Silk polypeptides with improved properties

Abstract

The present invention relates to new silk polypeptides. The present invention further relates to articles produced therefrom and uses thereof.

Description

[0001] SILK POLYPEPTIDES WITH IMPROVED PROPERTIES

[0002] The present invention relates to silk polypeptides with improved properties. The present invention further relates to articles produced therefrom and uses thereof.

[0003] BACKGROUND OF THE INVENTION

[0004] Silk polypeptides such as spider silk polypeptides represent a unique family of structural molecules. They share several common features such as a core sequence of highly repetitive units flanked by non-repetitive carboxyl- (C-) and / or amino- (N-) terminal domains. Silk polypeptides exhibit superior mechanical properties. For example, silk polypeptide fibers have exceptional mechanical properties such as high toughness, elasticity and low density, which reach maximum values compared to other fiber materials. They are superior even compared to Kevlar and steel. These extraordinary properties stem from the specific polypeptide structure which is repetitive in nature. Silk polypeptides are promising materials for drug delivery and tissue engineering due to their biocompatibility, biodegradability, self-assembly, and controllable structure and morphology. Additionally, silk materials exhibit high encapsulation efficiency and controllable drug release kinetics due to the adjustment of crystalline P-sheet formation. Moreover, fibers comprising silk polypeptides are useful in textile applications due to their excellent mechanical properties.

[0005] Early issues with bacterial expression systems to recombinantly produce silk polypeptides included low levels of production and poor stability of the silk polypeptide gene. Transcriptional errors, in addition to base deletions within the DNA sequence presented further problems. These problems were attributable to the highly repetitive nature of the core domain amino acid sequence prompting depletion of the alanyl- and glycyl-tRNA pool. By adapting the silk polypeptide sequence, it has now been possible for some time to produce silk polypeptides in larger quantities biosynthetically.

[0006] However, biosynthetically produced silk polypeptides such as spider silk polypeptides are less tear-resistant than natural silk polypeptides. For high-performance textiles, though, the highest possible tensile strength is desirable.

[0007] Thus, there is a need to synthesize new silk polypeptides such as spider silk polypeptides that come close to or even exceed the properties of natural silk polypeptides.

[0008] The present inventors found that by targeted mutation of the repetitive units of silk polypeptides, the tensile strength of the fibers produced therefrom could be altered. The experiments were done with a silk polypeptide comprising a repetitive unit having the amino acid sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2). This repeat unit is a polyalanine containing consensus sequence, derived from the spider silk polypeptide Araneus diadematus fibroin 4 (ADF-4), containing 8 alanine residues, GPGXX, and GGX motifs.

[0009] In total, 120 silk polypeptides with variations in the above amino acid sequence were cloned. In the end, 25 of the 120 cloned silk polypeptide variants were selected, recombinantly produced, purified, and spun into fibers. These 25 biosynthetic silk polypeptide variants typically have 1 to 6 mutations per repeat unit. In the tensile strength tests, it was surprisingly found that 3 of these 25 biosynthetically produced silk polypeptide variants formed a fiber with higher tensile strength and tear-resistance. Common to all 3 higher tensile strength silk polypeptide variants is that mutations in one or more of the first three amino acids (GSS) flanking the poly-alanine N- terminal region resulted in a more hydrophobic silk polypeptide amino acid stretch / sequence than the corresponding silk polypeptide amino acid stretch / sequence in the wild-type repeat unit. Specifically, the serine residues (S) flanking the poly-alanine N-terminal region were replaced by a smaller amino acid or by a more hydrophobic amino acid such as glycine (G) or alanine (A). Overall, it can be said that the stronger hydrophobicity in this region seems to be responsible or the reason for the higher tensile strength and tear-resistance.

[0010] Fibers comprising these new silk polypeptides are, due to their high tensile strength and tearresistance, ideal for the textile industry. In addition, films comprising these new silk polypeptides are very stable and have a hydrophobic character useful in diverse applications.

[0011] SUMMARY OF THE INVENTION

[0012] In a first aspect, the present invention relates to a silk polypeptide comprising at least two repeat units, wherein each repeat unit is independently selected from a peptide having the amino acid sequence X1X2X3AAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 1) or a peptide variant thereof, wherein the amino acid sequence X1X2X3 is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2), wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 1 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit. In a second aspect, the present invention relates to a nucleic acid molecule encoding the silk polypeptide of the first aspect.

[0013] In a third aspect, the present invention relates to a method for producing a silk polypeptide comprising the step of:

[0014] (i) expressing the nucleic acid molecule of the second aspect in a cell or synthesizing the nucleic acid molecule of the second aspect in an in vitro transcription / translation system, thereby producing the silk polypeptide.

[0015] In a fourth aspect, the present invention relates to an article comprising the silk polypeptide of the first aspect.

[0016] In a fifth aspect, the present invention relates to a method for producing an article comprising the steps of:

[0017] (i) providing a solution comprising the silk polypeptide of the first aspect, and

[0018] (ii) forming an article out of / from the solution provided in (i).

[0019] In a sixth aspect, the present invention relates to the use of the silk polypeptide of the first aspect or the article of the fourth aspect in the textile industry.

[0020] In a seventh aspect, the present invention relates to the use of the silk polypeptide of the first aspect or the article of the fourth aspect for the coating of surfaces.

[0021] This summary of the invention does not necessarily describe all features of the present invention. Other embodiments will become apparent from a review of the ensuing detailed description.

[0022] DETAILED DESCRIPTION OF THE INVENTION

[0023] Definitions

[0024] Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0025] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (TUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland. Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, GenBank Accession Number sequence submissions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

[0026] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and / or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0027] The term “comprise” or variations such as “comprises” or “comprising” according to the present invention means the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The term “consisting essentially of’ according to the present invention means the inclusion of a stated integer or group of integers, while excluding modifications or other integers which would materially affect or alter the stated integer. The term “consisting of’ or variations such as “consists of’ according to the present invention means the inclusion of a stated integer or group of integers and the exclusion of any other integer or group of integers.

[0028] The terms “a” and “an” and “the” and similar reference used in the context of describing the invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0029] The terms “polypeptide” and “protein” are used interchangeably in the context of the present invention. They refer to a long peptide-bond-linked chain of amino acids, e.g. one that is > 35 amino acids long.

[0030] The term “peptide”, as used herein, refers to a short peptide-linked chain of amino acids, e.g. one that is < 35 amino acids long.

[0031] The term “silk polypeptide”, as used herein, refers to a structural molecule. The structural molecule comprises a core amino acid sequence of highly repetitive units, optionally flanked by non-repetitive carboxyl- (C-) and / or amino- (N-) terminal domains. The repetitive units can also be designated as repeat units. The repeat units further comprise a limited set of distinct shorter peptide motifs. The peptide motifs can be grouped into four major categories: GPGXX, GGX, Axor (GA)nand spacers. These categories of peptide motifs in silk polypeptides have been assigned structural roles. For example, it has been suggested that the GPGXX motif is involved in a P-turn spiral, probably providing elasticity. The GGX motif is known to be responsible for a glycine- rich 3i-helix. Both GPGXX and GGX motifs are thought to be involved in the formation of an amorphous matrix that connects crystalline regions, thereby providing elasticity of the fiber. Alanine-rich motifs typically contain 6 to 9 residues and have been found to form crystalline P- sheets. The spacers are often glycine rich. The glycine rich spacers typically contain charged groups and separate the iterated poly- Ala peptide motifs into clusters. A special characteristic of silk polypeptides is that they can perform self-assembly.

[0032] The term “self-assembly”, as used herein, refers to a process in which a disordered system of pre-existing silk polypeptides forms an organized structure or pattern as a consequence of specific, local interactions (e.g. van der Waals forces, hydrophobic interactions, hydrogen bonds, and / or salt-bridges, etc.) among the silk polypeptides themselves, without external direction or trigger although external factors might influence speed and nature of self-assembly. This particularly means that when two or more disordered and / or unfolded silk polypeptides are brought into contact, they interact with each other and consequently form an ordered three- dimensional structure. This three-dimensional structure can also be considered as a silk polypeptide aggregate. The change from a disordered system to an organized structure or pattern during self-assembly is characterized by a transition from a fluid state to a fibrillary or gelatinous / gel-like and / or solid state and a corresponding increase in viscosity. The transition from a fluid state to a gelatinous / gel-like and / or solid state can be monitored, for example, by optical measurement or rheology. These techniques are known to the skilled person. The transition from a fluid state to a gelatinous / gel-like and / or solid state can be monitored, for example, using optical methods.

[0033] Natural silk polypeptide fibers have exceptional mechanical properties such as high toughness, elasticity and low density, which reach maximum values compared to other fiber materials. They are superior even compared to Kevlar and steel. To make use of the utmost properties of natural silk polypeptides, different approaches have attempted to express fragments of natural silk polypeptide genes in recombinant fashion. Early issues with bacterial expression systems to produce recombinant silk polypeptides included low levels of production and poor stability of the silk polypeptide gene. Transcriptional errors, in addition to base deletions within the DNA sequence presented additional problems. These problems were attributable to the highly repetitive nature of the core domain amino acid sequence prompting depletion of the alanyl- and glycyl-tRNA pool. By adapting the silk polypeptide sequence, it has now been possible for some time to produce silk polypeptides in larger quantities biosynthetically.

[0034] The silk polypeptide comprising the amino acid sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2) as repeat unit (also known under the name “module C”) belongs to one of the first recombinantly produced silk polypeptides. In this recombinant silk polypeptide, individual modules C have been combined to produce a mimic of Araneus diadematus fibroin 4 (ADF-4).

[0035] However, the properties of recombinant silk polypeptides, e.g. of silk polypeptides comprising module C repetitions, often do not match those of naturally occurring silk polypeptides. For example, the recombinant silk polypeptides, e.g. the silk polypeptides comprising module C repetitions, are less tear-resistant than natural silk polypeptides. For high- performance textiles, though, the highest possible tensile strength is desirable.

[0036] The present inventors found that by targeted mutation of the repetitive units of silk polypeptides, the tensile strength of fibers produced therefrom can be altered. The experiments were done with a silk polypeptide comprising a repetitive unit having the amino acid sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2). Several new silk polypeptides were designed, selected, recombinantly produced, and tested. Out of these, the present inventors identified new silk polypeptides whose fibers have a tensile strength that is comparable to or even higher than that of naturally occurring silk polypeptides. Fibers comprising these new silk polypeptides are, due to their high tensile strength, ideal for the textile industry. In addition, films comprising these new silk polypeptides are very stable and have a hydrophobic character useful in diverse applications. These new silk polypeptides are designated in the following as silk polypeptides of the present invention.

[0037] An exemplarily process for producing the silk polypeptides of the present invention is described in WO 2006 / 008163.

[0038] The term “silk polypeptide fiber”, as used herein, refers to an object that is significantly longer than it is wide. It comprises or consists of the silk polypeptide of the present invention. The term “silk polypeptide fiber”, as used herein, also encompasses a monofilament or multifilament silk polypeptide fiber. A monofilament silk polypeptide fiber is composed of a single (mono)filament. A multifilament silk polypeptide fiber is composed of a number of (mono)filaments. For example, a multifilament fiber may be composed of between 2 and 1000 (mono)filaments, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 (mono)filaments. The silk polypeptide fibers can be produced by wet or dry spinning. Preferably, the silk polypeptide fibers of the present invention are produced by wet spinning.

[0039] The term “silk polypeptide fiber”, as used herein, further encompasses single-drawn and multidrawn (e.g. double-drawn) silk polypeptide fibers. Said silk polypeptide fibers have been stretched one or more times during their preparation process. An exemplarily process for processing a silk polypeptide into fibers which may be used in the present invention is described in WO 2014 / 037453.

[0040] The term “wet spinning”, as used herein, refers to the production process of silk polypeptide fibers from polymer solutions that are injected or extruded into a precipitation / coagulation bath comprising a solvent. The polymer precipitates and one or more filaments can be wound up as a fiber, specifically after several further processing steps, e.g. one or more drawing steps. The wet spinning process is used for polymers that either cannot be melted, are very temperature-sensitive, or can only be processed in special solvent systems. The silk polypeptide fibers can subsequently be drawn / stretched to increase strength and orientation. This may be done while the polymer is still solidifying or after it has been formed. The solvent used for the precipitation / coagulation bath may be a molecule containing at least one hydroxyl group, e.g. methanol, ethanol, Hexafluorisopropanol (HFIP), or polyethylene gycol (PEG).

[0041] The term “drawing”, as used herein, refers to a technique in which the cross-sectional area of the silk polypeptide fiber is reduced. It is preferred that drawing leads to a reduction of the cross-sectional area of the silk polypeptide fiber of at least 10%, at least 20%, at least 30%, at least 40%, of least 50%, at least 60%, at least 70%, or at least 80%. An extruded silk polypeptide fiber can be drawn. Alternatively, an already drawn silk polypeptide fiber can be drawn again (e.g. in a multi-stage drawing). The drawing of the silk polypeptide fibers leads to a silk polypeptide fiber extension. It is preferred that the extension is by at least 2-fold, at least 4-fold, at least 5 -fold, at least 6-fold, at least 7-fold, or at least 8-fold in comparison to the length of the silk polypeptide as extruded or as already drawn. Preferably, such extension / drawing is carried out in the presence of the coagulation solution, washing solution, or during drying of the fiber, e.g. the silk polypeptide is at least partially submerged in the coagulation solution. This solution may also be designated as stretching solution. It can have the same composition as the coagulation solution. The coagulation solution may be a solution comprising a molecule containing at least one hydroxyl group, e.g. methanol, ethanol, Hexafluorisopropanol (HFIP), or polyethylene gycol (PEG).

[0042] Without wishing to be bound by any theory it is believed that the silk polypeptide fiber drawing leads to an alignment and more regular distribution of the silk polypeptide molecules within the fiber and, thereby, improves the mechanical properties of the fiber. Such extension can be carried out in a continuous or discontinuous process. In the continuous process it is preferred that the fiber is exposed to a pulling force.

[0043] The term “silk polypeptide yam”, as used herein, refers to an object composed of silk polypeptide fibers of the present invention. The silk polypeptide yam can be spun from silk polypeptide fibers, e.g. wet using a chemical-technical wet spinning process or dry using a mechanical spinning process. The silk polypeptide yam can also be produced by twisting single silk polypeptide fibers. During twisting, individual silk polypeptide fibers are turned together in order to achieve a silk polypeptide yarn with improved tensile strength. The yarn comprising the silk polypeptide of the present invention can be used in the textile industry, e.g. in order to produce fabrics, e.g. woven and non-woven fabrics. It has an excellent tensile strength and tearresistance.

[0044] The term “article”, as used herein, refers to any object which is produced from the silk polypeptide of the present invention. The article comprises or consists of the silk polypeptide of the present invention. The article is preferably a fiber, a yarn, more preferably a fabric comprising the yarn, or a film comprising the silk polypeptide of the present invention. The fabric may be a woven or non-woven fabric.

[0045] As mentioned above, silk polypeptide fibers such as spider silk polypeptide fibers have remarkable mechanical properties. Spider silk polypeptide fibers are produced by spiders. The problem is that spiders cannot be farmed because of their cannibalistic and territorial nature. Hence, large amounts of spider silk cannot be produced from spiders. Genetic engineering is an alternative approach to produce large quantities of spider silk polypeptides. The term “recombinant silk polypeptide”, as used herein, refers to molecules which are biotechnologically or recombinantly produced in host organisms. The recombinant production of silk polypeptides is, for example, described in Current Biology, Vol. 14, 2070-2074, 2004 and Current Biology, Vol. 14, 2070-2074, November 23, 2004. They have been created using transformed host cells or transiently transfected cell cultures. In turn, the term “synthetic silk polypeptide”, as used herein, refers to molecules that are produced chemically or biochemically.

[0046] Chemical silk polypeptide production allows the precise control of the synthesis of desired sequences. For example, silk polypeptides can be synthesized chemically via a two-step chemical synthesis method, that is, chemoenzymatic polymerization, e.g. using papain, followed by postpolycondensation, e.g. using polyphosphoric acid as a condensing agent.

[0047] Biochemical silk polypeptide production may be carried out in an in vitro transcription / translation system. This system is cell-free. Cell-free protein synthesis means the in vitro production of proteins without living cells. Instead, special cell extracts or lysates are used, which contain the protein synthesis apparatus and all other necessary components. In principle, extracts for cell-free protein expression can be obtained from any type of cell. However, systems based on E. coli, wheat germ, rabbit reticulocytes and insect cells are most frequently used. After the addition of amino acids, an energy source, salts and other cofactors, theoretically any protein encoded by a DNA or RNA template can be synthesized. The in vitro synthesis of proteins in cell-free extracts is an important tool for molecular biologists and has a variety of applications, including the rapid identification of gene products (e.g. proteomics), localization of mutations through synthesis of truncated gene products, protein folding studies, and incorporation of modified or unnatural amino acids for functional studies.

[0048] The use of in vitro translation systems can have advantages over in vivo gene expression when the over-expressed product is toxic to the host cell, when the product is insoluble or forms inclusion bodies, or when the protein undergoes rapid proteolytic degradation by intracellular proteases.

[0049] Diverse in vitro protein synthesis kits are available on the market. The firm BioLabs, for example, offers a PURExpress® in vitro protein synthesis kit. The PURExpress® in vitro protein synthesis kit is a cell-free transcription / translation system and consists of defined, reconstituted and highly purified components of the E. coli transcription / translation apparatus. All protein factors are produced recombinantly as His-tag fusions, the ribosomes and tRNAs are highly purified from E. coli. This means that the system is free of contaminating exonucleases, RNases and proteases. The firm Promega provides, for example, a TNT® Quick coupled transcription / translation system which might also be an option. The LEXSY in vitro translation system (Jena Bioscience) applies T7 RNA polymerase to generate target mRNA and cell extracts with functional ribosomes and other essential components for cell-free protein synthesis.

[0050] The term “tensile strength”, as used herein, refers to a measurement of the force required to pull the silk polypeptide fiber or yarn of the present invention to the point where it breaks. The tensile strength of a silk polypeptide fiber or yam of the present invention is the maximum amount of tensile stress that it can take before failure, for example breaking. There are three typical definitions of tensile strength:

[0051] Yield strength - The stress a material can withstand without permanent deformation. This is not a sharply defined point. Yield strength is the stress which will cause a permanent deformation of 0.2% of the original dimension.

[0052] Ultimate strength - The maximum stress a material can withstand.

[0053] Breaking strength - The stress coordinate on the stress-strain curve at the point of rupture.

[0054] The skilled person knows techniques to determine the tensile strength and tear-resistance of a fiber. For example, for a given single silk polypeptide fiber, to assess its strength, a monotonic longitudinal load is applied to the fiber until its failure. This failure load is then normalised by the cross-section, possibly at the breaking point, to obtain the tensile strength of the fiber. The testing of the tensile strength of a silk polypeptide fiber can, for example, be performed according to DIN EN ISO 2062:04 / 2010. For further information, it is referred to the examples of the present patent application.

[0055] As mentioned above, the present inventors designed new silk polypeptides. These new silk polypeptides are based on the silk polypeptide comprising a repetitive unit having the amino acid sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2). This repeat unit is a polyalanine containing consensus sequence, derived from the spider silk polypeptide Araneus diadematus fibroin 4 (ADF-4), containing 8 alanine residues, GPGXX and GGX motifs. Said new silk polypeptides are designated as silk polypeptides of the present invention.

[0056] Common to all silk polypeptides of the present invention is that mutations in one or more of the first three amino acids (GSS) flanking the poly-alanine N-terminal region resulted in a more hydrophobic amino acid stretch / sequence than the corresponding amino acid stretch / sequence in the wild-type repeat unit. Specifically, the serine residues (S) flanking the poly-alanine N- terminal region were replaced by a small amino acid without a hydroxyl group.

[0057] The term “hydrophobicity”, as used herein, indicates the degree to which an amino acid prefers a non-polar medium (such as ethanol) to a polar one (such as water). In contrast thereto, term “hydrophilicity”, as used herein, indicates the degree an amino acid attracts water.

[0058] The determination whether an amino acid stretch / sequence is more hydrophobic than the corresponding amino acid stretch / sequence in the wild-type repeat unit can easily be made by the skilled person. Suitable computational tools are available which allow to answer this question (see, for example, https: / / web.expasy.org / protscale / ). These tools are usually based on hydrophobicity scales. Hydrophobicity scales are values that define the relative hydrophobicity or hydrophilicity of amino acid residues. Several hydrophobicity scales have been published for various uses, e.g. the Hopp-Woods scale, Kyte-Doolittle scale, the Engelman scale, or the Eisenberg scale. The present inventors preferably used the Hopp-Woods scale to determine the hydrophobicity of regions in silk polypeptides and to determine the hydrophobicity of entire silk polypeptides. In this respect, it is referred to the experimental section of this specification.

[0059] The hydrophobicity or hydrophilicity of an amino acid stretch / sequence or of an entire polypeptide can specifically be determined using a hydrophilicity plot. The term “hydrophilicity plot”, as used therein, refers to a quantitative analysis of the degree of hydrophobicity or hydrophilicity of amino acids. It can be used to characterize or identify possible structures or domains of a protein. The plot has the amino acid sequence of a protein on its x-axis, and the degree of hydrophobicity and hydrophilicity on its y-axis. There are a number of methods to measure the degree of interaction of polar solvents such as water with specific amino acids. For instance, the above mentioned Hopp-Woods scale or the Kyte-Doolittle scale may be used. Analyzing the shape of the plot gives information about partial structure of the protein.

[0060] The Hopp-Woods scale is used for detecting charged and polar residues in proteins or to determine the hydrophobicity of entire proteins. It serves as a hydrophobicity index where hydrophobic residues are indicated by negative values and hydrophilic residues are indicated by positive values. Accordingly, the lower the value, the more hydrophobic the amino acid / amino acid segment. For example, the amino acid serine (S) has a Hopp-Woods hydrophobicity score of 0.3 and is, thus, hydrophilic. Further, the amino acid glutamine (Q) has a Hopp-Woods hydrophobicity score of 0.2 and is, thus, less hydrophilic than the amino acid serine (S). Furthermore, the amino acid glycine (G) has a Hopp-Woods hydrophobicity score of 0.0 and is, thus, less hydrophilic than the amino acid serine (S) and / or glutamine (Q). In addition, the amino acid alanine (A) has a Hopp-Woods hydrophobicity score of - 0.5 and is, thus, hydrophobic, specifically in relation to the amino acid serine (S), the amino acid glutamine (Q), and / or the amino acid glycine (G).

[0061] Thus, different amino acids contribute to the hydrophobic character of an amino acid stretch / sequence to varying degrees. In the context of the present invention, it is, however, important that the mutations in one or more of the first three amino acids (GSS) flanking the poly-alanine N-terminal region collectively result in a more hydrophobic amino acid stretch / sequence than the corresponding amino acid stretch / sequence in the wild-type repeat unit. The present inventors used the Hopp-Woods score to determine the hydrophobicity of regions in silk polypeptides and to determine the hydrophobicity of entire silk polypeptides. In this respect, it is referred to the experimental section of this specification.

[0062] The higher hydrophobicity of the amino acid stretch / sequence preferably also affects the entire repeat unit, and more preferably the entire silk polypeptide. Accordingly, it is preferred, that the entire repeat unit is more hydrophobic than the corresponding repeat unit without mutation(s) (i.e. wild-type repeat unit). In addition, it is more preferred that the entire silk polypeptide is more hydrophobic than the corresponding silk polypeptide without mutation(s) (wild-type silk polypeptide). The higher hydrophobicity has also an effect on articles such as fibers made therefrom.

[0063] Thus, the term “hydrophobicity”, as used herein, also refers to the ability of the silk polypeptide fiber of the present invention to repel respectively to not attract water. Hydrophobicity is to be brought back to the chemical structure of the silk polypeptide fibers of the present invention. So, it is that most of the hydrophobic materials are nonpolar, meaning the electronegativity between the bonded atoms is below a certain threshold. Hydrophobicity is not to be confused with lipophilicity, despite those terms being somewhat related. Also, hydrophobicity is the exact opposite of hydrophilicity, which refers to materials that highly attract water.

[0064] The skilled person knows techniques to determine the hydrophobicity of a silk polypeptide fiber or fabric produced therefrom. For example, the hydrophobicity of a fiber or fabric can be determined with a wicking test. Vertical or horizontal wicking tests are known.

[0065] For a vertical wicking test, AATCC 197 is one of the standard methods. In this standard, the bottom of the specimen comes into contact with water. Then, the wicking distance by specified time intervals is recorded. The higher the wicking distance at the same interval, the better the fiber or fabric is in wicking. The lower the wicking distance at the same interval, the more hydrophobic is the fiber or fabric.

[0066] For a horizontal wicking test, AATCC 198 is one of the standard methods. A specific amount of water is given from a specific height. The time of the water spreading out from the center to the edge of the circle (100 mm in diameter) is recorded. In this method, the shorter the time, the better the fiber or fabric is in wicking. The longer the time, the more hydrophobic is the fiber or fabric.

[0067] In other words, a silk polypeptide fiber that can transport moisture well (good wicking properties) has a higher hydrophilicity than a fiber that is less able to transport moisture. In contrast thereto, a silk polypeptide fiber that is poor at transporting moisture (bad wicking properties) has a higher hydrophobicity than a fiber that is better at transporting moisture.

[0068] For further information, it is referred to the examples of the present patent application.

[0069] The term “coating”, as used herein, refers to a covering that is applied to a support or substrate to be coated. The coating itself may be an all-over coating of the support or substrate, completely covering of the support or substrate, or it may only cover parts of the support or substrate.

[0070] In one embodiment, the coating covers at least 1%, preferably at least 30%, more preferably at least 50%, even more preferably at least 80%, and most preferably at least 90% or even 100% of the surface of the support or substrate. In one preferred embodiment, the coating is a uniform and / or homogenous coating. It has preferably a thickness of between 10 nm and 1 mm and more preferably a thickness of between 50 nm and 0.5 pm.

[0071] The coating may be achieved by dip coating and / or spray coating.

[0072] Residues in two or more amino acid sequences are said to “correspond” to each other if the residues occupy an analogous position in the different amino acid structures. It is well known in the art that analogous positions in two or more amino acid sequences can be determined by aligning the amino acid sequences based on amino acid sequence or structural similarities. Such alignment tools are well known to the person skilled in the art and can be, for example, obtained on the World Wide Web, for example, ClustalW (see www.ebi.ac.uk / clustalw) or Align (see http: / / www.ebi.ac.uk / emboss / align / index.html) using standard settings, preferably for Align EMBOSS: rneedle, Matrix: Blosum62, Gap Open 10.0, Gap Extend 0.5.

[0073] Embodiments of the invention

[0074] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous, unless clearly indicated to the contrary.

[0075] The present inventors found that by targeted mutation of the repetitive units of silk polypeptides, the tensile strength of the fibers produced therefrom could be altered. The experiments were done with a silk polypeptide comprising a repetitive unit having the amino acid sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2). This repeat unit is a polyalanine containing consensus sequence, derived from the spider silk polypeptide Araneus diadematus fibroin 4 (ADF-4), containing 8 alanine residues, GPGXX, and GGX motifs.

[0076] In total, 120 silk polypeptides with variations in the above amino acid sequence were cloned. In the end, 25 of the 120 cloned silk polypeptide variants were selected, recombinantly produced, purified, and spun into fibers. These 25 biosynthetic silk polypeptide variants typically have 1 to 6 mutations per repeat unit. In the tensile strength tests, it was surprisingly found that 3 of these 25 biosynthetically produced silk polypeptide variants formed a fiber with higher tensile strength and tear-resistance. Common to all 3 higher tensile strength silk polypeptide variants is that mutations in one or more of the first three amino acids (GSS) flanking the poly-alanine N- terminal region resulted in a more hydrophobic silk polypeptide amino acid stretch / sequence than the corresponding silk polypeptide amino acid stretch / sequence in the wild-type repeat unit. Specifically, the serine residues (S) flanking the poly-alanine N-terminal region were replaced by a small amino acid without a hydroxyl group such as glycine (G) or alanine (A). Overall, it can be said that the stronger hydrophobicity in this region seems to be responsible or the reason for the higher tensile strength and tear-resistance.

[0077] Fibers comprising these new silk polypeptides are, due to their high tensile strength and tearresistance, ideal for the textile industry. In addition, films comprising these new silk polypeptides are very stable and have a hydrophobic character useful in diverse applications. Thus, in a first aspect, the present invention relates to a (recombinant / synthetic) silk polypeptide comprising at least two repeat units, e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

[0078] 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,

[0079] 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,

[0080] 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,

[0081] 92, 93, 94, 95, 96, 97, 98, 99, or 100 repeat units, wherein each repeat unit is independently selected from a peptide having the amino acid sequence X1X2X3AAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 1) or a peptide variant thereof, wherein the amino acid sequence X1X2X3 is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2), wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 1 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit. However, the peptide variant may encompass amino acid exchanges which increase the hydrophobicity of the resulting repeat unit.

[0082] Specifically, the (recombinant / synthetic) silk polypeptide comprises between 2 to 100 repeat units. More specifically, the (recombinant / synthetic) silk polypeptide comprises between between 2 to 80 repeat units. Even more specifically, the (recombinant / synthetic) silk polypeptide comprises between 4 to 60 repeat units. Still even more specifically, the (recombinant / synthetic) silk polypeptide comprises between 8 to 48 repeat units.

[0083] Thus, the present invention relates to a (recombinant / synthetic) silk polypeptide specifically comprising between 2 to 100 repeat units, more specifically comprising between 2 to 80 repeat units, even more specifically comprising between 4 to 60 repeat units, and still even more specifically between 8 to 18 repeat units, wherein each repeat unit is independently selected from a peptide having the amino acid sequence

[0084] X1X2X3AAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 1) or a peptide variant thereof, wherein the amino acid sequence X1X2X3 is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2), wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 1 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit. Hydrophobicity of an amino acid indicates the degree to which an amino acid prefers a non-polar medium (such as pentane) to a polar one (such as water). The determination whether the amino acid sequence X1X2X3 is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit

[0085] GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP can easily be made by the skilled person. Suitable computational tools are available which allow to answer this question (see, for example, https: / / web.expasy.org / protscale / ). These tools are usually based on hydrophobicity scales. Hydrophobicity scales are values that define the relative hydrophobicity or hydrophilicity of amino acid residues. Several hydrophobicity scales have been published for various uses, e.g. the Hopp-Woods scale, Kyte-Doolittle scale, Engelman scale, or Eisenberg scale.

[0086] The hydrophobicity of the amino acid sequence X1X2X3 can specifically be determined using a hydrophilicity plot. A hydrophilicity plot refers to a quantitative analysis of the degree of hydrophobicity or hydrophilicity of amino acids. It can be used to characterize or identify possible structures or domains of a protein. The plot has the amino acid sequence of a protein on its x-axis, and the degree of hydrophobicity and hydrophilicity on its y-axis. There are a number of methods to measure the degree of interaction of polar solvents such as water with specific amino acids. For instance, the above mentioned Hopp-Woods scale or the Kyte-Doolittle scale may be used.

[0087] The present inventors preferably used the Hopp-Woods scale to determine the hydrophobicity. The Hopp-Woods scale is used for detecting charged and polar residues in proteins or to determine the hydrophobicity of entire proteins. It serves as a hydrophobicity index where, hydrophobic residues are indicated by negative values and hydrophilic residues are indicated by positive values. Accordingly, the lower the value, the more hydrophobic the amino acid / amino acid segment. For example, the amino acid serine (S) has a Hopp-Woods hydrophobicity score of 0.3 and is, thus hydrophilic. Further, the amino acid glutamine (Q) has a Hopp-Woods hydrophobicity score of 0.2 and is thus, less hydrophilic than the amino acid serine (S). Furthermore, the amino acid glycine (G) has a Hopp-Woods hydrophobicity score of 0.0 and is thus, less hydrophilic than the amino acid serine (S) and / or glutamine (Q). In addition, the amino acid alanine (A) has a Hopp-Woods hydrophobicity score of - 0.5 and is, thus, hydrophobic, specifically in relation to the amino acid serine (S), the amino acid glutamine (Q), and / or the amino acid glycine (G).

[0088] Thus, different amino acids contribute to the hydrophobic character of an amino acid sequence to varying degrees. In the context of the present invention, it is, however, important that the entire amino acid sequence X1X2X3 is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2). The present inventors preferably used the Hopp-Woods score to determine the hydrophobicity of the amino acid sequence X1X2X3. In this respect, it is referred to the experimental section of this specification.

[0089] In view of the above, it is particularly preferred that the amino acid sequence X1X2X3 which is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit has a hydrophobicity score of less than 0.2 according to Hopp-Woods. In contrast thereto, the amino acid sequence GSS in the wild-type repeat unit has a hydrophobicity score of 0.2 according to Hopp-Woods (Hopp-Woods score for GSS: 0.0 (G) + 0.3 (S) + 0.3 (S) = 0.6 / 3 (number of amino acids) = 0.2).

[0090] The higher hydrophobicity of the amino acid sequence X1X2X3 preferably also affects the entire repeat unit, and more preferably the entire silk polypeptide. Accordingly, it is preferred, that the entire repeat unit is more hydrophobic than the corresponding repeat unit without mutation(s) (i.e. wild-type repeat unit). In this case, the repeat unit having the amino acid sequence X1X2X3AAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 1) is more hydrophobic than the corresponding wild-type repeat unit GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2). Accordingly, it is more preferred that the entire silk polypeptide is more hydrophobic than the corresponding silk polypeptide without mutation(s) (wild-type silk polypeptide). In this case, the entire silk polypeptide comprising at least two repeat units, wherein the repeat unit has the amino acid sequence X1X2X3AAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 1) is more hydrophobic than the corresponding wild-type silk polypeptide comprising at least two repeat units having the amino acid sequence GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2).

[0091] As mentioned above, the peptide variants have 1, 2, or 3 amino acid exchanges in SEQ ID NO: 1 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit.

[0092] The 1, 2, or 3 amino acid exchanges in SEQ ID NO: 1 at amino acid positions 4 to 35 may be substitutions, additions, insertions, and / or deletions, preferably substitutions.

[0093] With regard to the different peptide variants mentioned above, the following is noted: The skilled person knows techniques to determine that the amino acid exchanges (e.g. substitutions, additions, insertions, and / or deletions, preferably substitutions) do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit. Suitable computational tools are available which allow to answer this question (see, for example, https: / / web.expasy.org / protscale / ). These tools are usually based on hydrophobicity scales which were determined for each amino acid. In addition, it is referred to the techniques described in the examples of the present specification.

[0094] In one preferred embodiment, the amino acids Xi, X2, and X3 are independently selected from the group consisting of Q, A, G, and S.

[0095] For example, in the amino acid sequence X1X2X3,

[0096] Xi is G, A or Q,

[0097] X2 is S, A, Q, or G, and / or

[0098] X3 is S, A, Q, or G.

[0099] Specifically, it should be noted that in case Xi is G, at least one of X2 or X3 is not S, in case X2 is S, Xi is not G and / or X3 is not S, and / or in case X3 is S, Xi is not G and / or X2 is not S.

[0100] Alternatively, it should be noted that in case Xi is G, X2 and / or X3 is A, Q, or G, in case X2 is S, Xi is A or Q and / or X3 is A, Q, or G, and / or in case X3 is S, Xi is A or Q and / or X2 is A, Q, or G]

[0101] In one more preferred embodiment, in the amino acid sequence X1X2X3,

[0102] Xi is G

[0103] X2 is A, Q, or G, and

[0104] X3is G.

[0105] In one even more preferred embodiment, in the amino acid sequence X1X2X3,

[0106] Xi is G

[0107] X2 is A, and

[0108] X3is G (V06).

[0109] In this case, the peptide has the amino acid sequence GAGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 3) or is a peptide variant thereof, wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 3 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit.

[0110] With a Hopp-Woods score of - 0.17, the amino acid sequence GAG is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit (Hopp-Woods score for GAG: 0.0 (G) + - 0.5 (A) + 0.0 (G) = - 0.5 / 3 (number of amino acids) = - 0.17). As mentioned above, the amino acid sequence GSS in the wild-type repeat unit has a hydrophobicity score of 0.2 according to Hopp-Woods. In one another more preferred embodiment, in the amino acid sequence X1X2X3,

[0111] Xi is Q or A,

[0112] X2 is G, and

[0113] X3is G.

[0114] In one another even more preferred embodiment, in the amino acid sequence X1X2X3,

[0115] Xi is Q,

[0116] X2 is G, and

[0117] X3is G (V23).

[0118] In this case, the peptide has the amino acid sequence QGGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 4) or is a peptide variant thereof, wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 4 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit.

[0119] With a Hopp-Woods score of 0.07, the amino acid sequence QGG is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit (Hopp-Woods score for QGG: 0.2 (Q) + 0.0 (G) + 0.0 (G) = 0.2 / 3 (number of amino acids) = 0.07). As mentioned above, the amino acid sequence GSS in the wild-type repeat unit has a hydrophobicity score of 0.2 according to Hopp-Woods.

[0120] In one another more preferred embodiment, in the amino acid sequence X1X2X3,

[0121] Xi is G,

[0122] X2 is S, and

[0123] X3is G (V47).

[0124] In this case, the peptide has the amino acid sequence GSGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 5), or is a peptide variant thereof, wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 5 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit.

[0125] With a Hopp-Woods score of 0.1, the amino acid sequence GSG is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit (Hopp-Woods score for GSG: 0.0 (G) + 0.3 (S) + 0.0 (G) = 0.3 / 3 (number of amino acids) = 0.1). As mentioned above, the amino acid sequence GSS in the wild-type repeat unit has a hydrophobicity score of 0.2 according to Hopp-Woods. The silk polypeptide may further comprise an amino acid sequence GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO: 6) and / or an amino acid sequence GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 7). A variant of the amino acid sequence according to SEQ ID NO: 6 may alternatively be comprised. Said variant has 1, 2, or 3 amino acid exchanges in said amino acid sequence at amino acid positions 1 to 24 and / or 26 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the silk polypeptide. The 1, 2, or 3 amino acid exchanges in SEQ ID NO: 6 at amino acid positions 1 to 24 and / or 26 to 35 may be substitutions, additions, insertions, and / or deletions, preferably substitutions.

[0126] In addition, a variant of the amino acid sequence according to SEQ ID NO: 7 may alternatively be comprised. Said variant has 1, 2, or 3 amino acid exchanges in said amino acid sequence at amino acid positions 1 to 19 and / or 21 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the silk polypeptide. The 1, 2, or 3 amino acid exchanges in SEQ ID NO: 7 at amino acid positions 1 to 19 and / or 21 to 35 may be substitutions, additions, insertions, and / or deletions, preferably substitutions.

[0127] As to the different peptide variants or variants mentioned above, the following is noted: The skilled person knows techniques to determine that the amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the repeat unit or entire silk polypeptide. Suitable computational tools are available which allow to answer this question (see, for example, http s : / / web . expasy . or g / protscal e / I . These tools are usually based on hydrophobicity scales which were determined for each amino acid. In addition, it is referred to the techniques described in the examples of the present specification.

[0128] In one still even more preferred embodiment, the (recombinant / synthetic) silk polypeptide comprises or consists of (SEQ ID NO: l)m, (SEQ ID NO: l)mSEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: l)m, (SEQ ID NO: l)mSEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO: l)m, wherein m is an integer of between 2 to 100, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

[0129] 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,

[0130] 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,

[0131] 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,

[0132] 95, 96, 97, 98, 99, or 100.

[0133] Specifically, the silk polypeptide comprises or consists of (SEQ ID NO: 3)m, (SEQ ID NO: 3)mSEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 3)m, (SEQ ID NO: 3)mSEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO: 3)m, wherein m is an integer of between 2 to 100, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100, the silk polypeptide comprises or consists of (SEQ ID NO: 4)m, (SEQ ID NO: 4)mSEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 4)m, (SEQ ID NO: 4)mSEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO:

[0134] 4)m, wherein m is an integer of between 2 to 100, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

[0135] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

[0136] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

[0137] 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

[0138] 94, 95, 96, 97, 98, 99, or 100, or the silk polypeptide comprises or consists of (SEQ ID NO: 5)m, (SEQ ID NO: 5)mSEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 5)m, (SEQ ID NO: 5)mSEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO:

[0139] 5)m, wherein m is an integer of between 2 to 100, e.g. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,

[0140] 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,

[0141] 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67,

[0142] 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,

[0143] 94, 95, 96, 97, 98, 99, or 100.

[0144] In one most preferred embodiment, the (recombinant / synthetic) silk polypeptide comprises or consists of (SEQ ID NO: 1)2, (SEQ ID NO: 1)4, (SEQ ID NO: 1)6, (SEQ ID NO: 1)8, (SEQ ID NO: l)i6, (SEQ ID NO: 1)32, (SEQ ID NO: 1)48, (SEQ ID NO: 1)2SEQ ID NO: 6, (SEQ ID NO: 1)4SEQ ID NO: 6 (SEQ ID NO: 1)6SEQ ID NO: 6, (SEQ ID NO: 1)8SEQ ID NO: 6, (SEQ ID NO: 1)I6SEQ ID NO: 6, (SEQ ID NO: 1)32SEQ ID NO: 6, (SEQ ID NO: 1)48SEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 1)2, SEQ ID NO: 6(SEQ ID NO: 1)4, SEQ ID NO: 6(SEQ ID NO: 1)6, SEQ ID NO: 6(SEQ ID NO: 1)8, SEQ ID NO: 6(SEQ ID NO: l)i6, SEQ ID NO: 6(SEQ ID NO: 1)32, SEQ ID NO: 6(SEQ ID NO: 1)48, (SEQ ID NO: 1)2SEQ ID NO: 7, (SEQ ID NO: 1)4SEQ ID NO: 7 (SEQ ID NO: 1)6SEQ ID NO: 7, (SEQ ID NO: 1)8SEQ ID NO: 7, (SEQ ID NO: 1)I6SEQ ID NO: 7, (SEQ ID NO: 1)32SEQ ID NO: 7, (SEQ ID NO: 1)48SEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO: 1)2, SEQ ID NO: 7(SEQ ID NO: 1)4, SEQ ID NO: 7(SEQ ID NO: 1)6, SEQ ID NO: 7(SEQ ID NO: 1)8, SEQ ID NO: 7(SEQ ID NO: l)i6, SEQ ID NO: 7(SEQ ID NO: 1)32, or SEQ ID NO: 7(SEQ ID NO: 1)48.

[0145] Specifically, the silk polypeptide comprises or consists of (SEQ ID NO: 3)2, (SEQ ID NO: 3)4, (SEQ ID NO: 3)6, (SEQ ID NO: 3)8, (SEQ ID NO: 3)i6, (SEQ ID NO: 3)32, (SEQ ID NO: 3)48, (SEQ ID NO: 3)2SEQ ID NO: 6, (SEQ ID NO: 3)4SEQ ID NO: 6 (SEQ ID NO: 3)6SEQ ID NO: 6, (SEQ ID NO: 3)8SEQ ID NO: 6, (SEQ ID NO: 3)i6SEQ ID NO: 6, (SEQ ID NO: 3)32SEQ ID NO: 6, (SEQ ID NO: 3)48SEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 3)2, SEQ ID NO: 6(SEQ ID NO:

[0146] 3)4, SEQ ID NO: 6(SEQ ID NO: 3)6, SEQ ID NO: 6(SEQ ID NO: 3)8, SEQ ID NO: 6(SEQ ID NO: 3)16, SEQ ID NO: 6(SEQ ID NO: 3)32, SEQ ID NO: 6(SEQ ID NO: 3)48, (SEQ ID NO:

[0147] 3)2SEQ ID NO: 7, (SEQ ID NO: 3)4SEQ ID NO: 7, (SEQ ID NO: 3)6SEQ ID NO: 7, (SEQ ID NO: 3)8SEQ ID NO: 7, (SEQ ID NO: 3)16SEQ ID NO: 7, (SEQ ID NO: 3)32SEQ ID NO: 7, (SEQ ID NO: 3)48SEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO: 3)2, SEQ ID NO: 7(SEQ ID NO:

[0148] 3)4, SEQ ID NO: 7(SEQ ID NO: 3)6, SEQ ID NO: 7(SEQ ID NO: 3)8, SEQ ID NO: 7(SEQ ID NO: 3)16, SEQ ID NO: 7(SEQ ID NO: 3)32, or SEQ ID NO: 7(SEQ ID NO: 3)48, the silk polypeptide comprises or consists of (SEQ ID NO: 4)2, (SEQ ID NO: 4)4, (SEQ ID NO:

[0149] 4)6, (SEQ ID NO: 4)8, (SEQ ID NO: 4)i6, (SEQ ID NO: 4)32, (SEQ ID NO: 4)48, (SEQ ID NO:

[0150] 4)2SEQ ID NO: 6, (SEQ ID NO: 4)4SEQ ID NO: 6, (SEQ ID NO: 4)6SEQ ID NO: 6, (SEQ ID NO: 4)8SEQ ID NO: 6, (SEQ ID NO: 4)i6SEQ ID NO: 6, (SEQ ID NO: 4)32SEQ ID NO: 6, (SEQ ID NO: 4)48SEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 4)2, SEQ ID NO: 6(SEQ ID NO:

[0151] 4)4, SEQ ID NO: 6(SEQ ID NO: 4)6, SEQ ID NO: 6(SEQ ID NO: 4)8, SEQ ID NO: 6(SEQ ID NO: 4)16, SEQ ID NO: 6(SEQ ID NO: 4)32, SEQ ID NO: 6(SEQ ID NO: 4)48, (SEQ ID NO: 4)2SEQ ID NO: 7, (SEQ ID NO: 4)4SEQ ID NO: 7, (SEQ ID NO: 4)6SEQ ID NO: 7, (SEQ ID NO: 4)8SEQ ID NO: 7, (SEQ ID NO: 4)i6SEQ ID NO: 7, (SEQ ID NO: 4)32SEQ ID NO: 7, (SEQ ID NO: 4)48SEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO: 4)2, SEQ ID NO: 7(SEQ ID NO:

[0152] 4)4, SEQ ID NO: 7(SEQ ID NO: 4)6, SEQ ID NO: 7(SEQ ID NO: 4)8, SEQ ID NO: 7(SEQ ID NO: 4)16, SEQ ID NO: 7(SEQ ID NO: 4)32, or SEQ ID NO: 7(SEQ ID NO: 4)48, or the silk polypeptide comprises or consists of (SEQ ID NO: 5)2, (SEQ ID NO: 5)4, (SEQ ID NO:

[0153] 5)6, (SEQ ID NO: 5)8, (SEQ ID NO: 5)i6, (SEQ ID NO: 5)32, (SEQ ID NO: 5)48, (SEQ ID NO:

[0154] 5)2SEQ ID NO: 6, (SEQ ID NO: 5)4SEQ ID NO: 6, (SEQ ID NO: 5)6SEQ ID NO: 6, (SEQ ID NO: 5)8SEQ ID NO: 6, (SEQ ID NO: 5)i6SEQ ID NO: 6, (SEQ ID NO: 5)32SEQ ID NO: 6, (SEQ ID NO: 5)48SEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 5)2, SEQ ID NO: 6(SEQ ID NO:

[0155] 5)4, SEQ ID NO: 6(SEQ ID NO: 5)6, SEQ ID NO: 6(SEQ ID NO: 5)8, SEQ ID NO: 6(SEQ ID NO: 5)16, SEQ ID NO: 6(SEQ ID NO: 5)32, SEQ ID NO: 6(SEQ ID NO: 5)48, (SEQ ID NO: 5)2SEQ ID NO: 7, (SEQ ID NO: 5)4SEQ ID NO: 7, (SEQ ID NO: 5)6SEQ ID NO: 7, (SEQ ID NO: 5)8SEQ ID NO: 7, (SEQ ID NO: 5)i6SEQ ID NO: 7, (SEQ ID NO: 5)32SEQ ID NO: 7, (SEQ ID NO: 5)48SEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO: 5)2, SEQ ID NO: 7(SEQ ID NO: 5)4, SEQ ID NO: 7(SEQ ID NO: 5)6, SEQ ID NO: 7(SEQ ID NO: 5)8, SEQ ID NO: 7(SEQ ID NO: 5)16, SEQ ID NO: 7(SEQ ID NO: 5)32, or SEQ ID NO: 7(SEQ ID NO: 5)48.

[0156] As mentioned above, peptide variants are also encompassed by the present invention. For example, a peptide variant of the amino acid sequence GSGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 5, V47) has the amino acid sequence GSGAAAAAAAASGPGGFGPENQGPSGPGGYGPGGP (SEQ ID NO: 8). This peptide variant is designated as V47’. The amino acid sequence GSGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 5) mentioned in the above embodiments may be replaced by the peptide variant V47’.

[0157] In a second aspect, the present invention relates to a nucleic acid molecule encoding the silk polypeptide of the first aspect.

[0158] It is preferred that the nucleic acid molecule is comprised in a vector. The vector may be any vector known to the skilled person. For example, the vector may be a plasmid vector or a viral vector such as an adenoviral or a baculoviral vector. Said vectors include expression as well as cloning vectors. Expression vectors generally contain the desired coding sequence and appropriate DNA sequences to control the expression of the operably linked coding sequence in a particular host organism (e.g. bacteria, yeast, plant, insect, or mammal) or in in vitro transcription / translation systems. Expression control sequences may be sequences which control (i) the expression, e.g. promoters, TATA-box, enhancers, (ii) post-transcriptional events, e.g. polyadenylation, and (iii) the translation of nucleic acid sequences. Cloning vectors are generally used to engineer and amplify a certain desired DNA fragment and may lack functional sequences needed for expression of the desired DNA fragment. The above-mentioned vectors are preferably recombinant vectors.

[0159] A cell may be transformed, transfected, or infected with the nucleic acid molecule or the vector comprising the nucleic acid molecule. The cell can be used for expressing the nucleic acid molecule or amplifying the nucleic acid molecule or the vector comprising the nucleic acid molecule. The cell may be a prokaryotic or a eukaryotic cell. The prokaryotic cell may be an E.coli cell, a Bacillus cell, or a Bacillus licheniformis cell. The eukaryotic cell may be a mammalian cell, a plant cell, a yeast cell, a fungi cell, or an insect cell. The mammalian cell may be a CHO, COS, HeLa, HEK293(T), or BHK cell. The yeast cell may be a Saccharomyces cerevisiae cell, a Schizosaccharomyces pombe cell, a Pichia pastoris cell, a Candida albicans cell, or a Hansenula polymorpha cell. The fungi cell may be an Aspergillus or a Trichoderma cell. The insect cell may be a Lepidoptera insect cell. The plant cell may be a tobacco cell, a potato cell, a corn cell, a pea cell, or a tomato cell. The above-mentioned cells may also be named host cells. They are preferably recombinant cells.

[0160] In a third aspect, the present invention relates to a method for producing a silk polypeptide comprising the step of:

[0161] (i) expressing the nucleic acid molecule of the second aspect in a cell, or synthesizing the nucleic acid molecule of the second aspect in an in vitro transcription / translation system, thereby producing the silk polypeptide.

[0162] In one embodiment, the silk polypeptide is produced recombinantly within a cell. The cell may also be named host cell. The cell may be a prokaryotic or eukaryotic cell. The prokaryotic cell may be an E. coli cell, a Bacillus cell or a Bacillus licheniformis cell. The eukaryotic cell may be a mammalian cell, a plant cell, a yeast cell, a fungi cell, or an insect cell. The mammalian cell may be a CHO, COS, HeLa, HEK293(T), or BHK cell. The yeast cell may be a Saccharomyces cerevisiae cell, a Schizosaccharomyces pombe cell, a Pichia pastoris cell, a Candida albicans cell, or a Hansenula polymorpha cell. The fungi cell may be an Aspergillus or a Trichoderma cell. The insect cell may be a Lepidoptera insect cell. The plant cell may be a tobacco cell, a potato cell, a corn cell, a pea cell, or a tomato cell. The above-mentioned cells are preferably recombinant cells.

[0163] The nucleic acid molecule may be found inside the cell (i) freely dispersed as such, (ii) incorporated in a vector, or (iii) integrated into the cell genome or mitochondrial DNA. Preferably, the vector, e.g. plasmid or viral vector, is an expression vector.

[0164] The cell may be transformed, transfected, or infected with the nucleic acid molecule or the vector comprising the nucleic acid molecule.

[0165] In one another embodiment, the silk polypeptide is produced in an in vitro transcription / translation system. This system is cell-free. Cell-free protein synthesis means the in vitro production of proteins without living cells. Instead, special cell extracts or lysates are used, which contain the protein synthesis apparatus and all other necessary components. In principle, extracts for cell-free protein expression can be obtained from any type of cell. However, systems based on E. coli, wheat germ, rabbit reticulocytes and insect cells are most frequently used. After the addition of amino acids, an energy source, salts and other cofactors, theoretically any protein encoded by a DNA or RNA template can be synthesized.

[0166] Diverse in vitro protein synthesis kits are available on the market. The firm BioLabs, for example, offers a PURExpress® in vitro protein synthesis kit. The PURExpress® in vitro protein synthesis kit is a cell-free transcription / translation system and consists of defined, reconstituted and highly purified components of the E. coli transcription / translation apparatus. All protein factors are produced recombinantly as His-tag fusions, the ribosomes and tRNAs are highly purified from E. coli. This means that the system is free of contaminating exonucleases, RNases and proteases. The firm Promega provides, for example, a TNT® Quick coupled transcription / translation system which might be an option. The LEXSY in vitro translation system (Jena Bioscience) applies T7 RNA polymerase to generate target mRNA and cell extracts with functional ribosomes and other essential components for cell-free protein synthesis. The silk polypeptide of the present invention may be produced as described in WO 2006 / 008163.

[0167] In one preferred embodiment, the method further comprises the step of:

[0168] (ii) isolating the silk polypeptide from the cell or in vitro transcription / translation system.

[0169] The isolation of the polypeptide from the cell or in vitro transcription / translation system may be achieved by separating said polypeptide from said cell or transcription / translation system via centrifugation, sedimentation, and / or filtration, e.g. via centrifugation and filtration, via sedimentation and filtration, via sedimentation and centrifugation, or via centrifugation, sedimentation, and filtration. Depending on the silk polypeptide to be harvested, the parameters for centrifugation, sedimentation, or filtration may vary. The person skilled in the art is able to easily adapt the appropriate separation parameters, e.g. the acceleration-force / g-force and / or time using centrifugation for separation, filter size using filtration for separation, and / or sedimentation time using sedimentation for separation, in order to harvest said polypeptide produced by said cells or said transcription / translation system.

[0170] After step (ii), further purification of the isolated silk polypeptide may be required. Said purification may be achieved via chromatography, preferably column chromatography, more preferably size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, affinity chromatography, or high pressure liquid chromatography, electrophoresis, preferably gel electrophoresis, or ultracentrifugation.

[0171] The silk polypeptide of the present invention may be isolated as described in WO 2011 / 120690 A2.

[0172] In a fourth aspect, the present invention relates to an article comprising / composed of / made from the silk polypeptide of the first aspect. Preferably, the article is a fiber, more preferably a fabric comprising the fiber, a yarn, more preferably a fabric comprising the yarn, or a film. The fabric may be a woven or non-woven fabric.

[0173] The present inventors found that fibers comprising the silk polypeptides of the present invention have a higher tensile strength and tear-resistance than fibers comprising the corresponding wild-type silk polypeptides. Overall, it can be said that the stronger hydrophobicity of the silk polypeptides of the present invention compared to the respective wildtype silk polypeptides seems to be responsible or the reason for the higher tensile strength and tear-resistance of the fibers produced therefrom.

[0174] The skilled person knows techniques to determine the tensile strength or tear-resistance of a fiber. For example, for a given single silk polypeptide fiber, to assess its strength, a monotonic longitudinal load is applied to the fiber until its failure. This failure load is then normalised by the cross-section, possibly at the breaking point, to obtain the tensile strength of the fiber. The testing of the tensile strength of a silk polypeptide fiber can, for example, be performed according to DIN EN ISO 2062:04 / 2010. For further information, it is referred to the examples of the present patent application.

[0175] In addition, the skilled person knows techniques to determine the hydrophobicity of a silk polypeptide fiber or fabric produced therefrom. For example, the hydrophobicity of a fiber or fabric produced therefrom can be determined with a wicking test. Vertical or horizontal wicking tests are known.

[0176] For a vertical wicking test, AATCC 197 is one of the standard methods. In this standard, the bottom of the specimen comes into contact with water. Then, the wicking distance by specified time intervals is recorded. The higher the wicking distance at the same interval, the better the fiber or fabric is in wicking.

[0177] For a horizontal wicking test, AATCC 198 is one of the standard methods. A specific amount of water is given from specific height. The time of the water spreading out from center to the edge of circle (100 mm in diameter) is recorded. In this method, the shorter the time, the better the fiber or fabric is in wicking.

[0178] In other words, a silk polypeptide fiber that can transport moisture well (good wicking properties) has a higher hydrophilicity than a fiber that is less able to transport moisture. In contrast thereto, a silk polypeptide fiber that is poor at transporting moisture (bad wicking properties) has a higher hydrophobicity than a fiber that is better at transporting moisture.

[0179] For further information, it is referred to the examples of the present patent application

[0180] In a fifth aspect, the present invention relates to a method for producing an article comprising the steps of:

[0181] (i) providing a solution comprising the silk polypeptide of the first aspect, and

[0182] (ii) forming an article out of / from the solution provided in (i).

[0183] In one preferred embodiment, the article is a fiber. The fiber is preferably formed by extruding the solution comprising the silk polypeptide of the first aspect.

[0184] Extrusion in this context means the application of pressure to the solution to force it through an opening, e.g. a nozzle. In the art of polymer technology, extrusion processes are often used to form fibers from molten thermoplasts, i.e. the entire extruded material solidifies after it has left the opening. In the context of the present invention, the extruded solution does not solidify entirely. Rather the dissolved polymers that are comprised in the extruded solution associate to form a fiber, which is typically smaller in diameter than the opening through which the solution is extruded, while the remaining solvent is separated from the fiber thus formed. In some embodiments, the fiber initially formed is attached to a fiber recovery device, e.g. a cylinder, spool or bobbin, onto which the fiber is continuously wound. Depending on the relative speed of the, e.g. cylinder, with respect to the speed of fiber formation during extrusion, there may also be a pulling force exerted onto the extruded fiber, i.e. the fiber in some embodiments may be considered to be drawn from the extruded solution.

[0185] The solution comprising the silk polypeptide may comprise formic acid, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or hexafluoroisopropanol (HFI) as a solvent. The solution for extruding the silk polypeptide fiber may also be designated as dope solution.

[0186] Preferably, the extrusion of the silk polypeptide fiber is carried out into a coagulation bath. The extrusion can take place directly into the coagulation bath or via an air gap into the coagulation bath. The coagulation solution may be selected from the group consisting of water (H2O), methanol, ethanol, (EtOH), l-Ethyl-3-methylimidazoliumchlorid (EMIMC1), and N- Methylmorpholin-N-oxid (NMMO). In one more preferred embodiment, the fiber is (after its formation) extended in a subsequent stretching step. The extension may be at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, or at least 10-fold. During the stretching step, the cross-sectional area of the fiber is reduced. It is preferred that stretching step leads to a reduction of the cross-sectional area of the silk polypeptide fiber of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, or at least 70%.

[0187] For example, the thickness (diameter) of the fiber upon extrusion may be in the range of 5 pm to 200 pm and / or the thickness (diameter) of the fiber after extension may be in the range of 2.5 pm to 100 pm.

[0188] Preferably, such stretching step is carried out in the presence of a coagulation solution, e.g. the silk polypeptide fiber is at least partially submerged in the coagulation solution. This solution is also referred to as stretching solution in the context of the present invention. It may have the same composition as the coagulation solution. The stretching solution may be selected from the group consisting of an aqueous solution, water (H2O), water vapor, methanol, and ethanol (EtOH).

[0189] In one even more preferred embodiment, the silk polypeptide fiber is subsequently spun into a yarn. The silk polypeptide yam can be spun from silk polypeptide fibers, e.g. wet using a chemical-technical wet spinning process or dry using a mechanical spinning process. Alternatively, the silk polypeptide yarn is produced by twisting single silk polypeptide fibers. During twisting, individual silk polypeptide fibers are turned together in order to achieve a silk polypeptide yam with improved tensile strength. The yam comprising the silk polypeptide of the present invention can be used in the textile industry, e.g. in order to produce fabrics, e.g. woven and non-woven fabrics.

[0190] In another preferred embodiment, the article is a film. The film is preferably formed by applying the solution comprising the silk polypeptide of the first aspect. The solution comprising the silk polypeptide may be applied onto a (solid) support or (solid) substrate. Afterwards, the solution comprising the silk polypeptide of the first aspect is usually dried. The solution comprising the silk polypeptide of the first aspect may be applied by casting, spraying, or dropping said solution onto the support material or substrate. The film may finally be removed from the support material.

[0191] The produced silk polypeptide film is specifically a self-supporting film, i.e. a silk polypeptide film that has the capacity for supporting itself without the help of additional materials such as carrier elements.

[0192] The solution comprising the silk polypeptide may comprise formic acid, dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), or hexafluoroisopropanol (HFIP) as a solvent. The solution for casting a silk polypeptide film may also be designated as casting solution.

[0193] For high-performance textiles, the highest possible tear resistance is desirable. The fibers comprising the silk polypeptides of the present invention are characterized by a high tensile strength and tear-resistance. Thus, in a sixth aspect, the present invention relates to the (in vitro) use of the silk polypeptide of the first aspect or article of the fourth aspect in the textile industry.

[0194] The films comprising the silk polypeptides of the present invention are very stable and have a hydrophobic character useful in diverse applications. Thus, in a seventh aspect, the present invention relates to the use of the silk polypeptide of the first aspect or article of the fourth aspect for the coating of surfaces, e.g. of a support material or substrate.

[0195] Various modifications and variations of the invention will be apparent to those skilled in the art without departing from the scope of invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in the art in the relevant fields are intended to be covered by the present invention.

[0196] BRIEF DESCRIPTION OF THE FIGURES

[0197] The following figures are merely illustrative of the present invention and should not be construed to limit the scope of the invention as indicated by the appended claims in any way.

[0198] Figure 1: Shows selected silk polypeptide variants of the present invention (one letter code), the tensile strength [cN / tex], and the wicking height of the respective selected silk polypeptide variants relative to the reference (wild-type silk polypeptide repeat unit) [%].

[0199] Figure 2: Shows the hydrophilicity of the of X1X2X3 motif (A) and the hydrophilicity of the entire protein repeat unit (B) of the selected silk polypeptide variants of the present invention compared to the corresponding wild-type silk polypeptide (wild-type repeat unit). The silk polypeptide variants V06, V23 and V47 show a lower hydrophilicity than the wild-type silk polypeptide (wild-type repeat unit).

[0200] Figure 3: Shows the tensile strength of fibers made from the selected silk polypeptide variants of the present invention compared to the wild-type silk polypeptide (wild-type repeat unit) (the stronger the better). The silk polypeptide variants V06, V23 and V47 show a higher tensile strength than the wild-type silk polypeptide (wild-type repeat unit). The silk polypeptide variant V51 is included for comparative reasons. It bears amino acid substitutions distant from the GSS motif. The tensile strength value of this variant is not as good as the tensile strength values of the silk polypeptide variants V06, V23 and V47 encompassed by the present invention.

[0201] Figure 4: Shows the results of the wicking test indicating the hydrophobicity of the fibers made from the selected silk polypeptide variants of the present invention compared to the wildtype silk polypeptide (wild-type repeat unit) (the lower the more hydrophobic). The silk polypeptide variants V06, V23 and V47 show a higher hydrophobicity than the wild-type silk polypeptide (wild-type repeat unit). The silk polypeptide variant V51 is included for comparative reasons. It bears amino acid substitutions distant from the GSS motif. The wicking test result of this variant is not as good as the wicking test results of the silk polypeptide variants V06, V23 and V47 encompassed by the present invention.

[0202] Figure 5: Shows a schematic representation of the wicking test. The height of the colored solution adsorbed by the fiber represents the hydrophilicity of the fiber. The higher the height of the colored solution, the more hydrophilic the fiber. EXAMPLES

[0203] The examples given below are for illustrative purposes only and do not limit the invention described above in any way.

[0204] 1 , Protein selection

[0205] In total, 120 silk polypeptides with variations in the above amino acid sequence were cloned. In the end, 25 of the 120 cloned silk polypeptide variants were selected, fermented, purified and spun into fibers. These 25 silk polypeptide variants typically have 1 to 6 mutations per repeat unit. In the tensile strength tests, it was surprisingly found that 3 of these 25 silk polypeptide variants formed a fiber with higher tensile strength. Common to all 3 higher tensile strength silk polypeptide variants is that mutations in one or more of the first three amino acids (GSS) flanking the poly-alanine N-terminal region resulted in a more hydrophobic silk polypeptide stretch than the corresponding silk polypeptide stretch in the wild-type repeat unit. Specifically, the serine residues (S) flanking the poly-alanine N-terminal region were replaced by a small amino acid without a hydroxyl group. Overall, it can be said that the stronger hydrophobicity seems to be responsible or the reason for the higher tensile strength.

[0206] The selected silk polypeptide variants are shown in Figure 1 together with the corresponding wild-type silk polypeptide. The selected silk polypeptide variants have a hydrophobicity which is higher than the hydrophobicity of the corresponding wild-type silk polypeptide (see Figure 2). The theoretical hydrophobicity was calculated using the Hopp-Woods scale (https: / / www.bachem.com / knowledge-center / peptide-calculator / ). The substitutions in the X1X2X3 motif led to a more hydrophobic silk protein both when the hydrophobicity of X1X2X3 was considered separately (Figure 2A) and within the whole repeat unit relative to the repeat unit of the wild-type reference (Figure 2B).

[0207] 2, Protein production

[0208] The silk polypeptides were prepared as described in WO 2006 / 008163 or WO2011 / 120690.

[0209] 3, Fiber production

[0210] The silk polypeptides were then processed into fibers as described in WO 2014 / 037453.

[0211] 4, Fiber analysis After silk polypeptide fiber production, the silk polypeptide fibers were analyzed using a tensile strength test and a wicking test.

[0212] 4.1 Tensile strength test

[0213] Tensile strength tests were performed by Trevira GmbH according to DIN EN ISO 2062:04 / 2010. This DIN standard determines the single-end breaking force and elongation at break using constant rate of extension. The results are shown in Figure 3. The more hydrophobic silk polypeptide variant V06, V23 and V47 fibers had a greater tensile strength relative to the silk polypeptide wild-type reference fiber.

[0214] The silk polypeptide V51 (GSSAAAAAAAASGPGGDGSYGPGPSGPGGYGPGGP) (SEQ ID NO: 9) was used for comparative purposes. The silk polypeptide V51 bears amino acid substitutions distant from the GSS motif and resulted in fibers with decreased tensile strength relative to the wild-type reference fiber and relative to the silk polypeptides variant V06, V23 and V47 fibers encompassed by the present invention (Figure 3).

[0215] 4.2 Wicking test

[0216] After silk polypeptide fiber production by wet-spinning, the fibers were subjected to wicking tests to measure their hydrophobicity. After fixing the fibers to a rod, they were weighted with a nut. The weighted end of the fiber was later fully immersed into a 0.1 % (w / v) patent blue V Na- salt dye solution, allowing the fibers to interact with the liquid. During 90 min, the height of the colored solution adsorbed by the fiber was measured with a ruler. The results are shown in Figure 4. A schematic representation of the wicking test is shown in Figure 5. It is clear from Figures 4 and 5 that the silk polypeptides variant V06, V23 and V47 fibers encompassed by the present invention are poor in transporting moisture (bad wicking property). They have a higher hydrophobicity than the wild-type reference fiber or the silk polypeptide V51 fiber.

[0217] 5, Further variants

[0218] Peptide variants of the silk polypeptides described herein are also encompassed by the present invention. For example, a peptide variant of the amino acid sequence GSGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 5, V47) has the amino acid sequence GSGAAAAAAAASGPGGFGPENQGPSGPGGYGPGGP (SEQ ID NO: 8). This peptide variant is designated as V47’. It is comparable with respect to its hydrophobicity and mechanical parameters with V47 or is even better (data not shown).

Claims

CLAIMS1. A silk polypeptide comprising at least two repeat units, wherein each repeat unit is independently selected from a peptide having the amino acid sequence X1X2X3AAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 1) or a peptide variant thereof, wherein the amino acid sequence X1X2X3 is more hydrophobic than the corresponding amino acid sequence GSS in the wild-type repeat unit GSSAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 2), wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 1 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit.

2. The silk polypeptide of claim 1, wherein the amino acids Xi, X2, and X3 are independently selected from the group consisting of Q, A, G, and S.

3. The silk polypeptide of claims 1 or 2, wherein in the amino acid sequence X1X2X3, Xi is G, A or Q,X2 is S, A, Q, or G, and / or X3 is S, A, Q, or G.

4. The silk polypeptide of any one of claims 1 to 3, wherein in the amino acid sequence X1X2X3,Xi is GX2 is A, Q, or G, andX3 is G, and wherein preferably Xi is G X2 is A, and X3 is G.

5. The silk polypeptide of claim 4, wherein the peptide has the amino acid sequence GAGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 3) or is a peptide variant thereof,wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 3 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit.

6. The silk polypeptide of any one of claims 1 to 3, wherein in the amino acid sequence X1X2X3,Xi is Q or A,X2 is G, andX3is G, and wherein preferablyXi is Q,X2 is G, andX3is G.

7. The silk polypeptide of claim 6, wherein the peptide has the amino acid sequence QGGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 4) or is a peptide variant thereof, wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 4 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit.

8. The silk polypeptide of any one of claims 1 to 3, wherein in the amino acid sequence X1X2X3,Xi is G,X2 is S, andX3is G.

9. The silk polypeptide of claim 8, wherein the peptide has the amino acid sequence GSGAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQ ID NO: 5), or is a peptide variant thereof, wherein the peptide variant has 1, 2, or 3 amino acid exchanges in SEQ ID NO: 5 at amino acid positions 4 to 35, wherein said amino acid exchanges do not increase the hydrophilicity or do not reduce the hydrophobicity of the resulting repeat unit.

10. The silk polypeptide of any one of claims 1 to 9, wherein the silk polypeptide further comprises an amino acid sequenceGSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO: 6).

11. The silk polypeptide of any one of claims 1 to 10, wherein the silk polypeptide further comprises an amino acid sequenceGSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 7).

12. The silk polypeptide of any one of claims 1 to 11, wherein the silk polypeptide comprises or consists of (SEQ ID NO: 1)m, (SEQ ID NO: 1)mSEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 1)m, (SEQ ID NO: 1)mSEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO: 1)m, wherein m is an integer of between 2 to 100.

13. The silk polypeptide of claim 12, wherein the silk polypeptide comprises or consists of (SEQ ID NO: 1)2, (SEQ ID NO: 1)4, (SEQ ID NO: 1)6, (SEQ ID NO: 1)8, (SEQ ID NO: 1)16, (SEQ ID NO: 1)32, (SEQ ID NO: 1)48, (SEQ ID NO: 1)2SEQ ID NO: 6, (SEQ ID NO: 1)4SEQ ID NO: 6 (SEQ ID NO: 1)6SEQ ID NO: 6, (SEQ ID NO: 1)8SEQ ID NO: 6, (SEQ ID NO: 1)I6SEQ ID NO: 6, (SEQ ID NO: 1)32SEQ ID NO: 6, (SEQ ID NO: 1)48SEQ ID NO: 6, SEQ ID NO: 6(SEQ ID NO: 1)2, SEQ ID NO: 6(SEQ ID NO: 1)4, SEQ ID NO: 6(SEQ ID NO: 1)6, SEQ ID NO: 6(SEQ ID NO: 1)8, SEQ ID NO: 6(SEQ ID NO: 1)16, SEQ ID NO: 6(SEQ ID NO: 1)32, SEQ ID NO: 6(SEQ ID NO: 1)48, (SEQ ID NO: 1)2SEQ ID NO: 7, (SEQ ID NO: 1)4SEQ ID NO: 7 (SEQ ID NO: 1)6SEQ ID NO: 7, (SEQ ID NO: 1)8SEQ ID NO: 7, (SEQ ID NO: 1)I6SEQ ID NO: 7, (SEQ ID NO: 1)32SEQ ID NO: 7, (SEQ ID NO: 1)48SEQ ID NO: 7, SEQ ID NO: 7(SEQ ID NO: 1)2, SEQ ID NO: 7(SEQ ID NO: 1)4, SEQ ID NO: 7(SEQ ID NO: 1)6, SEQ ID NO: 7(SEQ ID NO: 1)8, SEQ ID NO: 7(SEQ ID NO: 1)i6, SEQ ID NO: 7(SEQ ID NO: 1)32, or SEQ ID NO: 7(SEQ ID NO: 1)48.

14. A nucleic acid molecule encoding the silk polypeptide of any one of claims 1 to 13.

15. A method for producing a silk polypeptide comprising the step of:(i) expressing the nucleic acid molecule of claim 14 in a cell or synthesizing the nucleic acid molecule of claim 14 in an in vitro transcription / translation system, thereby producing the silk polypeptide.

16. The method of claim 15, wherein the method further comprises the step of:(ii) isolating the silk polypeptide from the cell or in vitro transcription / translation system.

17. An article comprising the silk polypeptide of any one of claims 1 to 13.

18. The article of claim 17, wherein the article is a fiber, preferably a fabric comprising the fiber, a yarn, preferably a fabric comprising the yam, or a film.

19. A method for producing an article comprising the steps of:(i) providing a solution comprising the silk polypeptide of any one of claims 1 to 13, and(ii) forming an article out of / from the solution provided in (i).

20. The method of claim 19, wherein the article is a fiber or film.

21. The method of claim 20, wherein the fiber is formed by extruding the solution comprising the silk polypeptide of any one of claims 1 to 13.

22. The method of claim 21, wherein the solution comprising the silk polypeptide is extruded into a coagulation bath.

23. The method of claim 22, where the fiber is extended in a subsequent stretching step.

24. The method of any one of claims 20 to 23, wherein the fiber is subsequently spun into a yam.

25. The method of claim 20, wherein the film is formed by applying, preferably casting, the solution comprising the silk polypeptide of any one of claims 1 to 13.

26. The method of claim 25, wherein the solution comprising the silk polypeptide is applied, preferably casted, onto a support or substrate.

27. Use of the silk polypeptide of any one of claims 1 to 13 or article of claims 17 or 18 in the textile industry.

28. Use of the silk polypeptide of any one of claims 1 to 13 or article of claims 17 or 18 for the coating of surfaces.

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