubility when compared to the material before step ii).

33. The process according to any one of claims 1 to 32, wherein following the process the material has at least about a 10% increase in solvent stability when compared to the material before step ii).

34. The process according to any one of claims 1 to 33, wherein following the process the material has at least about a 10% increase in toughness when compared to the material before step ii).

35. A product comprising material produced by the process according to any one of claims 1 to 34.

36. A solid material comprising coiled coil silk proteins which is insoluble in one or more of 2% SDS, 8M urea or 6M guanadium.

37. A solid material comprising coiled coil silk proteins, wherein each silk protein is cross-linked to at least two other silk proteins by an amide and/or ester crosslink.