Silk protein coatings

Abstract

The present invention relates to a method for coating an inert or naturally occurring material with a silk polypeptide. It further relates to a coated inert or naturally occurring material obtainable by said method and to uses thereof. It also relates to products comprising said coated material.

Claims

1. A method of coating hair or skin with a silk polypeptide, the method comprising the steps of: i) providing an aqueous solution of the silk polypeptide, and ii) applying the solution on the hair or skin, thereby coating the hair or skin with the silk polypeptide and improving hydrophilicity of the hair or skin, wherein the silk polypeptide consists of (C).sub.m, (C).sub.mNR.sub.z, NR.sub.z(C).sub.m, or NR.sub.z(C).sub.mNR.sub.z; or the silk polypeptide consists of (C).sub.m, (C).sub.mNR.sub.z, NR.sub.z(C).sub.m, or NR.sub.z(C).sub.mNR.sub.z linked to an artificial tag, wherein the tag is a lipid, a dye, a conjugated metal, an activated carbon, or a tag to facilitate purification of the silk polypeptide, wherein C is a repetitive unit having an amino acid sequence of SEQ ID NO: 21, m is an integer from 2 to 80, NR is a non-repetitive (NR) unit NR3 having the amino acid sequence of SEQ ID NO: 41 or NR4 having the amino acid sequence of SEQ ID NO: 42, and z is an integer from 1 to 3.

2. The method of claim 1, wherein the hair is human hair or animal hair.

3. The method of claim 1, where the hair is hair for extensions, periwigs, hair pieces, or toupees.

4. The method of claim 1, wherein the solution is applied using dip coating, spray coating or padding.

5. The method of claim 1, wherein the concentration of the silk polypeptide in the solution is in the range of 0.1 wt %/vol to 30 wt %/vol.

6. A naturally occurring material coated with a silk polypeptide and obtainable by the method of claim 1, wherein the natural occurring material is hair or skin and wherein the silk polypeptide consists of (C).sub.m, (C).sub.mNR.sub.z, NR.sub.z(C).sub.m, or NR.sub.z(C).sub.mNR.sub.z; or the silk polypeptide consists of (C).sub.m, (C).sub.mNR.sub.z, NR.sub.z(C).sub.m, or NR.sub.z(C).sub.mNR.sub.z linked to an artificial tag, wherein the tag is a lipid, a dye, a conjugated metal, an activated carbon, or a tag to facilitate purification of the silk polypeptide, wherein C is a repetitive unit having the amino acid sequence of SEQ ID NO: 21, m is an integer from 2 to 80, NR is a non-repetitive (NR) unit NR3 having the amino acid sequence of SEQ ID NO: 41 or NR4 having the amino acid sequence of SEQ ID NO: 42, and z is an integer from 1 to 3.

7. The method of claim 1, wherein the artificial tag to facilitate the purification of said silk polypeptide is a T7 tag.

8. The material of claim 6, wherein the artificial tag to facilitate the purification of said silk polypeptide is a T7 tag.

9. The method of claim 1, wherein the silk polypeptide is C.sub.16.

10. The method of claim 6, wherein the silk polypeptide is C.sub.16.

11. The method of claim 1, wherein the aqueous solution of the silk polypeptide is prepared by first dissolving the silk polypeptide in guanidine thiocyanate or urea solution and then dialyzed against water or a Tris buffer.

12. The method of claim 6, wherein the aqueous solution of the silk polypeptide is prepared by first dissolving the silk polypeptide in guanidine thiocyanate or urea solution and then dialyzed against water or a Tris buffer.

13. The method of claim 1, wherein the silk polypeptide coating on the hair or skin has a thickness range from 1 nm to 50 m.

14. The method of claim 6, wherein the silk polypeptide coating on the hair or skin has a thickness range from 1 nm to 50 m.

15. The method of claim 13, wherein the silk polypeptide coating on the hair or skin has a thickness range from 0.5 m to 10 m.

16. The method of claim 14, wherein the silk polypeptide coating on the hair or skin has a thickness range from 0.5 m to 10 m.

17. The method of claim 15, wherein the silk polypeptide coating on the hair or skin has a thickness range from 1.0 m to 5 m.

18. The method of claim 16, wherein the silk polypeptide coating on the hair or skin has a thickness range from 1.0 m to 5 m.

19. A method of coating hair or skin with a silk polypeptide, the method comprising the steps of: i) providing an aqueous solution of the silk polypeptide, and ii) applying the solution on the hair or skin, thereby coating the hair or skin with the silk polypeptide and improving hydrophilicity of the hair or skin, wherein the silk polypeptide consists of (C).sub.m, (C).sub.mNR.sub.z, NR.sub.z(C).sub.m, or NR.sub.z(C).sub.mNR.sub.z, wherein C is a repetitive unit having an amino acid sequence of SEQ ID NO: 21, m is an integer from 2 to 80, NR is a non-repetitive (NR) unit NR3 having the amino acid sequence of SEQ ID NO: 41 or NR4 having the amino acid sequence of SEQ ID NO: 42, and z is an integer from 1 to 3, wherein the silk polypeptide directly adheres to the hair or skin.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: Technical processes to coat a thread with a silk polypeptide. According to FIG. 1, the silk polypeptide can be applied on a thread via spray coating or via dip coating.

(2) FIG. 2: Electron microscopy of a spider silk coated aramid (Kevlar) filament. FIG. 2 shows that the spider silk coating is uniformly distributed over the aramid (Kevlar) filament.

(3) FIG. 3: Technical process to covalently attach molecules to the spider silk coating. FIG. 3 shows the coating of an untreated inert synthetic fiber with a spider silk polypeptide and the subsequently coating of the coated inert synthetic fiber with an agent or a chemical molecule.

(4) FIG. 4: Electron microscopy of an untreated already woven fabric and a coated already woven fabric at different magnifications.

(5) FIG. 5: Laser scanning microscope image (laser-intensity-magnification 50) of a European blond bleached hair (A, B), before (A) and after coating (B) with spider silk polypeptide C.sub.16 (3%) and of a virgin European hair (C, D), before (C) and after (D) coating with AQ.sub.24NR.sub.3 (0.85%). The quadrates shown in FIG. 5 A and FIG. 5 B designate specific distinctive spots on the hair sample to allow comparison of the same section of the hair sample before and after coating.

(6) FIG. 6: Laser scanning microscope images (Laser-intensity-Magnification 150) of nylon (PA) fiber before (A) and after coating (B) with C16 (3%).

(7) FIG. 7: Fluorescence images (Proxima-Imager) under fluorescence light (EpiVex) and Cy5 filter. In FIG. 7 A, western-blot analysis of AQ.sub.24NR3 with and without Cy5 is shown. Human hair coated by Cy5-labeled AQ.sub.24NR3 is shown in FIGS. 7 B and C.

(8) FIG. 8: Untreated human skin (FIG. 8 A) and human skin after coating with silk protein (FIG. 8 B). After coating with silk protein, the hydrophobicity of the skin was significantly decreased, resulting in a much higher wetting behavior of the water droplet

EXAMPLES

(9) In order to perform coating reactions, the inventors exemplarily designed the synthetic silk polypeptides C.sub.16, C.sub.32, C.sub.16NR4, (AQ).sub.24 and (AQ).sub.24NR3 which are derived from the dragline silk proteins ADF-3 and ADF-4 from the European garden cross spider Araneus diadematus. The proteins were chosen based on previous observations that ADF-3 and ADF-4 as well as their variants display an efficient assembly behaviour.

Example I: Coating of an Aramid Fiber with the Spider Silk Polypeptide C.SUB.16

(10) A single aramid (Kevlar) fiber was incubated for 5 seconds in a HFIP (Hexafluoroisopropanol) solution containing 2 wt %/vol of the spider silk polypeptide C.sub.16 at room temperature (25 C.). After evaporation of the solvent, the spider silk polypeptide C.sub.16 formed a transparent film around the aramid fiber. The thickness of the film measured via electron microscopy was 3 m (see FIG. 2).

(11) The same results can be obtained with other solvents such as formic acid and water. Similar experiments were performed using nylon fibers, glass fibers, carbon fibers, cellulose fibers, PTFE (teflon) fibers, elastane (spandex) fibers and human hair. All examined fibers could be coated accordingly, using the described method.

(12) To test the chemical stability of the coated fiber, spider silk polypeptide C.sub.16 coatings were incubated for 24 hours in 8M urea, 6M guanidinium hydrochloride and 6M guanidinium thiocyanate. Spider silk polypeptide C.sub.16 coatings processed with 1M potassium phosphate or 100% methanol could only be dissolved in guanidinium thiocyanate. This remarkable chemical stability of spider silk polypeptide C.sub.16 coatings is identical to that of natural dragline silk and to that of recombinantly produced and assembled ADF-4. Previous studies could correlate assembly properties and stabilities of assembled structures directly with the amino acid sequences of the silk proteins. Thus, properties of spider silk coatings can directly be modified by altering the primary structure of the silk protein via manipulation of the corresponding silk gene.

Example II: Covalent Coupling Via Chemically Crosslinking of a Substance to the Silk Polypeptide C.SUB.16 .Coated on an Aramid Fiber

(13) Many applications of fibers require the presence of specific functionalities on the fiber surface. In order to demonstrate that the spider silk coatings can be subsequently modified with a substance, the chromophor fluorescein and the enzyme -galactosidase were chemically coupled to a silk polypeptide C.sub.16 coated on an aramid fiber as a proof of principle (see FIG. 3). The coupling was achieved by activating surface exposed carboxyl groups of the spider silk polypeptide C.sub.16 using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). The spider silk polypeptide C.sub.16 coated aramid fibers were further incubated with ethylenediamine leading to the formation of an amide. The remaining free amino group of ethylenediamine was subsequently coupled to fluoresceinisothiocyanate resulting in the efficient covalent linkage of fluorescein via formation of a stable thiourea derivative.

(14) Similarly, incubation of -galactosidase with EDC-activated C.sub.16 films led to the formation of amide bonds between carboxyl groups of the silk polypeptide C.sub.16 and primary amines (e.g. from lysine residues) of -galactosidase which were accessible at the enzyme's surface. After repeated washing of such modified fibers, -galactosidase activity could be detected using 5-bromo-4-chloro-3-indolyl--D-galactopyranoside (X-Gal) as a substrate.

Example III: Non-Covalent Coupling of a Substance to the Silk Polypeptide C.SUB.16 .Coated on an Aramid Fiber

(15) In addition to the above mentioned covalent coupling of a substance to spider silk polypeptide C.sub.16 coated aramid fibers, non-covalent coupling was also performed. A single aramid fiber was incubated for 5 seconds in a formic acid containing 2 wt %/vol of the spider silk polypeptide C.sub.16 and copper chloride or cobalt chloride at room temperature (25 C.). After separation from the bath and evaporation of the solvent, the cobalt chloride or the copper chloride formed a colored around the aramid fiber coated with the spider silk polypeptide C.sub.16.

Example IV: Coating of a Woven Fabric of Cotton with the Spider Silk Polypeptide C.SUB.16

(16) Not only single fibers but also already woven fabrics are suitable templates for coating with spider silk. An already woven fabric of cotton and a cotton fibre were separately incubated in a 2 wt %/vol spider silk polypeptide C.sub.16 solution. After drying, the woven fabric of cotton and the cotton fibre showed a comparable coating behaviour (see FIG. 4).

(17) The direct coating of prefabricated fabrics, thus, results in a comparable, but different, coating pattern in comparison to fabrics made out of already silk-coated fibers. With fabric-coating, for example, the interspaces can be coated, whereas otherwise only the fibers and the intersections, but not the interspaces are engulfed by the spider silk coating. This shows that either coating of the fibers before weaving or treating of the already woven fabric results in evenly coated materialsuitable for different applications.

Example V: Dip Coating of an Aramid Thread with the Spider Silk Polypeptide C.SUB.16

(18) To perform the dip coating of an aramid thread with the spider silk polypeptide C.sub.16, the spider silk polypeptide C.sub.16 was dissolved in an aqueous solution (10 mM Tris, pH 7.5). The concentration of the silk polypeptide C.sub.16 in the aqueous solution was 2 wt %/vol. The dip coating procedure (see also FIG. 1) included: i) immersion: the aramid thread was immersed in the spider silk polypeptide C.sub.16 solution at a constant speed of 5 m/s; ii) incubation: the substrate was incubated in the coating solution for 2 minutes to allow for the coating material to adhere to the substrate iii) withdrawal: the excess of the substrate was removed from the aramid thread at a constant speed of 5 m/s; and iv) post treatment: the coating was dried at room temperature (25 C.).

Example VI: Spray Coating of an Aramid Thread with the Spider Silk Polypeptide C.SUB.16

(19) To perform the spray coating of an aramid thread with the spider silk polypeptide C.sub.16, the spider silk polypeptide C.sub.16 was dissolved in an aqueous solution (10 mM Tris, pH 7.5). The concentration of the silk polypeptide C.sub.16 in the aqueous solution was 2 wt %/vol. The spray coating procedure (see also FIG. 1) included: i) Preparation: the spider silk polypeptide C.sub.16 solution was transferred into a spray can or spraying device: ii) Coating: the silk polypeptide C.sub.16 solution was uniformly distributed onto the aramid thread by the spray can iii) Post treatment: the coating was dried at room temperature (25 C.).

Example VII: Padding of an Aramid Thread with the Spider Silk Polypeptide C.SUB.16

(20) Spider silk polypeptide C.sub.16 was dissolved in formic acid. The concentration of the silk polypeptide C.sub.16 in formic acid was 10 mg/ml. Different types of thread (Teflon (Goodfellow, diameter of fiber: 0.0211 mm), cellulose (Goodfellow, diameter of fiber: 0.015 mm), cotton wool, Kevlar (Goodfellow, diameter of fiber: 0.017 mm), elastane (spandex), untreated human hair and treated (free of dandruff) human hair) have been coated according the dip coating method. Spandex and cotton wool threads have been washed in deionized H.sub.2O and dried before coating. The threads were incubated in the in the spider silk polypeptide C.sub.16/formic acid solution for 5 seconds to allow for the coating material to adhere to the substrate. The coating was dried at room temperature (25 C.).

Example VIII: Coating of a Nylon Thread with the Spider Silk Polypeptide

(21) Spider silk polypeptide C.sub.16 was dissolved in aqueous solution (10 mM Tris pH 7,5). The concentration of the silk polypeptide C.sub.16 in formic acid was 1 wt %/vol. The nylon thread (Goodfellow, diameter of fiber: 0.01 mm) was incubated in the in the coating solution for 5 seconds to allow for the coating material to adhere to the substrate. The coating was dried at room temperature (25 C.).

Example IX: Coating of a Material with Different Spider Silk Proteins

(22) Different proteins C.sub.16, AQ.sub.24NR.sub.3, C.sub.16NR.sub.4, AQ.sub.24, C.sub.8 and C.sub.32 were used in order to coat a material (coated glass slide, particularly glass slide coated with a material having a silicium matrix, Sciences Services, Munich, Germany). The coating of a glass slide with different proteins C.sub.16, AQ.sub.24NR.sub.3, C.sub.16NR.sub.4, AQ.sub.24, C.sub.8 and C.sub.32 has been exemplarily shown with a coated glass slide, particularly glass slide coated with a silicium matrix. The results of the coating of the coated glass slide, particularly glass slide coated with a silicium matrix, with different proteins C.sub.16, AQ.sub.24NR.sub.3, C.sub.16NR.sub.4, AQ.sub.24, C.sub.8 and C.sub.32 are comparable to the coating of other glass slides (which are not coated, particularly not coated with a material having a silicium matrix) with different proteins C.sub.16, AQ.sub.24NR.sub.3, C.sub.16NR.sub.4, AQ.sub.24, C.sub.8 and C.sub.32. The final protein concentrations of C.sub.16, AQ.sub.24NR.sub.3 and C.sub.16NR.sub.4 in an aqueous Tris buffered solution were 3%, 0.5%, and 2.1%, respectively. The dip-coating method was used. Said method comprised the following steps: 60s dipping in the solution, 30 s drying out the solution, re-plunging the material in the solution 30 times and rinsing with purified water (Milli-Q). AQ.sub.24, C.sub.8 and C.sub.32 were dissolved in formic acid at the concentration of 2%. The spin-coating method was used at 1000 rpm for 1 min. The surface of the material (coated glass slide, particularly glass slide coated with a material having a silicium matrix,) was then scratched using a small needle. The height difference between the coating and the raw material was analyzed on different spots using a laser scanning microscope (VK 9700 Keyence, Neu Isenburg, Germany).

(23) Table 1 shows that the material (glass slide) could be homogenously coated with different spider silk proteins. The thickness of the coating depends on the nature of the proteins (hydrophilic & charged), the wettability of the solvent and the coating method.

(24) TABLE-US-00001 TABLE 1 Spider silk protein Coating Coating thickness (nm) Homogeneity C.sub.16 yes 316 14 AQ.sub.24NR.sub.3 yes 262 7 C.sub.16NR.sub.4 yes 130 21 AQ.sub.24 yes 113 10 C.sub.8 yes 66 8 C.sub.32 yes 80 8 (: highly homogenous coating, : homogenous coating)

Example X: Coating of a Material with the Spider Silk Polypeptide C.SUB.16 .Using Different Solvents

(25) Different solvents (Tris 100 mM, pH 8; Trifluoroacetic acid; Formic acid; Hexafluoro isopropanol) were used in order to dissolve C.sub.16 and coat a material (coated glass slide, particularly glass slide coated with a material having a silicium matrix, Sciences Services; Munich, Germany). The coating of a glass slide with different proteins C.sub.16, AQ.sub.24NR.sub.3, C.sub.16NR.sub.4, AQ.sub.24, C.sub.8 and C.sub.32 has been exemplarily shown with a coated glass slide, particularly glass slide coated with a silicium matrix. The results of the coating of the coated glass slide, particularly glass slide coated with a silicium matrix, with different proteins C.sub.16, AQ.sub.24NR.sub.3, C.sub.16NR.sub.4, AQ.sub.24, C.sub.8 and C.sub.32 are comparable to the coating of other glass slides (which are not coated, particularly not coated with a material having a silicium matrix) with different proteins C.sub.16, AQ.sub.24NR.sub.3, C.sub.16NR.sub.4, AQ.sub.24, C.sub.8 and C.sub.32. For the coating, an aqueous solution (Tris (100 mM, pH 8) was used. The final protein concentration of C.sub.16 in said solution was 3%. The dip-coating method was used. Said method comprised the following steps: 60 s dipping in the solution, 30 s drying out the solution, re-plunging the material in the solution 30 times and rinsing with millipore water. C.sub.16 was also dissolved directly in Trifluoroacetic acid, Formic acid and Hexafluoro isopropanol at a final concentration of 1%. The spin-coating method was used at 1000 rpm for 1 min. The surface of the coated material (coated glass slide, particularly glass slide coated with a material having a silicium matrix,) was then scratched using a small needle. The height difference between the coating and the raw material was analyzed on different spots using a laser scanning microscope (VK 9700 Keyence, Neu-Isenburg, Germany).

(26) Table 2 shows that the material (glass slide) could be coated with C.sub.16. The thickness depends on the nature of the protein, the wettability of the solvent and the coating method.

(27) TABLE-US-00002 TABLE 2 Solvent Coating Coating thickness (nm) Homogeneity Tris 100 mM, pH 8 yes 316 14 Trifluoroacetic acid yes 134 8 Formic acid yes 44 3 Hexafluoroisopropanol yes 53 14 (: highly homogenous coating, : homogenous coating)

Example XI: Coating of Naturally Occurring Materials with C.SUB.16

(28) C.sub.16 and AQ.sub.24NR.sub.3 were used to coat different naturally occurring materials (organic materials) such as human hair, cotton (Obergarn fiber), rubber, wool and cellulose (Good fellow d=0.015 mm, Huntington, Great Britain). European blond bleached hair was drop-coated using an aqueous solution of C.sub.16 in Tris buffer (100 mM, pH 8) at a protein concentration of 3% until drying and rinsed with purified water (Milli Q). European virgin hair was drop-coated using a solution of AQ.sub.24NR.sub.3 dissolved in purified water (Milli Q) at a protein concentration of 0.85% until drying. Cotton, rubber, wool and cellulose were coated using an aqueous solution of C.sub.16 in Tris buffer (100 mM, pH 8) at a protein concentration of 1.35%. The dip-coating method was used. Said method comprised the following steps: 120 s dipping in the solution, 120 s drying out the solution, re-plunging the material in the solution 10 times and rinsing with purified water (Milli Q). Each coated fiber (coating) was compared to a non-coated reference fiber (reference). The radii of the coated fiber and the non-coated fiber of human hair, cotton, wool and cellulose were compared to quantify the thickness of the coating. The coatings were analyzed on different spots using a laser scanning microscope (VK 9700 Keyence; Neu-Isenburg, Germany). The results are summarized in Table 3.

(29) TABLE-US-00003 TABLE 3 Radius Radius Coating Roughness Reference Coating thickness Ra* (m) Material Coating (m) (m) (m) Coating Reference Homogeneity Treated Hair yes 30.8 34.25 3.45 0.287 0.32 Virgin Hair yes 35 38.7 3.7 0.256 0.371 Cotton yes ~2.45 x Rubber yes Wool yes 10.7 0.5 11.8 0.3 1 Cellulose yes 9.42 0.1 12.7 0.1 3.27 x (*Ra: arithmetic middle height; : highly homogenous coating, : homogenous coating; x: slightly homogenous coating)

(30) Table 3 shows the coating of different naturally occurring materials (organic materials). The materials showed an increased thickness after coating. Treated and Virgin hair showed a decrease in roughness. Cotton fibers rolled together to form a bigger fiber. Therefore, it was difficult to image a single fiber and estimate the diameter difference. Although the thickness could not be exactly estimated as the fibers did not have a constant diameter over their whole length, the coating was clearly visible. Wool could be coated homogeneously with C.sub.16. Cellulose could be coated with C.sub.16. Because of the characteristics of cellulose, no homogenous coating could be detected by the coating method in this experiment. As cellulose is not a round smooth fiber, the coating was not thick enough to be homogeneous. However, an increase of the coating cycle number can increase the homogeneity of the coating.

(31) Laser scanning images in FIG. 5 show differences in the coating of hair keratin fibers. For both bleached and virgin hair, the cuticles disappeared under the coatings and the fiber surfaces were more homogeneous. The roughness decreased with the thickness of the coating. The addition of material smoothed the hair fibers. The light reflection could be measured as laser intensity. For the reference bleached hair, the average value over the full length was 13198 and for the coated hair the average value was about 13632 (over 16384 grey steps). For the virgin hair, the reference average value was about 13517, while the average value for coated hair was 13746. The laser intensity is specified in grey steps. The minimum represents the value 0 and the maximum represents the value 16384. The higher the value the higher the reflection/brilliance of the sample/material. In case of bleached hair and virgin hair, the value for the coated hair was higher compared to a reference, respectively. The coating enhanced the brilliance of the fibers.

Example XII: Coating of Inorganic Inert Materials with Spider Silk Polypeptide C.SUB.16

(32) C.sub.16 was used to coat different inorganic inert materials such as glass (glass slide, Roth; Karlsruhe, Germany), carbon (carbon fiber plate, R&G GmbH) and metal (metal plate). For the glass slide, C.sub.16 was dissolved directly in formic acid (final protein concentration 1%). The spin-coating method was used at 1000 rpm for 1 min. For the carbon and metal plate, the coating was performed with C.sub.16 in an aqueous solution (Tris 100 mM, pH 8). The final concentration was 1.35%. The dip-coating method for the carbon plate comprised the following steps: 120 s dipping in the solution, 120 s drying out the solution, re-plunging the material in the solution 10 times and rinsing with millipore water. The dip-coating method for the metal plate comprised the following steps: 120 s dipping in the solution, 120 s drying out the solution, re-plunging the material in the solution, 1000 times, and rinsing with purified water (Milli Q). The surface of the coated material (metal plate, carbon plate) was then scratched using a small needle. The surface of the coated material (metal plate, carbon plate) was analyzed on different spots using a laser scanning microscope (VK 9700 Keyence, Neu-Isenburg, Germany). Coated and non-coated materials were compared. The height difference between the coating and the raw material estimated the thickness of the coating. The coating smoothed the substrate surface. The results are summarized in Table 4.

(33) TABLE-US-00004 TABLE 4 Coating Inorganic thickness Roughness* (nm) Homo- inert material Coating (nm) Coating Reference geneity Glass yes 76.5 11 Ra = 18 Ra = 30 Rq = 22 Rq = 37 Carbon fiber yes x plate Metal plate yes x (*Roughness: Ra: arithmetic middle height; Rq: quadratic height average, : homogenous coating; x: slightly homogenous coating)

(34) Table 4 shows the thickness of the C.sub.16 coating on inorganic materials. Glass, carbon and metal could be coated. In case of glass, homogenous coating could be detected. Coated glass showed a decreased roughness compared to non-coated glass (reference).

Example XIII: Coating of Synthetic Inert Materials with Spider Silk Polypeptide C.SUB.16

(35) C.sub.16 was used to coat different synthetic inert materials such as polyester (PET, Syngarn fiber), polyamide (PA, Good fellow d=0.01 mm), polytetrafluorethylene (PTFE, Good Fellow d=0.0211 mm), polypropylene (PP, Good Fellow plate), ultra high molecular weight polyethylene (UHMW PE, Good Fellow plate), elastane and polyaramid. The coating of polyester, polyamide, PTFE, polypropylene, UHMW polyethylene was performed with C.sub.16 in an aqueous solution (Tris 100 mM, pH 8). The final protein concentration was 1.35%. The dip-coating method was used. Said method comprised the following steps: 60 s dipping in the solution, 30 s drying out the solution, and re-plunging the sample in the solution 30 times and rinsing with millipore water. Elastane and polyaramid were coated using a solution of C.sub.16 in Tris buffer (100 mM, pH 8) with a final protein concentration of 14%. The fibers were dipped 3 times in the solution and rinsed with millipore water. Each material was compared to a non-coated reference. PE, PA, PTFE, elastane, and polyaramid were in form of a fiber. The radius of the fiber can be compared to estimate the thickness of the coating. It was not possible to estimate the coating thickness over the whole length of the fiber. Thus, the fibers were analyzed on different spots using a laser scanning microscope (VK 9700 Keyence, Neu-Isenburg, Germany). The results are shown in Table 5.

(36) TABLE-US-00005 TABLE 5 Synthetic Radius Radius Coating inert Coat- ref coated thickness Homo- material ing (m) (m) (m) geneity PET yes 4.7 0.4 5.8 0.4 1.16 PA yes 5 0.02 5.75 0.3 0.74 x PTFE yes 5.4 0.05 9.7 0.5 4.3 x PP yes UHMW PE yes Elastane yes 299.3 3 348.8 40.sup. 49.5 Polyaramid yes 79 1 84 1 5 x

(37) Table 5 shows the thickness of the C.sub.16 coating on synthetic inert materials. All materials could be coated. In case of PET, PA, PTFE, elastane, and polyaramid, an increase in the thickness of the coated synthetic inert material compared to the non-coated reference was detected. In case of PTFE fibers, the coating was slightly homogeneous. The coating thickness value of 4.3 m is an average. For PP and UHMW PE plates, a coating could be clearly detected. Elastane consists of many fibers which are rolled together. Thus, the measured thickness value represents the coating thickness on several fibers which are rolled together. The coating of the smooth surface of the polyamid (PA) fiber resulted in a less homogenous surface. This is shown by laser scanning microscope images (FIG. 6) of a polyamid (PA, nylon) fiber. A fiber before (FIG. 6 A) and after coating (FIG. 6 B) with C.sub.16 is shown. The coating was detectable all over the fiber. After coating, the fiber surface was less homogeneous (FIG. 6 B).

Example XIV: Coating of Human Hair with AQ.SUB.24.NR.SUB.3 .Crosslinked with a Cy5 Fluorescent Tag

(38) A solution of AQ.sub.24NR.sub.3 (protein concentration 4 mg/ml) in guanidine thiocyanate (5M) was prepared and dialyzed against Tris buffer 100 mM at pH 8 resulting in a protein solution at 1 mg/ml (the coating with this protein is shown in FIG. 7 B). In a further experiment, a solution of AQ.sub.24NR.sub.3 (protein concentration 2 mg/ml) in 5M Urea was prepared and dialyzed against 100 mM Tris buffer, at pH 8 (the coating with this protein is shown in FIG. 7 C). The protein solution was labeled with Amersham Cy5 Maleimide Mono-Reactive Dye (GE Healthcare, cat. no. PA25031). The bioconjugation with the fluorescent tag was done using the cysteine sulphydryl group (one per molecule). A solution of TCEP (10 L, Tris(2-carboxyethyl)phosphine) at 18 mg/ml in Tris, pH 7), used to reduce the disulphide bonds, was added to the protein solution. The protein solution was incubated for 10 min at room temperature (RT). The dye solution of Cy5maleimide (GE Healthcare) was mixed with 50 L of anhydrous Dimethyformamide (DMF); 40 L was added to the protein solution. The solution was incubated for 2 hours at RT and overnight at 4 C. The protein solution (3 g) was used directly for western-blot analysis.

(39) An untreated human hair was plunged in the protein solution for 10 min and rinsed with pure water (milli Q) (FIG. 7 B). The tip of an Indian hair was plunged in the labeled protein solution for 10 min and then air-dried (FIG. 7 C). The hair and the gel were imaged using Proxima with Epi-Vex light and Cy5 filter (FIG. 7 A). On the western-blot gel, labeled AQ.sub.24NR.sub.3 protein and non-labeled AQ.sub.24NR.sub.3 protein were compared. Only the labeled protein can be detected under the Cy5 filter. As a result, the fluorescent labeled protein emitted light, while the non-labeled could not be seen. The proteins were covalently bound to the fluorescent dye. Silk in solution can be chemically modified using the different addressable amino groups. Cy5-labeled AQ.sub.24NR.sub.3 protein can be used to coat different substrates, such as hair, without altering the coating characteristics as well as the fluorescence characteristics. The hair coating is visible under Cy5-filter (FIG. 7).

(40) As a general conclusion, the experiments show exemplarily that the method of the present invention allows coating of synthetic inert materials, inorganic inert materials, and naturally occurring materials. The coating homogeneity depends on the chemical nature of the silk (hydrophilicity and charges), the chemical nature of the material, the geometry of the material, the chosen solvent and the coating method. Chemically modified silk protein can also be coated on different substrates.

Example XV: Coating of Human Indian and Human Virgin Hair with AQ.SUB.24.NR.SUB.3

(41) The experiment shows the effects of protein-coating on European virgin hair (never treated with chemicals) and Indian hair (treated with chemicals; used for extensions). The protein AQ.sub.24NR.sub.3 was used in different protein concentrations for the treatment of hair. For a 0.2% protein solution, AQ.sub.24NR.sub.3 was dissolved in 5M Urea and dialyzed against pure water (Milli Q). For a 0.6% and 0.85% protein solution, AQ.sub.24NR.sub.3 was directly dissolved in pure water (Milli Q) without any dialyzing step. The European virgin hair or the Indian hair was plunged for about 10 min in the above mentioned protein solutions and then air-dried. Exactly the same spot on the European virgin hair or the Indian hair (marked by adhesive strip and permanent marker) was analyzed before and after treatment with the above mentioned protein solutions using a laser scanning microscope (VK 9700 Keyence; Neu Isenburg, Germany). The results are shown in Tables 6 to 9. The analysis was performed with a VK Analyzer of Keyence.

(42) TABLE-US-00006 TABLE 6 Thickness coating on Indian hair Radius Radius Coating Conditioner untreated hair treated hair thickness concentration [m] [m] [m] 0.2% 30.3 31.8 1.5 0.6% 35 37.7 2.7 0.85% 34.3 37.1 2.8

(43) TABLE-US-00007 TABLE 7 Average surface roughness (Ra) of Indian hair Ra Ra Roughness Conditioner untreated hair treated hair reduction concentration [m] [m] [m] 0.2% 0.271 0.149 0.122 0.6% 0.531 0.264 0.267 0.85% 0.525 0.149 0.376

(44) TABLE-US-00008 TABLE 8 Thickness coating on European virgin hair Radius Radius Coating Conditioner untreated hair treated hair thickness concentration [m] [m] [m] 0.2% 28.2 29.7 1.5 0.6% 35.7 38.7 3 0.85% 35 38.7 3.7

(45) TABLE-US-00009 TABLE 9 Average surface roughness (Ra) of European virgin hair Ra Ra Roughness Conditioner untreated hair treated hair reduction concentration [m] [m] [m] 0.2% 0.5213 0.415 0.098 0.6% 0.894 0.781 0.113 0.85% 0.371 0.256 0.115

(46) An increase of the concentration of the protein-solution (i.e. from 0.2% to 0.85%) resulted in an increase of the radius of the coated hair. The roughness of the hair surface decreased with the increasing thickness of the coating. In addition, the smoothness of the hair surface increased with the increasing thickness of the coating.

Example XVI: Coating of Human Skin with Spider Silk Protein C.SUB.16

(47) Untreated human skin (FIG. 8 A) and human skin after coating with silk protein (FIG. 8 B). A water droplet (dyed, for clarification) normally does not wet human skin due to the hydrophobic nature of intact skin. After coating with a silk protein, the hydrophobicity of the skin was significantly decreased, resulting in a much higher wetting behavior of the water droplet.

(48) In detail, a solution of the spider silk protein C.sub.16 (1 mg/ml) in Urea (5%) was prepared. After washing the skin of a hand with a sodium lauryl sulfate free soap and subsequent air drying, 40 l of the protein-Urea solution (B) and 40 l of a Urea (5%) control solution (without protein) (A) were applied on a defined skin area (see FIG. 8). The silk coating results in a continuous invisible film which increases the hydrophilic properties of skin and effects protection of skin. After drying 40 l pure water was applied on the skin (FIG. 8). Due to the increase of hydrophilic properties of the silk coated skin, water was able to permeate immediately into the skin (FIG. 8 B), whereas the water drop in FIG. 8 A did not permeate the uncoated skin. FIG. 8 B (silk coated skin) shows a water drop spread over a large area in contrast to FIG. 8 A (uncoated skin) showing a distinct water drop. The protective properties of the coated invisible film resisted several cycles of washing with water/soap and drying.