RU2545331C2 - Method of obtaining polymer-containing fibres - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to method of spinning fibre, containing polypeptide polymer, as well as to products, including said polymer fibre. Method of fibre spinning includes draft of fibre from dope solution, containing polymer, preferably silk polypeptide which can be introduced into water solution with concentration constituting at least 0.15 mg/ml, polyacrylamide (PAA), which increases longitudinal viscosity of dope solution, and solvent. Invention makes it possible to obtain fibres, including living and non-living biological material, which could perform function of framework material for fabric engineering and growing artificial organs.

EFFECT: application of PAA in dope solution results in obtaining smooth and homogeneous fibres, non-biodegradable and long-lasting, in addition, application of very low concentrations of polymers and/or very low concentrations of improvers of PAA longitudinal viscosity facilitates spinning of fibres from dope solution.

24 cl, 4 dwg, 7 ex

Description

The present invention relates to a method for spinning a polymer fiber. In addition, it relates to a polymer fiber obtained by the aforementioned method, and to variants of its application. The invention also relates to products comprising said polymer fiber.

State of the art

Conventional methods for producing polymer fiber have significant disadvantages with respect to both methods and products. Methods typically have requirements that are excessively stringent to incorporate biological material, such as proteins or cells, regarding conditions regarding temperature, pressure, or the use of chemicals and solvents. Products are usually designed to demonstrate excellence in one property, such as strength, resilience, or mass. However, the combination of all these properties was difficult.

Natural spider silk can take various forms depending on the gland in which it forms (Gosline et al., J. Exp. Biol. (202): 3295, 1999). Its properties are noteworthy: its tensile strength can exceed the corresponding steel characteristic and be equal to the tensile strength of aramid elementary fibers, for example, Kevlar brand. Spider silk can also be very malleable when stretched up to about 300% of its length without breaking. Among other things, it is light in weight.

Since obtaining native spider silk is impractical due to the territorial and cannibalistic nature of spiders, scientific and commercial interest has initiated a study of the artificial production of spider silk with the aim of possible production on an industrial scale. However, finding a commercially competitive method for mass production of spider silk was difficult. Artificial production was faced with the problems of achieving both sufficient protein yield and high-quality thread assembly. The solution of the protein source problem is directed to the production of recombinant spider silk proteins using bacteria (Scheibel, Microb. Cell. Fact., (1): 14, 2004). However, drawing the fiber from aqueous solutions of these proteins is not possible, since such a solution, by itself, does not exhibit sufficient longitudinal viscosity.

Spinning is a method by which strains in the direction of the fiber axis compete with the surface tension of the spinning syrup. For example, it is not possible to draw a stable flowing elementary fiber from water due to the high surface tension of water.

This illustrates Rayleigh instability, which causes the water jet that flows from the faucet to break into drops. This effect is caused by surface tension of water.

The relationship between the viscous forces and the surface tension forces describes the Onesorg number Oh.

$$Oh=\frac{\eta }{\sqrt{\rho \sigma R}}$$

A small value of the Oh number (Oh << 1) means that the flowing elementary fiber will break into small droplets, which stimulates surface tension. A large value of the Oh number means that the flowing elementary fiber will remain stable (Edmond, Schofield et al. 2006). As a result, this means that the spinning syrup must demonstrate the great importance of the Onesorge number. Obviously, the value of the Oh number can be increased as a result of an increase in viscosity. For fluids characterized by a high Truton coefficient, longitudinal viscosity will dominate in the longitudinal flow processes and therefore large values of the Oh number can be achieved for fluids that are characterized by a rather low shear viscosity η, but a high Truton coefficient Tr.

Recombinant spider silk protein can be obtained using bacteria, its assembly has been extensively studied (Scheibel, Microb. Cell. Fact., (1): 14, 2004; Huemmerich et al., Curr. Biol., (22): 2070- 4, 2004; Rammensee et al., PNAS, (105): 6590-6595, 2008). Artificial spinning syrups having low protein concentrations (10-20 mg / ml eADF3 and eADF4) do not show the ratio Tr >> 1. The surface tension is still much greater than the effects of viscosity, so Oh << 1 also takes place, so obtaining fiber from these solutions having a low protein concentration was not possible. Fiber production was provided only by solutions of recombinant spider silk protein having very high protein concentrations (~ 200 mg / ml) (Exier et al., Angewandte Chemie-Intemational Edition, (19): 3559-3562, 2007). However, an increase in protein concentration leads to an increase in the shear viscosity of the solution, and the processability of a fluid characterized by a very high (> 1 Pa-sec) shear viscosity is rather poor.

The inventors of the present invention, to their surprise, found that only very low polymer concentrations are required to spin the fibers of the process of the present invention. The use of very low polymer concentrations has the advantage of not increasing the shear viscosity of the solution, which in turn improves the processability of the fluid.

Surprisingly, obtaining fibers from very low concentration polymers, for example silk polypeptides, such as spider silk polypeptides, under mild conditions was facilitated by the inclusion of longitudinal viscosity improvers. Although spider silk spinning syrups were described by their nature as being characterized by very high percentage levels of spider silk protein, the present method surprisingly requires the use of only very low concentrations of silk polypeptides, starting from concentrations of only 0.15 mg / ml.

In addition, the present method, surprisingly, requires the use of only very low concentrations of the aforementioned longitudinal viscosity improvers. Low concentrations of the aforementioned improvers, on the one hand, are sufficient to increase the longitudinal viscosity of the solution, but, on the other hand, do not substantially increase the shear viscosity of the solution, so that the formation of fibers improves.

Thus, the use of very low concentrations of polymers and / or very low concentrations of longitudinal viscosity improvers in the process of the present invention facilitates the spinning of fibers from a dope. In addition, technical obstacles to the method are small, since they directly draw fibers from a solution of silk polypeptide under environmental conditions or conditions close to them.

SUMMARY OF THE INVENTION

In a first aspect, the present invention relates to a method for spinning fibers from a spinning solution, comprising the steps of:

(a) preparing a spinning solution containing (i) a polymer that can be introduced into the aqueous solution at a concentration of at least 0.15 mg / ml, (ii) a compound that increases the longitudinal viscosity of the spinning solution, and ( iii) a solvent; and

(b) drawing the fiber from the dope, or combining extrusion and drawing the fiber from the dope, resulting in a fiber.

In a second aspect, the present invention relates to a spinning dope for implementing the method of the present invention, comprising (i) a polymer that can be added to an aqueous solution at a concentration of at least 0.15 mg / ml, (ii) a compound that increases the longitudinal viscosity of the dope, and (iii) the solvent.

In a third aspect, the present invention relates to a fiber obtained by the method of the first aspect, and to its use.

In a fourth aspect, the present invention provides products comprising a fiber of the second aspect.

This summary of the invention does not necessarily describe all features of the invention.

DETAILED DESCRIPTION OF THE INVENTION

Before providing a detailed description of the present invention below, it is necessary to understand that this invention is not limited to the specific methodology, protocols and reagents described herein, as they may vary. You must also understand that the terminology in accordance with the use in this document is intended only for the purpose of describing specific embodiments and is not intended to limit the scope of the present invention, which will be limited only by the attached claims. Unless otherwise specified, all technical and scientific terms, as used herein, have the same meanings as commonly understood by those skilled in the art.

Preferably, the terms as used herein are defined as described in A multilingual glossary of biotechnological terms: (IUPAC Recommendations), Leuenberger, H. G. W., Nagel, B. and Kölbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

In the course of the entire presentation of the text of this description of the invention, several documents are cited. Each of the documents cited in this document (including all patents, patent applications, scientific publications, technical descriptions from the manufacturer, instructions, presentation of sequences with GenBank access numbers and the like), be higher or lower in this document, in in its entirety, by reference is incorporated herein. Nothing in this document should be construed as an assumption that the invention is not entitled to the claimed priority date based on the priority application.

In the following, elements of the present invention will be described. These elements are listed in conjunction with specific embodiments, however, it must be understood that to create additional embodiments, they can be combined in any way and in any quantity. The variously described examples and preferred embodiments are not to be construed as limiting the present invention to the detailed embodiments only. This description of the invention should be understood as justifying and encompassing embodiments that combine the detailed embodiments with any number of described and / or preferred elements. In addition, any permutations and combinations of all the described elements in this application should be considered disclosed in the description of this application, unless the context indicates otherwise.

Throughout the entire presentation of this description of the invention and the claims that follows, unless the context requires otherwise, the word “include” and variations such as “include” and “including” will be understood as implying the inclusion of the indicated integer or stage or a group of integers or stages, but not the exclusion of any other integer or stage or group of integers or stages.

In accordance with the use in this description of the invention and the attached claims, the singular (for example, one, one and this) include plural matches, unless the content clearly dictates another.

Residues in two or more polypeptides are considered “corresponding” to each other if the residues occupy a similar position in the polypeptide structures. As is well known in the art, similar positions in two or more polypeptides can be determined by aligning polypeptide sequences based on amino acid sequence similarity or structural similarity. Means for such alignment are well known to those skilled in the art and can, for example, be obtained on the Internet, for example, ClustalW (www.ebi.ac.uk/clustalw) or Align (http://www.ebi.ac.uk/ /emboss/align/index.html) when using standard settings, preferably for aligning EMBOSS: needle, Matrix: Blosum62, Penalty for opening a space: 10.0, Penalty for continuing a space of 0.5.

In a first aspect, the present invention relates to a method for spinning fibers from a spinning dope, comprising essentially consisting of or consisting of the following steps:

(a) preparing a spinning solution containing (i) a polymer that can be introduced into the aqueous solution at a concentration of at least 0.15 mg / ml, (ii) a compound that increases the longitudinal viscosity of the spinning solution, and ( iii) a solvent; and

(b) drawing the fiber from the dope, or combining extrusion and drawing the fiber from the dope, resulting in a fiber.

In the context of the present invention, the term "polymer" refers to a molecule formed from repeating structural units, usually connected by covalent chemical bonds.

In one preferred embodiment, the polymer is a biopolymer, such as a polypeptide or a synthetic polymer.

The term “biopolymer” as used herein refers to a molecule comprising monomeric units that are covalently linked to form larger structures. Classification of biopolymers can be divided into three main classes based on the different monomer units used and the structure of the resulting biopolymers. The first class of biopolymers are polynucleotides, which are polymers formed from nucleotide monomers, for example, polymers formed from 10 or more nucleotide monomers. The second class of biopolymers are polypeptides that are polymers of amino acids. The third class of biopolymers is polysaccharides, which are often linear linked polymeric carbohydrate structures. It is preferred that the biopolymer be a polypeptide.

Preferably, the biopolymer is biodegradable, and more preferably also compostable. Biodegradable biopolymers decompose to form COz and water under the influence of microorganisms. Biodegradable biopolymers, which are also compostable, can be introduced into the industrial composting process and will decompose over a period of months.

It is also preferred that the biopolymer be biocompatible. This means that the polymer is non-toxic, non-mutagenic and causes a zero or only minimal inflammatory reaction.

Thus, also fibers spun from biopolymers according to the method of the present invention can exhibit the above-mentioned advantageous characteristics, namely they can be biodegradable, compostable and / or biocompatible, or can be mainly biodegradable, compostable and / or biocompatible. In particular, since the number of longitudinal viscosity improvers in the spinning dope of the method of the present invention is preferably very low.

As mentioned above, in a polypeptide, repeating structural units are amino acids joined by covalent amide bonds (peptide bonds).

Unless otherwise indicated, the terms “polypeptide” and “protein” are used interchangeably herein to mean any peptide-linked amino acid chain, regardless of length or post-translational modification.

Preferably, the polypeptide is a silk polypeptide comprising at least two identical repeating units, bovine serum albumin (BSA), zein or casein. The polypeptide may also be collagen, fibronectin or keratin.

Bovine serum albumin (BSA), also known as "Fraction V", is a serum albumin protein. Zein is a prolamine protein found in corn. Casein (from the Latin caseus “cheese”) is the predominant phosphoprotein (αS1, αS2, β, κ), which is responsible for almost 80% of the proteins in cow's milk and cheese.

In the context of the present invention, the term "silk polypeptide" refers to a polypeptide or silk protein (it should be noted that, unless otherwise indicated, these two terms, as used herein, are interchangeable) that are expressed in a recombinant expression system ( for example, microorganisms, yeast, plants, insects or mammals), that is, separated from its natural environment in the gland of a spider (recombinant polypeptide or silk protein). Preferably, the "silk polypeptide" is isolated or purified. In particular, “purified silk polypeptide” or “isolated silk polypeptide” is free or substantially free of cellular material or other contaminating proteins from a cellular or tissue source from which the protein is isolated or produced. The phrase “substantially free of cellular material” includes silk polypeptide preparations in which the silk polypeptide is separated from the cellular components of the cells from which it is recombinantly derived. Thus, a silk polypeptide that is substantially free of cellular material includes preparations of silk polypeptides containing less than about 30%, 20%, 10%, 5%, or 1% (based on dry weight) of contaminating protein. If silk polypeptide is expressed in cell culture, it will also be free or substantially free of culture medium, i.e., the culture medium will be less than about 20%, 10%, 5%, or 1% of the volume of the polypeptide preparation.

"Silk polypeptide" in accordance with the context of the present invention further relates to a polypeptide comprising an amino acid sequence that includes or consists of at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, preferably at least 95%, and most preferably 100%, of multiple copies of one identical repeating unit (for example, A ₂ , Q ₆ or C ₁₆ , where positions 2, 6 or 16 represent the number of repeating units) or multiple copies of two or more different repeating units (e.g., (AQ) ₂₄ or (AQ) ₁₂ , C ₁₆ ).

The terms “repeating unit” and “repeat unit” in the context of the present invention can be used interchangeably,

In the context of the present invention, the term "silk polypeptide" also refers to a silk polypeptide, which includes or consists of at least two identical repeating units, which include or consist of identical copies of the amino acid sequences of silk polypeptides found in nature, or variations of the amino acid sequences of silk polypeptides found in nature, or combinations of both.

In the context of the present invention, a “repeating unit” refers to a region that in the amino acid sequence corresponds to a region that comprises or consists of at least one peptide motif (eg, AAAAAA (SEQ ID NO: 13) or GPGQQ (SEQ ID NO: 4) ), which is repeatedly present in a naturally occurring silk polypeptide (e.g., MaSpI, ADF-3, ADF-4 or Flag) (i.e., an identical amino acid sequence), or an amino acid sequence substantially similar to it (i.e. to the variant amino acid pic research). In this regard, the term "substantially similar" refers to the degree of amino acid identity for at least 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 even 99.9%, preferably along the entire length, corresponding to the reference amino acid sequence found in nature. A “repeating unit” including an amino acid sequence that is “substantially similar” to the corresponding amino acid sequence in a naturally occurring silk polypeptide (ie, a wild-type repeating unit) is also similar in terms of its functional properties, for example, a silk polypeptide, including a “substantially similar repeating unit”, still has the ability to form fiber. Preferably, from a silk polypeptide comprising a “substantially similar repeating unit”, it is still possible to draw a smooth and uniform fiber at a speed of at least 0.1 cm / sec, preferably 10 cm / sec, and more preferably 10 m / sec One of skill in the art can visually assess whether fiber production will still occur. The smooth and uniform appearance of said fiber can be controlled using electron microscopy.

A “repeating unit” comprising an amino acid sequence that is “identical” to the amino acid sequence of a naturally occurring silk polypeptide, for example, may be a portion of a silk polypeptide corresponding to one or more peptide motifs MaSpI (SEQ ID NO: 43), MaSpII (SEQ ID NO: 44), ADF-3 (SEQ ID NO: 1) and / or ADF-4 (SEQ ID NO: 2). A “repeating unit” comprising an amino acid sequence that is “substantially similar” to the naturally occurring amino acid sequence of a silk polypeptide, for example, may be a portion of a silk polypeptide corresponding to one or more peptide motifs MaSpI (SEQ ID NO: 43), MaSpII (SEQ ID NO: 44), ADF-3 (SEQ ID NO: 1) and / or ADF-4 (SEQ ID NO: 2), but including one or more amino acid substitutions at specific amino acid positions.

The “repeating unit” does not include non-repeating hydrophilic amino acid domains, generally believed to be naturally occurring at the carboxy and amino ends of alkali polypeptides.

A “repeating unit” in accordance with the present invention further relates to an amino acid sequence having a length of from 3 to 200 amino acids or from 5 to 150 amino acids, preferably having a length of from 10 to 100 amino acids or from 15 to 80 amino acids, and more preferably having a length of 18 to 60 amino acids or from 20 to 40 amino acids. For example, the repeating unit of the present invention may have a length of 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, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195 or 200 amino acids. Most preferably, the repeating unit of the invention consists of 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 18, 20, 24, 27, 28, 30, 31, 32, 33, 34, 35, 36, 37, 38, or 39 amino acids.

The silk polypeptide used in the method of the present invention preferably consists of amino acids in an amount of from 6 to 1500, amino acids in an amount of from 50 to 1500, or amino acids in an amount of from 200 to 1300, and most preferably amino acids in an amount of from 250 to 1200 or amino acids in an amount in from 500 to 1000.

Preferably, the silk polypeptide used in the method of the present invention comprises or consists of 2-80 repeating units, 3-80 repeating units or 4-60 repeating units, more preferably from 8 to 48 repeating units or from 10 to 40 repeating units, and most preferably from 16 to 32 repeating units. For example, the silk polypeptide used in the method of the present invention may include or consist of: 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, or 80 repeating units. Most preferably, the silk polypeptide comprises 4, 8, 12, 16, 24, 32, or 48 repeating units. As mentioned above, at least two repeating units included in the silk polypeptide used in the method of the present invention are identical repeating units. Thus, the silk polypeptide used in the method of the present invention may include multiple copies of one identical repeating unit (for example, A ₂ or C ₁₆ , where positions 2 or 6 represent the number of repeating units) or multiple copies of two or more different repeating units (for example , (AQ) ₂₄ or (QAQ) ₈ ). For example. 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, or 80 of the 80 repeating units that can be included in the silk polypeptide used in the method of the present invention are identical repeating units.

The silk polypeptide used in the method of the present invention may include or consist of the amino acid sequence of any silk polypeptide known to a person skilled in the relevant field of technology. It is preferred that the silk polypeptide used in the method of the present invention comprise or consists of the amino acid sequence of an insect silk polypeptide, preferably an arthropod polypeptide or a spider silk polypeptide. The silk polypeptide used in the method of the present invention may also include or consist of the amino acid sequence of a mitilide silk polypeptide.

Preferably, the spider silk polypeptide would comprise or consist of the amino acid sequence of a large ampulla gland polypeptide (MaSp), such as a spider silk skeleton polypeptide, a small ampulloid gland polypeptide (MiSp), a flagelliform tartar polypeptide, a spider silk polypeptide from tree gland (aggregate), spider silk polypeptide from the lobular gland (aciniform) or pear silk spider polypeptide from the piriform gland (pyriform). Most preferably, the spider silk polypeptide comprises or consists of the amino acid sequence of a spider silk polypeptide of a frame yarn or a spider silk polypeptide of a spider silk. In the general case, it is preferred that the amino acid sequence of the carcass filament polypeptide or lucid spider polypeptide is selected from the polypeptide of the carcass filament or the lynx spider polypeptide Araneidae or Araneoid.

It is preferred that the insect silk polypeptide turn off or consist of the amino acid sequence of the Lepidoptera silk polypeptide. More preferably, the insect silk polypeptide comprises or consists of the amino acid sequence of the Bombycidae silk polypeptide, most preferably Bombyx mori.

Preferably, the aforementioned silk polypeptides are obtained recombinantly, that is, they are recombinant silk polypeptides. For example, the silk polypeptides used in the method of the present invention are recombinant spider silk polypeptides, such as wire-thread spider silk polypeptides or lucid spider silk polypeptides, insect recombinant silk polypeptides or mitylid recombinant silk polypeptides.

The repeating unit of the silk polypeptide used in the method of the present invention may include or consist of the amino acid sequence of any region that includes or consists of at least one peptide motif that is repetitively present in the naturally occurring silk polypeptide known to those skilled in the art. areas of technology. Preferably, the repeating unit of the silk polypeptide used in the method of the present invention comprises or consists of the amino acid sequence of a region that includes or consists of at least one peptide motif that is repeatedly present in the insect silk polypeptide, more preferably in the arthropod silk polypeptide or spider silk polypeptide. The repeating unit of the silk polypeptide used in the method of the present invention may also include or consist of the amino acid sequence of a region that includes or consists of at least one peptide motif that is repeatedly present in the mitilide silk polypeptide.

It is preferred that the repeating spider silk link include or consist of the amino acid sequence of a region that includes or consists of at least one peptide motif that is repetitively present in the naturally occurring large ampuloid gland (MaSp) polypeptide, such as the polypeptide silk spider carcass filament, small ampulliform gland polypeptide (MiSp), polypeptide lucid spiral filament, spider silk polypeptide from tree gland, spider silk polypeptide from lobular jelly PS or polypeptide of a spider silk from a piriform gland. Most preferably, the repeating unit comprises or consists of the amino acid sequence of a region that includes or consists of at least one peptide motif that is repetitively present in the naturally occurring spider silk polypeptide of the wireframe thread or the spider silk polypeptide of the spider silk thread.

It is preferred that the repeating insect silk link include or consist of the amino acid sequence of a region that includes or consists of at least one peptide motif that is repetitively present in the naturally occurring Lepidoptera silk polypeptide. More preferably, the repeating insect silk unit comprises or consists of the amino acid sequence of a region that includes or consists of at least one peptide motif that is repetitively present in the naturally occurring insect silk polypeptide Bombycidae, most preferably Bombyx mori.

The term "consensus sequence" as used in the context of the present invention refers to an amino acid sequence that includes amino acids that are often present at a particular position (for example, "G") and where other amino acids that are not further defined are replaced with the placeholder " X. "

Preferably, the silk polypeptide used in the method of the present invention includes essentially consists of or consists of at least two identical repeating units, each of which includes at least one, preferably one, a consensus sequence selected from the group consisting of:

i) GPGXX (SEQ ID NO: 3), where X is any amino acid, preferably in each case independently selected from A, S, G, Y, P, N and Q;

ii) GGX, where X is any amino acid, preferably in each case independently selected from Y, P, R, S, A, T, N and Q, more preferably in each case independently selected from Y, P and Q; and

iii) A _x , where x is an integer in the range from 5 to 10.

It is also preferred that the silk polypeptide used in the method of the present invention comprise or consists of at least two identical repeating units, each of which includes at least one, preferably one, amino acid sequence selected from the group consisting of: GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19).

The repeating (peptide) motifs GPGXX (SEQ ID NO: 3) and GGX, that is, glycine-rich motifs, give flexibility to the silk polypeptide and thus the thread obtained from the silk protein including the motifs mentioned. In detail, the repeating motif GPGXX (SEQ ID NO: 3) forms β-turn helical structures that give the silk polypeptide elasticity. Both silk of the large ampulliform gland and silk of the hunting spiral thread include the GPGXX motif (SEQ ID NO: 3). The repeating GGX motif is associated with a spiral structure comprising three amino acids per turn, and is found in most spider silk. GGX motif can give silk additional elastic properties. The repeating polyalanine A _x (peptide) motif forms the β-sheet crystal structure, which gives the silk polypeptide strength, (WO 03/057727). The (peptide) motifs GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19) were selected from resilin (WO 08/155304). Resilin is an elastomeric protein found in most arthropods (arthropodd). It is located in specialized areas of the cuticle, imparting low stiffness and high strength (Elvin et al. Nature (473): 999-1002, 2005).

Thus, in one preferred embodiment of the present invention, the silk polypeptide comprises or consists of repeating units, each 14 of which includes at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, or 9), preferably one, an amino acid sequence selected from the group consisting of GPGAS (SEQ ID NO: 5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7). GPGGP (SEQ ID NO: 8), GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGGG (SEQ ID NO: 10), GPGQG (SEQ ID NO: 40) and GPGGS (SEQ ID NO: 4) :eleven). In another preferred embodiment of the present invention, the silk polypeptide comprises or consists of repeating units, each of which includes at least one (for example, 1, 2, 3, 4, 5, 8, 7, or 8), preferably one, an amino acid sequence selected from the group consisting of GGY, GGP, GGA, GGR, GGS, GGT, GGN and GGQ. In one additional preferred embodiment of the present invention, the silk polypeptide comprises or consists of repeating units, each of which includes at least one (for example, 1, 2, 3, 4, 5 or 6), preferably one, an amino acid sequence selected from the group consisting of AAAAA (SEQ ID NO: 12), AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ ID NO: 15), AAAAAAAAA (SEQ ID NO: 16) and AAAAAAAAAA (SEQ ID NO: 17).

In another preferred embodiment, the silk polypeptide comprises or consists of repeating units, each of which includes at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), preferably one amino acid sequence selected from the group consisting of GPGAS (SEQ ID NO: 5) , GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO: 8), GPGGA (SEQ ID NO: 9), GPGQQ (SEQ ID NO: 4), GPGGG (SEQ ID NO: 10), GPGQG (SEQ ID NO: 40), GPGGS (SEQ ID NO: 11), GGY, GGP, GGA, GGR, GGS, GGT, GGN, GGQ, AAAAA (SEQ ID NO: 12), AAAAAA ( SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ ID NO: 15), AAAAAAAAAA (SEQ ID NO: 16), AAAAAAAAA A (SEQ ID NO: 17), GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19).

Most preferably, the silk polypeptide used in the method of the present invention includes, essentially consists of or consists of repeating units, which include or consist of:

(i) GPGAS (SEQ ID NO: 5), AAAAAA (SEQ ID NO: 13), GGY and GPGSG (SEQ ID NO: 6) as the amino acid sequence, preferably in this order,

(ii) AAAAAAAA (SEQ ID NO: 15), GPGGY (SEQ ID NO: 7), GPGGY (SEQ ID NO: 7) and GPGGP (SEQ ID NO: 8) as the amino acid sequence, preferably in this order,

(iii) GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4) and GPGQQ (SEQ ID NO: 4) as the amino acid sequence, preferably in this order,

(iv) GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9) and GGP as the amino acid sequence, preferably in this order,

(v) AAAAAAAA (SEQ ID NO: 15), GPGQG (SEQ ID NO: 40) and GGR as an amino acid sequence, preferably in that order,

(vi) AAAAAAAA (SEQ ID NO: 15), GPGGG (SEQ ID NO: 10), GGR, GGN and GGR as an amino acid sequence, preferably in that order,

(vii) GGA, GGA, GGA, GGS, GGA and GGS as the amino acid sequence, preferably in that order,

(viii) GPGGA (SEQ ID NO: 9), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11) and GPGGY ( SEQ ID NO: 7) as an amino acid sequence, preferably in that order.

It should be noted that at least two repeating units included in the aforementioned silk polypeptides are identical repeating units.

Preferably, the silk polypeptide used in the method of the present invention comprises essentially consists of or consists of 2-80 repeating units, 3-80 repeating units or 4-60 repeating units, more preferably from 8 to 48 repeating units or from 10 to 40 repeating units, and most preferably from 16 to 32 repeating units, i.e. 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 or 80 in toryayuschihsya units each of which comprises at least one, preferably one, a consensus sequence selected from the group consisting of:

i) GPGXX (SEQ ID NO: 3), where X is any amino acid, preferably in each case independently selected from A, S, G, Y, P, N and Q;

i) GGX, where X is any amino acid, preferably in each case independently selected from Y, P, R, S, A, T, N and Q, more preferably in each case independently selected from Y, P and Q; and

iii) A _x , where x is an integer in the range from 5 to 10.

As mentioned above, at least two repeating units included in the silk polypeptide used in the method of the present invention are identical repeating units.

It is also preferred that the silk polypeptide used in the method of the present invention comprise or consists of 2-80 repeating units, 3-80 repeating units or 4-60 repeating units, more preferably from 8 to 48 repeating units or from 10 to 40 repeating units, and most preferably from 16 to 32 repeating units, each of which includes at least one, preferably one, amino acid sequence selected from the group consisting of: GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19).

Thus, the silk polypeptide used in the method of the present invention preferably includes, essentially consists of or consists of 2-80 repeating units, 3-80 repeating units or 4-60 repeating units, more preferably from 8 to 48 repeating units or from 10 to 40 repeating units, and most preferably from 16 to 32 repeating units, each of which includes at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25), preferably one amino acid sequence a bone selected from the group consisting of GPGAS (SEQ ID NO: 5), GPGSG (SEQ ID NO: 6), GPGGY (SEQ ID NO: 7), GPGGP (SEQ ID NO: 8), GPGGA (SEQ ID NO: 8) 9), GPGQQ (SEQ ID NO: 4), GPGQG (SEQ ID NO: 40), GPGGG (SEQ ID NO: 10), GPGGS (SEQ ID NO: 11), GGY, GGP, GGA, GGR, GGS, GGT , GGN, GGQ, AAAAA (SEQ ID NO: 12), AAAAAA (SEQ ID NO: 13), AAAAAAA (SEQ ID NO: 14), AAAAAAAA (SEQ ID NO: 15), AAAAAAAAA (SEQ ID NO: 16), AAAAAAAAAA (SEQ ID NO: 17), GGRPSDTYG (SEQ ID NO: 18) and GGRPSSSYG (SEQ ID NO: 19).

Most preferably, the silk polypeptide used in the method of the present invention includes, essentially consists of or consists of:

(i) repeating units that include or consist of: GPGAS (SEQ ID NO: 5), AAAAAA (SEQ ID NO: 13), GGY and GPGSG (SEQ ID NO: 6) as an amino acid sequence, preferably in that order,

(ii) repeating units that include or consist of: AAAAAAAA (SEQ ID NO: 15), GPGGY (SEQ ID NO: 7), GPGGY (SEQ ID NO: 7) and GPGGP (SEQ ID NO: 8) as amino acid sequences, preferably in that order,

(iii) repeating units that include or consist of: GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4), GPGQQ (SEQ ID NO: 4) and GPGQQ (SEQ ID NO: 4) as amino acid sequences, preferably in that order,

(iv) repeating units that include or consist of: GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9), GGP, GPGGA (SEQ ID NO: 9) and GGP as the amino acid sequence, preferably in this order

(v) repeating units that include or consist of: AAAAAAAA (SEQ ID NO: 15), GPGQG (SEQ ID NO: 40) and GGR as an amino acid sequence, preferably in this order,

(vi) repeating units that include or consist of: AAAAAAAA (SEQ ID NO: 15), GPGGG (SEQ ID NO: 10), GGR, GGN and GGR as an amino acid sequence, preferably in that order,

(vii) repeating units that include or consist of: GGA, GGA, GGA, GGS, GGA and GGS as an amino acid sequence, preferably in that order, and / or

(viii) repeating units that include or consist of: GPGGA (SEQ ID NO: 9), GPGGY (SEQ ID NO: 7), GPGGS (SEQ ID NO: 11), GPGGY (SEQ ID NO: 7), GPGGS ( SEQ ID NO: 11) and GPGGY (SEQ ID NO: 7) as the amino acid sequence, preferably in that order.

It should be noted that at least two repeating units included in the aforementioned silk polypeptides are identical repeating units.

Preferably, the silk polypeptide used in the method of the present invention includes, essentially consists of or consists of:

(i) (GPGXX) _n (SEQ ID NO: 3) as a repeating unit, where X is any amino acid, preferably in each case independently selected from A, S, G, Y, P, N and Q, and n is 2, 3, 4, 5, 6, 7, 8 or 9;

(ii) (GGX) _n as a repeating unit, where X is any amino acid, preferably in each case independently selected from Y, P, R, S, A, T, N and Q, more preferably in each case independently selected from Y, P and Q, and n is 2, 3, 4, 5, 6, 7 or 8; and / or

(iii) (A _x ) _n as a repeating unit, where x is an integer in the range from 5 to 10, and n is 2, 3, 4, 5, 6, 7, 8, 9, or 10.

As mentioned above, at least two repeating units included in the silk polypeptides used in the method of the present invention are identical repeating units.

It is preferred that the repeating units be independently selected from module A (SEQ ID NO: 20), module C (SEQ ID NO: 21), module Q (SEQ ID NO: 22), module K (SEQ ID NO: 23), module sp (SEQ ID NO: 24), module S (SEQ ID NO: 25), module R (SEQ ID NO: 26), module X (SEQ ID NO: 27) or module Y (SEQ ID NO: 28) or their variants (i.e. variants of module A, variants of module C, variants of module Q, variants of module K, variants of module sp, variants of module S, variants of module R, variants of module X or variants of module Y). Modules A (SEQ ID NO: 20) and Q (SEQ ID NO: 22) basically have the amino acid sequence ADF-3 of the spider Araneus diadematus. Module C (SEQ ID NO: 21) basically has the amino acid sequence ADF-4 of the spider Araneus diadematus. Modules K (SEQ ID NO: 23), sp (SEQ ID NO: 24), X (SEQ ID NO: 27) and Y (SEQ ID NO: 28) basically have the amino acid sequence of the spider Nephila clavipes spider filament FLAG protein (WO 2006/008163). Modules S (SEQ ID NO: 25) and R (SEQ ID NO: 26) are basically based on resilin (Arthropoda) (WO 2008/155304).

Thus, in one preferred embodiment of the present invention, the repeating units of the silk polypeptide consist of module A: GPYGPGASAAAAAAGGYGPGSGQQ (SEQ ID NO: 20), module C: GSSAAAAAAAAASGPGGYGPENQGPSGPGGYGPGGP (SEQGGGGGGPPQQQGQGGGGPP: 21QQQGG: 21) ) modulus K: GPGGAGGPYGPGGAGGPYGPGGAGGPY (SEQ ID NO: 23), module sp: GGTTIIEDLDITIDGADGPITISEELTI (SEQ ID NO: 24), module S: PGSSAAAAAAAASGPGQGQGQGQGQGGRPSDTYG (SEQ ID NO: 25), module R: SAAAAAAAAGPGGGNGGRPSDTYGAPGGGNGGRPSSSYG (SEQ ID NO: 26) module X: GGAGGAGGAGGSGGAGGS (SEQ ID NO: 27) or module Y: GPGGAGPGGYGPGGSGPGGYGPGGSGPGGY (SEQ ID NO: 28) or their variants.

Preferably, the silk polypeptide used in the method of the present invention comprises essentially consists of or consists of 2-80 repeating units, 3-80 repeating units or 4-60 repeating units, more preferably from 8 to 48 repeating units or from 10 to 40 repeating units, and most preferably from 16 to 32 repeating units, i.e. 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 or 80 in trailing links that are independently selected from module A (SEQ ID NO: 20), module C (SEQ ID NO: 21), module Q (SEQ ID NO: 22), module K (SEQ ID NO: 23), module sp ( SEQ ID NO: 24), module S (SEQ ID NO: 25), module R (SEQ ID NO: 26), module X (SEQ ID NO: 27) or module Y (SEQ ID NO: 28) or their variants ( that is, variants of module A, variants of module C, variants of module Q, variants of module K, variants of module sp, variants of module S, variants of module R, variants of module X or variants of module Y). It should be noted that at least two repeating units included in the silk polypeptide used in the method of the present invention are identical repeating units (modules).

Thus, it is preferred that the silk polypeptide used in the method of the present invention comprise essentially consists of or consists of: (i) a repeating unit (units) consisting of module A and / or a repeating unit (units) consisting of variants of module A, (ii) repeating link (s), consisting of module C, and / or repeating link (links), consisting of variants of module C, (hi) repeating link (links), consisting of module Q, and / or a repeating link (s) consisting of options for the module Q, (i v) (a) a repeating link (s) consisting of module A and a repeating link (links) consisting of module Q, (b) a repeating link (links) consisting of module A, and a repeating link (links) consisting of the options for module Q, (c) a repeating link (s), consisting of options for module A, and a repeating link (links), consisting of module Q, (d) a repeating link (links), consisting of variants of module A, and a repeating link (links), consisting of variants of the module Q, (v) (a) a repeating link (links), consisting from module A, and a repeating link (s), consisting of module C, (b) a repeating link (s), consisting of module A, and a repeating link (links), consisting of variants of module C, (c) repeating link (links) ), consisting of variants of module A, and a repeating link (links), consisting of module C, (d) a repeating link (links), consisting of variants of module A, and a repeating link (links), consisting of variants of module C, (vi ) (a) a repeating link (s) consisting of module C and a repeating link (s) consisting of module Q, (b) repeating link (s), consisting of module C, and repeating link (s), consisting of variants of module Q, (c) repeating link (links), consisting of variants of module C, and repeating link (s), consisting of module Q, (d) repeating link (s), consisting of options for module C, and repeating link (s), consisting of options for module Q, or (vii) (a) repeating link (s) consisting of module A, repeating unit (s), consisting of module Q, and repeating units on (links), consisting of module C, (b) a repeating link (links), consisting of module A, a repeating link (links), consisting of module Q, and a repeating link (links), consisting of variants of module C, (c ) a repeating link (s), consisting of module A, a repeating link (s), consisting of options for module Q, and a repeating link (links), consisting of module C, (d) a repeating link (links), consisting of variants of module A repeating link (s), consisting of the module Q, and repeating link (links s), consisting of module C, (e) a repeating link (s), consisting of module A, a repeating link (s), consisting of variants of module Q, and a repeating link (links), consisting of variants of module C, (f) a repeating link (s) consisting of variants of module A, a repeating link (links) consisting of variants of module Q, and a repeating link (links) consisting of module C, (g) a repeating link (links) consisting of variants of module A repeating unit (s), consisting of the module Q, and repeating I link (s), consisting of options for module C, (h) repeating link (links), consisting of options for module A, repeating link (links), consisting of options for module Q, and repeating link (links), consisting of options for module FROM.

Modules A, C, Q, K, sp, S, R, X, or Y or their variants (i.e. module A variants, module C variants, module Q variants, module K variants, module sp options, module S variants, module variants R, variants of module X or variants of module Y) can also be combined with each other in any combination and for any quantity of each, that is, module (repeating unit) A can be combined with module (repeating unit) Q (that is, AQ combination) , module (repeating unit) C can be combined with module (repeating unit) Q (i.e., a combination of CQ), module (repeat link) Q can be combined with module (repeating link) A and module (repeating link) Q (i.e. QAQ combination), module (repeating link) A can be combined with module (repeating link) A and module (repeating link) Q (i.e., a combination of AAQ), and so on, provided that the silk polypeptide used in the method of the present invention includes or consists of at least two repeating units that are identical. For example, the silk polypeptide used in the method of the present invention may include or consist of: A _n , (AA) _n , (AQ) _n , (QA) _n , Q _n , (QQ) _n , (QAQ) _n , (AQA) _n , C _n , (CC) _n , (CCC) _n , (CQ) _n , (QC) _n , (QCQ) _n , (CQC) _n , (AA) _n Q _n , (QQ) _n A _n , ( AAA) _n Q _n , (QQQ) _n A _n , (AQQ) _n , (QQA) _n , K _n , sp _n , S _n , R _n , X _n , Y _n , (Ksp) _n , (spK) _n , (XY) _n , (YX) _n , (XX) _n , (YY) _n , (XXX) _n , (YYY) _n , (AX) _n , (XA) _n , (CX) _n , (XC) _n , (QX) _n , (XQ) _n , (YQ) _n , (QY) _n , (SS) _n , (SR) _n , (RS) _n or (RR) _n , where n is at least 2 preferably 4, 8, 9, 10, 12, 16, 20, 24 or 32. In the case of a silk polypeptide consisting of (AQ) ₁₂ , it should be noted that in the silk polypeptide the module (repeating unit) A p is present 12 times, and the module (repeating unit) Q is also present 12 times, and that, thus, the silk polypeptide consists of 24 modules (repeating units). The arrangement of the modules (repeating units) of the silk polypeptide consisting of (AQ) ₁₂ is as follows: AQAQAQAQAQAQAQAQAQAQAQAQ. In addition, in the case of a silk polypeptide formed from modules (repeating units) of a silk polypeptide and consisting of (QAQ) ₈ , it should be noted that a module (repeating unit) A is present 8 times in a silk polypeptide, and a module (repeating unit) Q present 16 times, and that, therefore, the silk polypeptide consists of 24 modules (repeating units). The arrangement of the modules (repeating units) of the silk polypeptide consisting of (QAQ) ₈ is as follows: QAQQAQQAQQAQQAQQAQQAQQAQ.

The silk polypeptide used in the method of the present invention may also include or consist of the following: (A * Q) _n , (AQ *) _n , (A * Q *) _n , (Q * A) _n , (QA *) _n , (Q * A *) _n , (QAQ *) _n , (QA * Q) _n , (Q * AQ) _n , (QA * Q *) _n , (Q * A * Q) _n , (Q * AQ *) _n , (Q * A * Q *) _n , (AQA *) _n , (AQ * A) _n , (A * QA) _n , (AQ * A *) _n , (A * Q * A) _n , (A * QA *) _n , (A * Q * A *) _n , where n is at least 2, preferably 4, 8, 9, 10, 12, 16, 20, 24 or 32, and where * indicates a variant of a module, that is, a variant of module A or Q.

The terms “combined with each other” or “coupled to each other” in the context of the present invention may mean that the modules (repeating units) are combined or linked to each other directly, or in the context of the present invention may mean that the modules (repeating units) ) are combined or linked to each other through one or more spacer amino acids. In preferred embodiments, the modules (repeating units) included in the silk polypeptide are combined or linked directly to each other. In other preferred embodiments, the modules (repeating units) included in the silk polypeptide are combined or linked to each other via spacer amino acids in an amount of from 1 to 25 or from 1 to 20, more preferably through spacer amino acids in an amount of from 1 to 15 or from 1 to 10, and most preferably through spacer amino acids in an amount of from 1 to 5, that is, through 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 spacer amino acids. Said spacer amino acids may be any amino acids naturally found in proteins. Preferably, said spacer amino acid is not proline. Preferably, the spacer amino acid (s) having charged groups is independently selected from the group consisting of asparaginate, glutaminate, histidine, and lysine. The spacer amino acids mentioned must be amino acids that do not adversely affect the ability of the silk polypeptide to form a coating, preferably to form a uniform coating, on an inert or naturally occurring material such as Kevlar or wool. Furthermore, the spacer amino acids mentioned must be amino acids that do not cause steric hindrance, for example, amino acids having a small size, such as lysine and cysteine.

In more preferred embodiments, the silk polypeptide comprises modules that are directly linked to each other and modules that are linked to each other through spacer amino acids in an amount of 1 to 25 or 1 to 20, more preferably via spacer amino acids in an amount of 1 to 15 or from 1 to 10, and most preferably through spacer amino acids in an amount of from 1 to 5, that is, through 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 spacer amino acids.

The module option A, C, Q, K, sp, S, R, X or Y differs from the reference module (wild type) A, C, Q, K, sp, S, R, X or Y from which it came from, up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acid changes in the amino acid sequence (i.e. substitutions, additions, insertions, deletions, cut-offs with N -end and / or cut-offs from the C-terminus). Such a variant of the module in an alternative or additional embodiment may be characterized by a certain degree of sequence identity to the reference module (wild type) from which it originated. Thus, a variant of module A, C, Q, K, sp, S, R, X or Y is characterized by sequence identity to the corresponding reference module (wild type) A, C, Q, K, sp, S, R, X or Y, at least 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 even 99.9%. Preferably, sequence identity is observed over a continuous length of at least 10, 15, 18, 20, 24, 27, 28, 30, 34, 35 or more amino acids, preferably along the entire length of the corresponding reference module (wild type) A, C , Q, K, sp, S, R, X or Y.

In particular, it is preferred that the sequence identity be at least 80% over the entire length, at least 85% over the entire length, at least 90% over the entire length, at least at least 95% of the entire length, would be at least 98% of the entire length or would be at least 99% of the entire length of the corresponding reference module (wild type) A, C, Q, K, sp , S, R, X, or Y. In addition, it is particularly preferred that the sequence identity be at least 80% by a continuous length of at least 10, 15, 18, 20, 24, 28, or 30 amino acids would be at least 85% of a continuous length of at least 10, 15, 18, 20, 24 , 28 or 30 amino acids, would be at least 90% for a continuous extension of at least 10, 15, 18, 20, 24, 28 or 30 amino acids, would be at least 95% for a continuous at least 10, 15, 18, 20, 24, 28 or 30 amino acids in length would be at least 98% over a continuous length of at least 10, 15, 18, 20, 24, 28 or 30 amino acids or would be at least 99% over a continuous length of at least 10, 15, 18, 20, 24, 28, or 30 amino acids of the corresponding reference module (wild type) A, C, Q, K, sp, S, R, X or Y.

A fragment (or deletion variant) of module A, C, Q, K, sp, S, R, X or Y preferably includes removal of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids from the N-terminus and / or from the C-terminus. Removal can also be internal.

In addition to this, a variant or fragment of module A, C, Q, K, sp, S, R, X or Y in the context of the present invention is considered a variant or fragment of module A, C, Q, K, sp, S, R, X or Y, only if modifications to the amino acid sequence on which the given variant or fragment is based do not adversely affect the ability of the silk polypeptide to form fiber. Preferably, from a silk polypeptide comprising a variant or fragment of module A, C, Q, K, sp, S, R, X or Y, it is still possible to draw a smooth and uniform fiber at a speed of at least 0.1 cm / sec, preferably 10 cm / sec, and more preferably 10 m / sec. One of skill in the art can visually assess whether fiber production will still occur. The smooth and uniform appearance of said fiber can be controlled using electron microscopy.

Thus, in one preferred embodiment of the present invention, the repeating units are independently selected from module A ^C (SEQ ID NO: 29), module A ^K (SEQ ID NO: 30), module C ^C (SEQ ID NO: 31), module C ^K1 (SEQ ID NO: 32), module C ^K2 (SEQ ID NO: 33) or module C ^KC (SEQ ID NO: 34). Modules A ^C (SEQ ID NO: 29), A ^K (SEQ ID NO: 30), C ^C (SEQ ID NO: 31), C ^K1 (SEQ ID NO: 32), C ^K2 (SEQ ID NO: 33) and C ^KC (SEQ ID NO: 34) are variants of module A, which basically has the amino acid sequence ADF-3 of the spider Araneus diadematus, and module C, which basically has the amino acid sequence ADF-3 of the spider Araneus diadematus, (WO 2007/025719). In module A ^C (SEQ ID NO: 29), the amino acid S (series) at position 21 is replaced by amino acid C (cysteine), in module A ^K (SEQ ID NO: 30) the amino acid S at position 21 is replaced by amino acid K (lysine), module C ^C (SEQ ID NO: 31), the amino acid S at position 25 is replaced by amino acid C, in module C ^K1 (SEQ ID NO: 32) the amino acid S at position 25 is replaced by amino acid K, in module C ^K2 (SEQ ID NO: 33) amino acid E (glutaminate) at position 20 is replaced by amino acid K and in module C ^KC (SEQ ID NO: 34) amino acid E at position 20 is replaced by amino acid K, and amino acid S at position 25 is replaced by amino acid C (WO 2007/02571 9).

Preferably, the repeating units in the silk polypeptide used in the method of the present invention consist of module A^C: GPYGPGASAAAAAAGGYGPGCGQQ (SEQ ID NO: 29), module A^K: GPYGPGASAAAAAAGGYGPGKGQQ (SEQ ID NO: 30), Module C^C: GSSAAAAAAAASGPGGYGPENQGPCGPGGYGPGGP (SEQ ID NO: 31), module C^K1: GSSAAAAAAAASGPGGYGPENQGPKGPGGYGPGGP (SEQ ID NO: 32), module C^K2: GSSAAAAAAAASGPGGYGPKNQGPSGPGGYGPGGP (SEQ ID NO: 33) or module C^Kc: GSSAAAAAAAASGPGGYGPKNQGPCGPGGYGPGGP (SEQ ID NO: 34).

It is also preferred that the silk polypeptide used in the method of the present invention comprise essentially consists of or consists of 2-80 repeating units, 3-80 repeating units or 4-60 repeating units, preferably from 8 to 48 repeating units or from 10 to 40 repeating units, and most preferably from 16 to 32 repeating units, i.e. 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 or 80 repeating units that are independently selected from module A ^C (SEQ ID NO: 29), module A ^K (SEQ ID NO: 30), module C ^C (SEQ ID NO: 31), module C ^K1 (SEQ ID NO: 32), module C ^K2 (SEQ ID NO: 33) or module C ^KC (SEQ ID NO: 34). It should be noted that at least two repeating units included in the silk polypeptide used in the method of the present invention are identical repeating units (modules).

For example, the silk polypeptide used in the method of the present invention may include the following or consist of: modules C ^C ₄ , C ^C ₈ , C ^C ₁₆ , C ^C ₃₂ , A ^C ₅ , A ^C ₈ or A ^C ₁₀ .

Modules A ^K , C ^C , C ^K1 , C ^K2, and C ^KC can also be combined with each other, i.e. a module (repeating unit) A ^K can be combined with a module (repeating unit) C ^C (i.e. a combination of A ^K C ^C ), a module (repeating unit) With ^K1 can be combined with a module (repeating unit) With ^K2 and with a module (repeating unit) With ^KC (i.e., a combination of C ^K1 C ^K2 C ^KC ) and so on, provided that the polypeptide the silk used in the method of the present invention includes or consists of at least two repeating units that are identical. Thus, the silk polypeptide used in the method of the present invention may also include or consist of: modules (A ^K ) _n , (C ^C ) _n , (C ^K1 ) _n , (C ^K2 ) _n , (C ^KC ) _n , (A ^K A ^C ) _n , (C C ^C C ^C ) _n , (C ^K1 C ^K2 ) _n , (C ^K2 C ^K1 ) _n , (C ^K1 C ^K2 C ^K1 ) _n , (C ^K2 C ^K1 C ^K2 ) _n , (C ^K1 C ^K2 C ^KC ) _n , (C ^KC C ^K2 C ^KC ) _n or (C ^KC C ^K2 C ^K1 ) _n , where n is at least 2, preferably 4, 5, 6, 7 , 8, 10, 12, 16 or 20. The term “combined with each other” is defined above.

In addition, it is preferable that the silk polypeptide used in the method of the present invention would comprise essentially consist of or consist of: 2-80 repeating units, 3-80 repeating units or 4-60 repeating units, preferably from 8 to 48 repeating units or from 10 to 40 repeating units, and most preferably from 16 to 32 repeating units, i.e. 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, or 80 repeating units that are independently selected from module A (SEQ ID NO: 20) or its variants, module C (SEQ ID NO: 21) or its variants, module Q (SEQ ID NO ; 22) or its variants, module K (SEQ ID NO; 23) or its variants, module sp (SEQ ID NO: 24) or its variants, module S (SEQ ID NO: 25) or its variants, module R (SEQ ID NO: 26) or its variants, module X (SEQ ID NO: 27) or its variants, module Y (SEQ ID NO: 28) or its variants, module A ^C (SEQ ID NO: 29), module A ^K ( SEQ ID NO: 30), module C ^C (SEQ ID NO: 31), module C ^K1 (SEQ ID NO: 32), module C ^K2 (SEQ ID NO: 33) or module C ^KC (SEQ ID NO: 34) . Again, it should be noted that at least two repeating units included in the polypeptide used in the method of the present invention are identical repeating units (modules).

Modules A ^K , C ^C , C ^K1 , C ^K2, and C ^KC can also be combined with modules A, C, Q, K, sp, S, R, X, or Y, that is, the module (repeating unit) A ^K can be combined with a module (repeating unit) C (i.e., a combination of A ^K C), or a module (repeating unit) C ^C can be combined with a module (repeating unit) C (that is, a combination of C ^C C) and so on, provided that the silk polypeptide used in the method of the present invention includes or consists of at least two repeating units that are identical. Thus, the silk polypeptide used in the method of the present invention may also include or consist of modules (AQA ^K ) _n , (QA ^K ) _n , (QA ^K Q) _n , (A ^K QA) _n , (A ^K QA ^K ) _n , (CC ^C ) _n , (CC ^C C) _n , (C C ^C C ^C C) _n , (CC ^C C ^{C C} ) _n , (C ^C Q) _n , (QC ^C ) _n , (QC ^C Q ) _n , (C ^C QC) _n , (CQC ^C ) _n , (C ^C QC ^C ) _n , (CC ^K1 ) _n , (C ^K1 C) _n , (C ^K1 CC) _n , (CC ^K1 C) _n , (C ^KC C ^KC C) _n , (CC ^KC C ^KC ) _n , (C ^KC Q) _n , (QC ^KC ) _n , (QC ^KC Q) _n , (A ^K C ^K1 Q) _n , (QC ^K2 A ^K ) _n or (C ^Kl C ^K2 C) _n , where n is at least. 2, preferably 4, 5, 6, 7, 8, 10, 12, 16 or 20. The term “combined with each other” is defined above.

For example, the silk polypeptide used in the method of the present invention includes or consists of modules C ₁₆ C ^C , C ^C C ₁₆ , C ₈ C ^C C ₈ , C ₈ C ^C ₈ , C ^C ₈ C ₈ , C ₄ C ^C ₈ C ₄ , C ^C ₄ C ₈ C ^C ₄ , C ^C (AQ) ₂₄ or (AQ) ₂₄ C ^C.

The silk polypeptide used in the method of the present invention may further include at least one non-repeating (NR) unit, i.e. 1, 2, 3, 4, 5, 6 or more NR units, preferably one NR unit. In the context of the present invention, the term “non-repeating (NR) unit” refers to the region of amino acids present in a naturally occurring silk polypeptide that does not exhibit any obvious pattern of repetition (non-repeating unit or NR unit). Preferably, the non-repeating amino acid sequence corresponds to the non-repeating amino acid sequence of the filament polypeptides found in nature, preferably ADF-3 (SEQ ID NO: 1) or ADF-4 (SEQ ID NO: 2), or an amino acid sequence substantially similar to it.

It is particularly preferred that the amino acid sequence of the non-repeating unit corresponds to the non-repeating amino acid sequence of the carboxy terminus of the filament polypeptides found in nature, preferably ADF-3 (SEQ ID NO: 1) or ADF-4 (SEQ ID NO: 2), or amino acid a sequence essentially similar to her. More preferably, the amino acid sequence of the non-repeating unit corresponds to the non-repeating amino acid sequence of the carboxy terminus of ADF-3 (SEQ ID NO: 1), which includes amino acids 513 to 636, or ADF-4 (SEQ ID NO: 2), which includes amino acids 302 to 410, or an amino acid sequence essentially similar to them.

In this regard, the term "substantially similar" refers to the degree of amino acid identity for at least 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 even 99.9%, preferably for 20, 30, 40, 50, 60, 70, 80 or more amino acids, preferably along the entire length, the corresponding reference non-repeating (carboxy-terminal) amino acid sequence of the filament polypeptides found in nature, preferably ADF-3 (SEQ U NO: 1) or ADF-4 ( SEQ ID NO: 2).

A “non-repeating unit” comprising an amino acid sequence that is “substantially similar” to the corresponding non-repeating (carboxy-terminal) amino acid sequence in a framework strand polypeptide found in nature (i.e., a non-repeating (carboxy-terminal) wild-type link), preferably within ADF-3 (SEQ ID NO: 1) or ADF-4 (SEQ ID NO: 2) is also similar in terms of its functional properties, for example, a silk polypeptide comprising a “substantially similar non-repeating unit” flushes the ability to form fibers. Preferably, from a silk polypeptide comprising a “substantially similar non-repeating unit”, it is still possible to draw a smooth and uniform fiber at a speed of at least 0.1 cm / sec, preferably 10 cm / sec, and more preferably 10 m / sec One of skill in the art can visually assess whether fiber production will still occur. The smooth and uniform appearance of said fiber can be controlled using electron microscopy.

Most preferably, the non-repeating (NR) unit is NR3 (SEQ ID NO: 41) or variants thereof or NR4 (SEQ ID NO: 42) or variants thereof. The NR3 link (SEQ ID NO: 41) basically has the amino acid sequence ADF-3 of the spider Araneus diadematus, and the NR4 link (SEQ ID NO: 42) basically has the amino acid sequence ADF-4 of the spider Araneus diadematus (WO 2006/008163) .

The variant of the NR3 or NR4 link differs from the reference NR3 (SEQ ID NO: 41) or NR4 (SEQ ID NO: 42) from which it originated, up to 1, 2, 3, 4, 5, 6, 7 8 9 10 11, 12, 13, 14, 15, 16, 17. 18, 19, 20, 25, or 30 amino acid changes in the amino acid sequence (i.e., substitutions, additions, insertions, deletions, N-terminal cuts and / or C cuts -end). Such a variant of the NR3 or NR4 link in an alternative or additional embodiment may be characterized by a certain degree of sequence identity to the reference NR3 or NR4 link from which it originated. Thus, a variant of an NR3 or NR4 unit is characterized by sequence identity with the corresponding reference NR3 or NR4 unit of at least 50%, 55%, 60%, 65%, 70%, 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 even 99.9%. Preferably, sequence identity is observed over a continuous length of at least 10, 20, 30, 40, 50, 60, 70, 80, 90 and more amino acids, preferably along the entire length of the corresponding NR3 or NR4 reference unit.

In particular, it is preferred that the sequence identity be at least 80% over the entire length, at least 85% over the entire length, at least 90% over the entire length, at least at least 95% of the entire length would be at least 98% of the entire length or would be at least 99% of the entire length of the corresponding NR3 or NR4 reference unit. In addition, it is particularly preferred that the sequence identity is at least 80% over a continuous length of at least 20, 30, 40, 50, 60, 70, or 80 amino acids, at least 85% for a continuous length of at least 20, 30, 40, 50, 60, 70, or 80 amino acids would be at least 90% for a continuous length of at least 20, 30, 40, 50, 60, 70, or 80 amino acids, would be at least 95% over a continuous length of at least 20, 30, 40, 50, 60, 70, or 80 amino acids, would be at least 98% of the continuous extent of at least 20, 30, 40, 50, 60, 70 or 80 amino acids or would be at least 99% of the continuous extent of at least 20, 30, 40, 50, 60, 70 or 80 amino acids of the corresponding NR3 or NR4 reference unit.

A fragment (or deletion variant) of the NR3 or NR4 link preferably includes removal of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids from the N-terminus and / or C-terminus. Removal can also be internal.

In addition, a variant or fragment of an NR3 or NR4 link in the context of the present invention is considered a variant or fragment of an NR3 or NR4 link only if modifications to the amino acid sequence on which this variant or fragment is based do not adversely affect the ability of the silk polypeptide to form fiber. Preferably, from a silk polypeptide comprising a variant or fragment of an NR3 or NR4 unit, it is still possible to draw a smooth and uniform fiber at a speed of at least 0.1 cm / sec, preferably 10 cm / sec, 20 cm / sec, 50 cm / s, 75 cm / s, 1 m / s, 2 m / s, 3 m / s, 4 m / s, 5 m / s, and more preferably 10 m / s. One of skill in the art can visually assess whether fiber production will still occur. The smooth and uniform appearance of said fiber can be controlled using electron microscopy.

Alternatively, a test can be conducted as to whether the variant or fragment of the NR3 or NR4 link will still allow polymerization and / or increase the solubility of the silk polypeptide in which it is included. One of ordinary skill in the art can easily assess whether a silk polypeptide comprising a variant or fragment of an NR3 or NR4 unit will have the aforementioned functional properties similar to those of a silk polypeptide including an appropriate reference NR3 or NR4 unit. Suitable assays are well known to those skilled in the art. For example, polymerization for silk polypeptides, including a variant or fragment of an NR3 or NR4 unit, and polymerization for silk polypeptides, including an appropriate reference unit NR3 or NR4, can be easily visualized using native gel electrophoresis. The solubility of a silk polypeptide comprising a variant or fragment of an NR3 or NR4 unit, and the solubility of a silk polypeptide comprising an appropriate NR3 or NR4 reference unit, can be subjected to a simple test by saturation of said silk polypeptides with an aqueous solution. The results, in conclusion, can be compared with each other.

Preferably, the silk polypeptide used in the method of the present invention is selected from the group consisting of ADF-3 (SEQ ID NO: 1) or its variants, ADF-4 (SEQ ID NO: 2) or its variants, MaSp I (SEQ ID NO : 43) or its variants, MaSp II (SEQ ID NO: 44) or its variants, (C) _m , (C) _n NR _z , NR _z (C) _m , (AQ) _n , (AQ) _n NR _z , NR _z (AQ) _n , (QAQ) _o , NR _z (QAQ) _o , (QAQ) _o NR _z , Y _p , X _p and K _P , where m is an integer in the range from 8 to 48 (then there are 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 or 48), n is an integer in the range from 4 to 24 (i.e. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15. 16, 17, 18, 19, 20, 21, 22, 23 or 24), o is an integer in the range from 2 to 20 (i.e. 2, 3.4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20), p is an integer in the range from 8 to 16 (i.e., 8, 9, 10, 11, 12 , 13, 14, 15 or 16), az is an integer ranging from 1 to 3 (i.e. 1, 2 or 3), preferably 1, and NR is a non-repeating unit. The above formulas are defined by one of the following: in the formula

(i) (C) _m — the number of “m” modules C is combined with each other, namely from 8 to 48 modules C, represented by the amino acid sequence corresponding to SEQ ID NO: 21,

(ii) (C) _m NR _z — the number of “m” modules C is combined with each other, namely from 8 to 48 modules C, represented by the amino acid sequence corresponding to SEQ ID NO: 21, where said modules C are additionally combined with the number “ z "non-repeating (NR) units, namely, non-repeating (NR) units in an amount ranging from 1 to 3, for example, non-repeating (NR) units NR3 represented by the amino acid sequence corresponding to SEQ ID NO: 41, or NR4 represented by the amino acid the sequence corresponding to SEQ ID NO: 42,

(iii) NR _z (C) _m - is present (z = 1) or combined with each other (z = 2 or 3) the number of “z” non-repeating (NR) links, namely from 1 to 3 non-repeating (NR) links, for example, non-repeating (NR) units of NR3 represented by an amino acid sequence corresponding to SEQ ID NO: 41, or NR4 represented by an amino acid sequence corresponding to SEQ ID NO: 42, wherein said non-repeating (NR) unit (s) is additionally combined with the number “m” modules C, namely with modules C represented by the amino acid sequence, corresponds to guide SEQ ID NO: 21, in an amount ranging from 8 to 48,

(iv) (AQ) _n - the number of “n” combinations of modules A and Q is combined with each other, namely from 6 to 24 combinations of modules A and Q, where module A represents the amino acid sequence corresponding to SEQ ID NO: 20, and the module Q represents an amino acid sequence corresponding to SEQ ID NO: 22,

(v) (AQ) _n NR _z - the number of “n” combinations of modules A and Q is combined with each other, namely from 6 to 24 combinations of modules A and Q, where module A represents the amino acid sequence corresponding to SEQ ID NO: 20, and module Q represents the amino acid sequence corresponding to SEQ ID NO: 22, and where said combinations of modules A and Q are further combined with the number of “z” non-repeating (NR) units, namely, non-repeating (NR) units in an amount ranging from 1 to 3, for example with non-repeating (NR) NR3 units represented by amino acids a valid sequence corresponding to SEQ ID NO: 41, or NR4 represented by the amino acid sequence corresponding to SEQ ID NO: 42,

(vi) NR _z (AQ) _n - present (z = 1) or combined with each other (z = 2 or 3) the number of "z" non-repeating (NR) links, namely from 1 to 3 non-repeating (NR) links, for example, non-repeating (NR) units of NR3 represented by an amino acid sequence corresponding to SEQ ID NO: 41, or NR4 represented by an amino acid sequence corresponding to SEQ ID NO: 42, where said non-repeating (NR) unit (s) is additionally combined with the number "n »Combinations of modules A and Q, namely with combinations of modules A and Q in an amount in the range from 6 to 24, where Module A is the amino acid sequence corresponding to SEQ ID NO: 20, and the modulus Q represents an amino acid sequence corresponding to SEQ ID NO: 22,

(vii) (QAQ) _o - the number of “o” combinations of modules Q, A and Q is combined with each other, namely from 8 to 16 combinations of modules Q, A and Q, where module Q represents the amino acid sequence corresponding to SEQ ID NO: 22, and module A represents the amino acid sequence corresponding to SEQ ID NO: 20,

(viii) (QAQ) _o NR _z - the number of “o” combinations of modules Q, A and Q is combined with each other, namely from 8 to 16 combinations of modules Q, A and Q, where module Q represents the amino acid sequence corresponding to SEQ ID NO: 22, and module A represents the amino acid sequence corresponding to SEQ ID NO: 20, and where the aforementioned combinations of modules Q, A and Q are further combined with the number of “z” non-repeating (NR) units, namely, non-repeating (NR) units in an amount in the range of 1 to 3, for example with non-repeating (NR) NR3 units represented by ami the acid sequence corresponding to SEQ ID NO: 41, or NR4 represented by the amino acid sequence corresponding to SEQ ID NO: 42,

(ix) NR _z (QAQ) o - there is (z = 1) or combined with each other (z = 2 or 3) the number of “z” non-repeating (NR) links, namely from 1 to 3 non-repeating (NR) links, for example, non-repeating (NR) NR3 units represented by an amino acid sequence corresponding to SEQ ID NO: 41 or NR4 represented by an amino acid sequence corresponding to SEQ ID NO: 42, wherein said non-repeating (NR) unit (s) is further combined with the number of “o” module combinations Q, A and Q, namely with combinations of modules Q, A and Q in an amount in the range from 8 to 16, g de module Q represents the amino acid sequence corresponding to SEQ ID NO: 22, and module A represents the amino acid sequence corresponding to SEQ ID NO: 20,

(x) Y _p - combined with each other the number of "p" modules Y, namely from 8 to 16 modules Y, represented by the amino acid sequence corresponding to SEQ ID NO: 28,

(xi) X _p - combined with each other the number of "p" modules X, namely from 8 to 16 modules X, represented by the amino acid sequence corresponding to SEQ ID NO: 27, and

(xii) K _p - the number of "p" K modules is combined with each other, namely from 8 to 16 K modules represented by the amino acid sequence corresponding to SEQ ID N0: 23.

More preferably

(i) z in (C) _m NR _z or NR _z (C) _m is 1, am is an integer in the range of 8 to 48 (i.e. 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 or 48), z in (C) _m NR _z or NR _z (C) _m is 2, am is an integer in the range from 8 to 48 (i.e. 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 or 48) or z in (C) _m NR _z or NR _z (C) _m is 3 , and m is an integer in the range from 8 to 48 (i.e. 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 or 48),

(ii) z in (AQ) _n NR _z or NR _z (AQ) _n is 1 and n is an integer in the range from 4 to 24 (i.e. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24), z in (AQ) _n NR _z or NR _z (AQ) _n is 2, an is an integer ranging from 4 to 24 (i.e. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24) or z in (AQ) _n NR _z or NR _z (AQ) _n is 3, and n is an integer in the range from 4 to 24 (i.e. 4, 5, 6, 7, 8, 9, 10 , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24),

(iii) z in NR _z (QAQ) _o or (QAQ) _o NR _z is 1, and o is an integer in the range from 2 to 20 (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20), z in NR _z (QAQ) _o or (QAQ) _o NR _z is 2, and o is an integer in a range from 2 to 20 (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) or z in NR _z (QAQ) _o or (QAQ) _o NR _z is 3, and o is an integer in the range from 2 to 20 (i.e. 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20),

where NR is a non-repeating unit, preferably NR3 or NR4.

Most preferably silk polypeptide used in the method of the present invention is a C ₁₆ NR3, C ₃₂ NR3, C ₁₆ NR4, C ₃₂ NR4, (AQ) _12, (AQ) _24, (AQ) ₁₂ NR3, (AQ) ₂₄ NR3 , (AQ) ₁₂ NR4, (AQ) ₂₄ NR4, C ₁₆ , C ₃₂ , Y ₈ , Y ₁₆ , X ₈ , X ₁₆ , K ₈ or K ₁₆ .

Variant ADF-3, ADF-4, MaSp I or MaSp II differs from the reference polypeptide (wild type) ADF-3 (SEQ ID NO: 1), ADF-4 (SEQ ID NO: 2), MaSp I (SEQ ID NO : 43) or MaSp II (SEQ ID NO: 44) from which it descended, up to 150 (up to 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120 , 130, 140 or 150) by amino acid changes in the amino acid sequence (i.e., substitutions, additions, insertions, deletions, cut-offs from the N-terminus and / or cuts from the C-terminus). Such an alternative or alternative embodiment may be characterized by a certain degree of sequence identity with the reference polypeptide (wild type) from which it originated. Thus, the variant ADF-3, ADF-4, MaSp I or MaSp II is characterized by sequence identity with the corresponding reference polypeptide (wild-type) ADF-3, ADF-4, MaSp I or MaSp II, comprising at least 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 even 99 ,9%. Preferably, sequence identity is observed over a continuous length of at least 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200, 250, 300, 350, 400 and more amino acids, preferably along the entire length of the corresponding reference polypeptide (wild type) of ADF-3, ADF-4, MaSp I or MaSp II.

In particular, it is preferred that the sequence identity be at least 80% over the entire length, at least 85% over the entire length, at least 90% over the entire length, at least at least 95% of the entire length, would be at least 98% of the entire length or would be at least 99% of the entire length of the corresponding reference polypeptide (wild-type) ADF-3, ADF-4, MaSp I or MaSp II. In addition, it is particularly preferred that the sequence identity be at least 80% over a continuous length of at least 20, 30, 50, 100, 150, 200, 250 or 300 amino acids, at least at least 85% for a continuous length of at least 20, 30, 50, 100, 150, 200, 250 or 300 amino acids, would be at least 90% for a continuous length of at least 20, 30, 50, 100, 150, 200, 250, or 300 amino acids would be at least 95% over a continuous length of at least 20, 30, 50, 100, 150, 200, 250, or 300 amino acids, would be at least 98% for a continuous extent of at least 20, 30, 50, 100, 150, 200, 250 or 300 amino acids, or would be at least 99% for a continuous extent, at least 20, 30, 50, 100, 150, 200, 250 or 300 amino acids of the corresponding reference polypeptide (wild type) ADF-3, ADF-4, MaSp I or MaSp II.

A fragment (or deletion variant) of the ADF-3 polypeptide (SEQ ID NO: 1) preferably includes the removal of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 150, 170, 200, 220, 250, 270, 300, 320, 350, 370, 400, 420, 450, 470, 500, 520, 550, 570, 600 or 610 amino acids from the N-terminus and / or C-terminus. Removal can also be internal.

A fragment (or deletion variant) of the ADF-4 polypeptide (SEQ ID NO: 2) preferably includes the removal of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70.75, 80, 85, 90, 95, 100, 120, 150, 170, 200, 220, 250, 270, 300, 320, 330, 340, 350, 360, 370, 380, or 390 amino acids from the N-terminus and / or C-terminus. Removal can also be internal.

A fragment (or deletion variant) of the MaSp I polypeptide (SEQ ID NO: 43) preferably includes the removal of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 620, 640 , 660, 670, 680 or 690 amino acids from the N-terminus and / or C-terminus. Removal can also be internal.

A fragment (or deletion variant) of the MaSp II polypeptide (SEQ ID NO: 44) preferably includes removal of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35 , 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 520, 540, 560 or 570 amino acids from the N-terminus and / or from the C-terminus. Removal can also be internal.

In addition, a variant or fragment of ADF-3, ADF-4, MaSp I or MaSp II in the context of the present invention is considered a variant or fragment of ADF-3, ADF-4, MaSp I or MaSp II, only if modifications in relation to the amino acid sequence on which this variant or fragment is based do not adversely affect the ability of the silk polypeptide to form fiber. Preferably, from a silk polypeptide comprising a variant or fragment of ADF-3, ADF-4, MaSp I or MaSp II, it is still possible to draw a smooth and uniform fiber at a speed of at least 0.1 cm / sec, preferably 10 cm / s, and more preferably 10 m / s. One of skill in the art can visually assess whether fiber production will still occur. The smooth and uniform appearance of said fiber can be controlled using electron microscopy.

Preferably, the method is carried out at temperatures in the range from 5 ° C. to 60 ° C., more preferably at temperatures in the range from 10 ° C. to 50 ° C., and most preferably at temperatures in the range from 20 ° C. to 40 ° C. Thus, the method can be implemented at a temperature of 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, or 60 ° C.

Preferably, the process is carried out at a pressure in the range from 10 kPa to 1000 kPa, more preferably from 40 kPa to 500 kPa, and most preferably from 50 kPa to 150 kPa. Thus, the method can be implemented at 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 950, 1000 kPa. It is particularly preferred that the method be implemented under standard conditions of temperature and pressure, that is, at 20 ° C and 101.325 kPa.

In preferred embodiments of the present invention, the dope obtained in step (a) comprises more than one polymer, preferably 2, 3 or 4 polymers, for example a polypeptide such as silk polypeptides, casein, zein or BSA. For example, the dope obtained in step (a) may contain (i) a silk polypeptide and casein, (ii) a silk polypeptide and Zein, (iii) a silk polypeptide and BSA, (iv) a silk polypeptide, Zein and casein, (v ) silk polypeptide, casein and BSA, (vi) silk polypeptide, BSA, casein and zein, (vi) Zein and casein, (viii) Zein and BSA, (ix) casein and BSA, or (x) Zein and casein and BSA. The polypeptides can be labeled, for example, enzyme labels, biotin, radioactive or fluorescent labels, for example fluorescein (FITC).

In other preferred embodiments, the spinning solution obtained in step (a) comprises more than one type of silk polypeptide. Preferably, the spinning solution obtained in step (a) of the method of the present invention contains 2, 3, 4, 5, 6, 7, 8, 9 or 10 different types of silk polypeptides, most preferably 2 different types of silk polypeptides. For example, a spinning solution may contain spider silk polypeptides of a filament spider that are different from each other with respect to their amino acid sequence. Spinning carried out in the method of the present invention may also include polypeptides of the spider silk of the carcass filament and the silk of the spider silk stalk, which differ from each other with respect to their natural origin. The spider silk polypeptide of the carcass filament arises in the large ampulla gland, while the lucid spiral filament polypeptide arises in the flagelliform gland.

The concentration of the polymer, for example, silk polypeptide, BSA, zein or casein, is preferably in the range from 0.15 mg / ml to 500 mg / ml, more preferably in the range from 0.15 mg / ml to 99.9 mg / ml, in the range from 0.15 mg / ml to 90 mg / ml, in the range from 0.15 mg / ml to 80 mg / ml, in the range from 0.15 mg / ml to 70 mg / ml, in the range from 0, 15 mg / ml to 60 mg / ml or in the range of 0.15 mg / ml to 50 mg / ml, most preferably in the range of 0.5 mg / ml to 50 mg / ml, in the range of 0.5 mg / ml to 40 mg / ml, in the range from 0.5 mg / ml to 30 mg / ml or in the range from 1 mg / ml to 20 mg / ml. It is particularly preferred that the concentration of the polymer, for example silk polypeptide, BSA, zein or casein, be in the range of 0.15 mg / ml to 20 mg / ml.

Thus, for example, the concentration of the polymer, for example, silk, casein, zein or BSA polypeptide, is (at least) 0.15 mg / ml, 0.2 mg / ml, 0.3 mg / ml, 0.4 mg / ml, 0.5 mg / ml, 0.6 mg / ml, 0.7 mg / ml, 0.8 mg / ml, 0.9 mg / ml, 1 mg / ml, 2 mg / ml, 3 mg / ml, 4 mg / ml, 5 mg / ml, 6 mg / ml, 7 mg / ml, 8 mg / ml, 9 mg / ml, 10 mg / ml, 11 mg / ml, 12 mg / ml, 13 mg / ml, 14 mg / ml, 15 mg / ml, 16 mg / ml, 17 mg / ml, 18 mg / ml, 19 mg / ml, 20 mg / ml, 21 mg / ml, 22 mg / ml, 23 mg / ml, 24 mg / ml, 25 mg / ml, 26 mg / ml, 27 mg / ml, 28 mg / ml, 29 mg / ml, 30 mg / ml, 31 mg / ml, 32 mg / ml, 33 mg / ml, 34 mg / ml, 35 mg / ml, 36 mg / ml, 37 mg / ml, 38 mg / ml, 39 mg / ml, 40 mg / ml, 41 mg / ml, 42 mg / ml, 43 mg / ml, 44 mg / ml, 45 mg / ml, 46 mg / l, 47 mg / ml, 48 mg / ml, 49 mg / ml, 50 mg / ml, 55 mg / ml, 60 mg / ml, 65 mg / ml, 70 mg / ml, 75 mg / ml, 80 mg / ml, 85 mg / ml, 90 mg / ml, 95 mg / ml, 100 mg / ml, 110 mg / ml, 120 mg / ml, 130 mg / ml, 140 mg / ml, 150 mg / ml, 160 mg / ml, 170 mg / ml, 180 mg / ml, 190 mg / ml, 195 mg / ml, 200 mg / ml, 250 mg / ml, 300 mg / ml or 350 mg / ml.

It should be noted that the method of the present invention requires the use of only very low concentrations of polymers, for example silk polypeptides, ranging from concentrations of only 0.15 mg / ml, and preferably ending in concentrations not greater than 90 mg / ml, more preferably not greater than 50 mg / ml, and most preferably not greater than 20 mg / ml. An advantage of this is the very low shear viscosity of the dope used in the process of the present invention, which in turn improves the processability of said dope.

The structure, increasing the viscosity, according to its structure, no restrictions are imposed as long as it will increase the longitudinal viscosity of the dope.

Preferably, the compound that increases the longitudinal viscosity of the solution is a linear or branched polymer having a molecular weight of at least 20 kDa, for example 25 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa , 90 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1 MDa, 1.5 MDa, 2 MDa, 2.5 MDa, 3 MDa, 3.5 MDa, 4 MDa, 4.5 MDa or 5 MDa. It is preferred that the linear or branched polymer have a molecular weight in the range from 20 kDa to 5 MDa or from 50 kDa to 4 MDa, more preferably from 100 kDa to 2 MDa or from 100 kDa to 1 MDa.

More preferably, the compound that increases the longitudinal viscosity of the solution is a linear or branched polymer having a molecular weight of at least 500 kDa, for example 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1 MDa, 1.5 MDa, 2 MDa, 2.5 MDa, 3 MDa, 3.5 MDa, 4 MDa, 4.5 MDa or 5 MDa. It is more preferred that the linear or branched polymer have a molecular weight in the range of 500 kDa to 5 MDa or 900 kDa to 5 MDa, most preferably 1 MDa to 4 MDa or 1 MDa to 3 MDa.

It is preferred that a compound that increases the longitudinal viscosity of the solution be selected from the group consisting of polyacrylamide (PAA), polyethylene (PE), polyethylene glycol (PEG), polysaccharides, dextrans, polyvinyl alcohols, nucleic acids and mixtures thereof. In one preferred embodiment, the dextran is dextran sulfate.

Preferably, the compound that increases the longitudinal viscosity of the solution is polyethylene glycol (PEG) having a molecular weight of at least 20 kDa, for example 25 kDa, 30 kDa, 40 kDa, 50 kDa, 60 kDa, 70 kDa, 80 kDa , 90 kDa, 100 kDa, 200 kDa, 300 kDa, 400 kDa, 500 kDa, 600 kDa, 700 kDa, 800 kDa, 900 kDa, 1 MDa, 1.5 MDa, 2 MDa, 2.5 MDa, 3 MDa, 3.5 MDa, 4 MDa, 4.5 MDa or 5 MDa. Preferably, polyethylene glycol (PEG) would have a molecular weight in the range from 20 kDa to 5 MDa or from 50 kDa to 4 MDa, more preferably from 100 kDa to 2 MDa or from 100 kDa to 1 MDa.

More preferably, the compound that increases the longitudinal viscosity of the solution is polyethylene glycol (PEG) having a molecular weight of at least 500 kDa, for example 550 kDa, 600 kDa, 650 kDa, 700 kDa, 750 kDa, 800 kDa, 850 kDa, 900 kDa, 950 kDa, 1 MDa, 1.5 MDa, 2 MDa, 2.5 MDa, 3 MDa, 3.5 MDa, 4 MDa, 4.5 MDa or 5 MDa. More preferably, polyethylene glycol (PEG) would have a molecular weight in the range of 500 kDa to 5 MDa or 900 kDa to 5 MDa, most preferably 1 MDa to 4 MDa or 1 MDa to 3 MDa.

The concentration of the compound, which increases the longitudinal viscosity of the solution, is preferably in the range from 0.1% (w / v) to 5% (w / v), more preferably in the range from 0.5% (w / v) .) up to 4% (w / v), and most preferably in the range from 1% (w / v) to 3% (w / v).

Thus, for example, the concentration of the compound that increases the longitudinal viscosity of the solution is 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9 , 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2 , 2, 2,3, 2,4, 2,5, 2,6, 2,7, 2,8, 2,9, 3,0, 3,1, 3,2, 3,3, 3,4 , 3,5, 3,6, 3,7, 3,8, 3,9, 4,0, 4,1, 4,2, 4,3, 4,4, 4,5, 4,6, 4 7, 4.8, 4.9 or 5% (w / v).

As, to their surprise, the inventors of the present invention found, already this low concentration of the compound, which increases the longitudinal viscosity of the solution, is sufficient for spinning the fibers using the method proposed in this document. The low concentration of said compound, on the one hand, increases the longitudinal viscosity of the solution, but, on the other hand, does not substantially increase the shear viscosity of the solution, so that the formation of fibers improves. Thus, the spinning solution used in the method of the present invention is mainly formed from a polymer, such as a silk polypeptide, zein or casein, that is, is relatively pure. In addition, due to the moderate use of compounds or substances in addition to a polymer, such as a silk polypeptide, zein, or casein, the method of the present invention is cost-effective and environmentally friendly.

The ratio in terms of% (mass.) Between the silk polypeptide and the longitudinal viscosity improver is preferably in the range from 0.1 to 5, from 0.2 to 5, from 0.3 to 5, or from 0.4 to 5, more preferably from 0.4 to 2, most preferably 0.8 to 1.5. Thus, in preferred embodiments, in which the silk polypeptide is present in the solution in an amount in the range of 1 to 20 mg / ml, the amount of longitudinal viscosity improver is selected so that the ratio in terms of% (mass) between the silk polypeptide and the longitudinal viscosity improver preferably would be in the range from 0.1 to 10, more preferably from 0.2 to 5, more preferably from 0.4 to 2, most preferably from 0.8 to 1.5.

For example, by adding high molecular weight polyacrylamide (5 MDa, 1% (w / v)), the Onesorg C ₁₆ dope may be sufficiently increased to provide improved fiber production on the air / liquid interface.

The solvent may be any solution in which a polymer is soluble, for example a polypeptide such as a spider silk polypeptide, Zein, BSA or casein, and a polymer that improves longitudinal viscosity. It is preferred that the solvent be selected from the group consisting of a polar solvent, preferably water, aqueous buffer, alcohol, preferably isopropanol, hexafluoroisopropanol, glycerol, ethanol (UN), propanol, butanol, octanol or acetone; a non-polar solvent, preferably hexane, dodecane or oil, other organic solvents, preferably ethyl acetate; and mixtures thereof. Thus, the solvent may also be a mixture of a polar solvent, such as water, and a non-polar solvent, such as hexane. In one preferred embodiment, the pH of the solvent is in the range of 4 to 10.

It should be noted that due to the combined use of very low concentrations of polymers, such as silk polypeptides, zein or casein, and very low concentrations of compounds that increase the longitudinal viscosity of the solution in the dope, using the method of the present invention can be facilitated to obtain fiber.

In addition, due to the combined use of very low concentrations of polymers, such as silk polypeptides, zein or casein, and very low concentrations of compounds that increase the longitudinal viscosity of the solution in the dope, the solution does not have a high viscosity consistency in contrast to other prior art dope solutions technicians. Thereby, the processability of the dope solution can be improved.

Preferably, the drawing of the fiber is initiated by introducing the air / liquid or liquid / liquid interface at the solution into contact with the drawing tool and drawing the drawing tool away from said interface. The term “air” as used herein preferably means a mixture of gases found naturally in the earth’s atmosphere. In the context of a liquid / liquid interface, where one of the liquids is a dope, it is preferred that the other liquid be poorly miscible with the dope. Accordingly, in the case of a dope solvent in the form of a polar solvent, the other liquid will preferably be non-polar. Alternatively, in the case of a dope solvent in the form of a non-polar solvent, preferably another liquid will be polar. A liquid / liquid interface can also be obtained at least temporarily by contacting two miscible liquids in contact with each other, for example, in a pipe where one liquid stream encompasses a stream of a second liquid. As you can imagine, without the desire to bind yourself with any theory, the initial introduction of the interface into contact with a drawing tool, such as a pipette tip, creates an attachment point for polymers in the dope solution. The pipette retraction leads to the orientation of the polymers in the solvent when their head or tail is turned to the drawing tool, as a result of which the polymers are aligned side by side with each other and are more and more associated with each other. Upon subsequent withdrawal, the polymers pass from an ordered state related to the type of solution or liquid crystal to a semi-solid or solid state. The term “drawing” in this context is used not only to indicate the initiation of a given alignment process, but also to mean the continuous removal of aligned or partially aligned polymer molecules from the spinning solution. During this condition of preferably continuous drawing, the drawing tool is no longer in contact with the spinning solution, but it “draws” further polymer molecules from the solution into a “separated solution”, that is, a solution containing aligned polymers that are no longer in contact with the bulk of the dope. Thus, in the context of the present invention, the term “drawing” means drawing as a process for producing fiber, which may also include stretching or hardening. In addition, drawing the fibers from the solution is not a process limited to drawing the fibers from the separated solution using a drawing tool, such as, for example, a glass needle, as shown in Example 1. The method may also include any suitable automated system, such as a pipe, through which solution is taken in the replenishing flow, with a nozzle at the end, where the spinning solution is extruded and withdrawn by a drawing tool, thereby thereby continuously drawing the fiber from the ext udirovannogo spinning solution. The drawing tool can be any type of tool with a tip corresponding to the drawing of the fiber and made of the corresponding material, for example, capable of providing a point of attachment to the polymers in the dope. Examples include, but are not limited to: pipette tips, needles, or thin rods made of plastic, metal, or glass. No restrictions are imposed on the fiber drawing speed until it is high enough to draw a thin flowing elementary fiber that will exist for a sufficiently long period of time to allow the solvent to evaporate and produce a fiber, preferably a thin, light and long fiber. However, in one preferred embodiment, the drawing is carried out by contacting the solution with the drawing tool and drawing the fiber from the surface. During the operation, that is, upon initial contact with the surface and drawing the fiber from the surface, the term “drawing” also refers to the removal of fiber from the solution, preferably at a constant speed. The ideal speed depends on the viscosity of the spinning solution, that is, on the type and concentration of the polymers used, and should be determined experimentally. Preferably, the hood is carried out at speeds of at least 0.1 cm / s, preferably in the range from 0.1 cm / s to 15 m / s, from 1 cm / s to 5 m / s, from 3 cm / s to 1 m / s, from 5 cm / s to 50 cm / s, from 5 cm / s to 15 cm / s, and more preferably 10 cm / s. For example, the hood is carried out at speeds of at least 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 20, 30, 40, 50, 60, 70, 80, 90 cm / s, 1, 1,5, 2, 2,5, 3, 3,5, 4, 4,5, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 m / s.

The method of the invention also provides for the extrusion of a dope to produce fiber. The term "extrusion" in this context refers to the application to the spinning solution of pressure to force it through an opening, for example a nozzle. In the field of polymer technology, “extrusion” methods are often used to produce fibers from molten thermoplastics, that is, the total extruded material hardens after leaving the hole. In the context of the present invention, the extruded dope does not completely harden. Instead, the dissolved polymers contained in the extruded dope are associated with the formation of a fiber, which is usually smaller in diameter than the hole through which the dope is extruded, while separating the residual solvent from the thus obtained fiber. In one preferred embodiment, the initially obtained fiber is connected to a fiber extraction device, for example a cylinder, spool or bobbin, onto which the fiber is continuously wound. Depending on the relative speed of, for example, the cylinder with respect to the speed of fiber production during extrusion, a pulling force can also be exerted on the extruded fiber, that is, the fiber in some embodiments can be considered to be “drawn” from the extruded solution.

In order to further improve the spinning ability of the spinning solution, the shear viscosity of the artificial spinning syrups can be increased simultaneously with the longitudinal viscosity to a value that does not harm the processing of the fluid, for example, by adding Newtonian viscosity improvers. Thus, spinnability indicates the suitability of the spinning solution for drawing fibers.

Thus, in one embodiment of the invention, the dope solution further comprises a compound that increases Newtonian viscosity, preferably monosaccharides, for example glucose, galactose or fructose; disaccharides, for example lactose or sucrose; polysaccharides, for example glycogen; or polyols, for example, inositol, sorbitol, glycerin or polyglycols; or mixtures thereof.

Preferably, the spinning solution further comprises dispersed material dispersed therein. Upon receipt of the fiber, this dispersed material is relatively uniformly embedded in the fiber. On the dispersed material in its composition, no restrictions are imposed as long as it is suitable for use when included in the fiber. It is preferred that the dispersed material be selected from the group consisting of granules, metals, conductive electricity (including gold, copper), crystallites, protein / protein complexes, enzymes (e.g., green fluorescent protein (PBS) or β-galactosidase) and cells (for example, human osteoblastic cells or human tendon precursor cells or any pluripotent cells or stem cells). Preferably, the cells are selected from cells secreting enzymes or hormones, for example Langerhans islet cells. Preferred crystallites are dyes that are water insoluble, fluorescent dyes (e.g. Sudan red) or azo dyes (e.g. Nile red).

There are no restrictions on the concentration of particulate material in the dope. Preferably, the particulate material in the dope is present in an amount in the range from 0.01% (w / v) to 10% (w / v), preferably from 0.5% (w / v) to 1% (mass./about.). Thus, the dispersed material in the dope may be present in an amount of 0.01, 0.05, 0.1, 02, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6, 5, 7.0, 7.5. 8.0, 8.5, 9.0, 9.5, 10.0% (w / v).

In one preferred embodiment, the spinning solution further comprises a compound that increases Newtonian viscosity, for example polyglycols, or dispersed material, for example granules. In another preferred embodiment, the spinning solution further comprises both a compound that increases Newtonian viscosity, for example polyglycols, and dispersed material, for example, granules.

The fiber after preparation may be processed by curing in a curing solution or crosslinking. Thus, it is preferred that the method of the present invention after step (b) comprise step (c) of curing the fiber in the curing solution or crosslinking the fiber.

Crosslinking may include joining other compounds with a fiber, or joining identical or different fibers to each other, whereby, for example, multi-fiber structures such as multilayer fibers or core-shell structures can be obtained.

The term “crosslinking,” as used herein, refers to a method for chemically bonding two molecules with a covalent bond. Crosslinking reagents or coupling reactions have terminal groups reactive with respect to specific functional groups (primary amines, sulfhydryl and the like) in polypeptides or other molecules. Preferably, the crosslinking or coupling reaction reagent used in the method of the present invention is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) or glutaraldehyde.

In yet another embodiment, the fiber is spooled while continuously drawing. This makes it possible to obtain and store long fibers.

In a second aspect, the present invention relates to a spinning solution for implementing the method of the present invention, comprising essentially consisting of or consisting of: (i) a polymer that can be introduced into an aqueous solution with a concentration of at least 0.15 mg / ml, (ii) a compound that increases the longitudinal viscosity of the dope, and (iii) a solvent.

In this context, it is particularly preferred that the polymer be the silk polypeptide as defined above.

In addition, it is particularly preferred that the concentration of the polymer, such as silk polypeptide, be at least 0.15 mg / ml and not more than 60 mg / ml, more preferably at least 0.15 mg / ml and not more than 50 mg / ml, and most preferably at least 0.15 mg / ml and not more than 20 mg / ml.

It is also particularly preferred that the concentration of the compound that increases the longitudinal viscosity of the dope is in the range of 0.1% (w / v) to 5% (w / v), more preferably in the range of 0, 5% (w / v) to 4% (w / v), and most preferably in the range from 1% (w / v) to 3% (w / v).

In particular, it is even more preferred that the concentration of the polymer, for example silk polypeptide, be at least 0.15 mg / ml and not more than 60 mg / ml, more preferably at least 0.15 mg / ml and not more than 50 mg / ml, and most preferably at least 0.15 mg / ml and not more than 20 mg / ml, and so that the concentration of the compound, which increases the longitudinal viscosity of the dope, would be in in the range of 0.1% (w / v) to 5% (w / v), more preferably in the range of 0.5% (w / v) to 4% (w / v) , and most prefer flax in the range of 1% (w. / v.) to 3% (wt. / vol.).

The spinning solution according to this aspect of the invention may also contain all the other components of the spinning solution mentioned above in the context of the description of the method of the invention. Similarly, this applies to all preferred and especially preferred embodiments of the various aforementioned components.

In a third aspect, the present invention provides a fiber obtained by the method of the first aspect.

In a fourth aspect, the present invention provides products comprising a fiber of the second aspect, such as

(i) a thread or

(ii) a woven fabric or non-woven fabric.

In one further aspect, the present invention provides the use of a fiber or filament comprising one or more cells for analyzing one or more cell functions and / or for preserving said cells. Preferably, the analysis of cell function is carried out before, during and / or after exposure to one or more cells with a test compound or stimulus, preferably a mechanical, thermal and / or electrical stimulus. Fibers, including cells, could also serve as a frame material for tissue engineering and the cultivation of artificial organs.

The present invention improves the spinnability of the polymer solution, making it possible to obtain the entire range of new materials. The present invention is particularly well suited for incorporating living and non-living biological material, since the method can be carried out under appropriate conditions using the temperature, pressure, solvent, and chemicals used.

Since in general all proteins can be incorporated into fibers in this way, in principle any enzymatic activity can be incorporated into such fibers. Perhaps more importantly, cells can also be introduced into the fibers as demonstrated for incorporation of micron-sized colloidal particles into the fibers. Since conditions favor cell viability, areas of application in bioengineering and tissue culture are possible. Fibers, including cells, could serve as a frame material for tissue engineering and the cultivation of artificial organs. In addition, the encapsulation of living cells makes it possible to influence various environmental conditions on these cells, for example, by pulling them through a reagent bath. With such approaches, new tools for cell biology may be available. In this spirit, the invention can be used for sequential as well as high-performance manipulation of cells in a continuous chain. One possibility is to draw the fibers during the introduction of cells from a spinning solution containing a biologically active compound, and screening the cells as they exit the solution. Thus, the position on the chain forms an index for cells, forming new approaches in unicellular high-throughput screening assays. In addition to this, areas of application for wound healing are possible. For example, fibers comprising cells secreting wound healing factors could be included in bandages or xerogels or supplemented, in particular in the context of wet wound healing. You can also imagine the options for use in obtaining skin grafts, reconstruction of ligaments or surgical mesh. In addition, the fibers obtained in accordance with the invention can be used in a wide range of other commercial products, for example, in the case of a rope, mesh, clothing fabric, building material, tent fabric, backpacks and much more, where strength, elasticity and low weight.

The following figures and examples are merely illustrations of the present invention and should in no way be taken as limiting the scope of the invention indicated in the attached claims.

Brief Description of the Figures

Figure 1 shows fibers drawn from a dope containing 5 mg / ml FITC-C ₁₆ and 0.5% (w / v) PAA. A needle made of colorless glass is drawn from an aliquot of dope (A), which as a result, a thin flowing elementary fiber (B) is obtained. (C) Fibers subjected to drawing at a speed of at least 10 cm / sec have a smooth and uniform appearance. (D) Fibers subjected to stretching at a speed less than the proper threshold speed. The liquid droplets still form a coating of the flowing elementary fiber and are retained after the fiber is deposited on a clean microscope slide. Figure 2 shows fibers drawn from a spinning solution containing approximately 10 ⁷ / ml polystyrene granules (1 μm in diameter) dispersed in solution at 5 mg / ml C ₁₆ and 0.5% (w / v) PAA. Granules are included in the fibers and distributed fairly evenly. Figure 3 shows an atomic force microscopy image of a fiber subjected to drawing from a dope solution containing polystyrene granules (1 μm in diameter) dispersed in solution at 5 mg / ml C ₁₆ and 0.5% (w / v) PAA . Figure 4 The granules are coated with fiber material and are tightly connected to the fiber. Demonstrates zein fiber, drawn from a dope solution containing 300 mg / ml zein and 0.5% (w / v) PAA in 63.75% ethanol. Zein fiber showed a smooth surface.

Examples

The following examples are for illustrative purposes only and in no way limit the invention described above.

Example 1: The principle of the method of drawing

Figure 1A shows an aliquot of a dope solution consisting of C ₁₆ as a polymer component, PAA as a polymer that improves longitudinal viscosity, and water as a solvent on a microscope slide in contact with a clear glass needle as a drawing tool. Pulling the needle, which is in contact with the spinning solution, from the solution results in a thin flowing elementary fiber. This elementary fiber with a sufficiently quick drawing exists for a sufficiently long period of time to ensure evaporation of water as a solvent and to obtain a thin and light fiber (Figure 1B). Fibers contain protein, which can be demonstrated by IR spectroscopy and fluorescence microscopy using labeled protein.

Example 2: Low Concentration Spider Silk Protein Fibers

100 μl of dope (10 mg / ml FITC-C ₁₆ , 1.0% (w / v) PAA) was placed on a microscope slide. A needle made of colorless glass (5 cm long and 1 mm in diameter) was brought into contact with a spinning solution, and then it was removed by hand from a microscope slide at variable speeds. This method as a result led to the drawing of the fiber from the dope, while the quality of the fiber depended on the speed used. At a drawing speed of 10 cm / sec or more, the fibers had a smooth and uniform appearance (Figure 1C). In the case of drawing at speeds lower than the threshold, liquid droplets still formed a coating of a flowing elementary fiber and remained after the fiber was deposited on a clean microscope slide (Figure 1D). At a drawing speed of 10 cm / sec or more, good fiber was obtained.

Example 3: Fibers Drawn by a Falling Steel Pellet

100 μl of a dope solution (10 mg / ml C ₁₆ , 1.0% (w / v) PAA) was placed near the edge of the microscope slide. Between the edge and the dope was placed a steel granule with a diameter of 1 mm in contact with the dope. While holding the microscope slide with a hand, the glass was slightly tilted, causing the granule to fall from the edge. Surprisingly, the falling granule pulled the fiber from the spinning dope in much the same way as the needle from Example 1. The method of dropping a ball and producing fiber can be compared to the falling of a spider producing fiber, although there is obviously a spinning syrup reservoir at different ends of the fiber.

Example 4: Embedding Granules in Fibers

About 10 ⁷ / ml of Dynabeads product (polystyrene or PS granules) 1 μm in diameter was dispersed in 100 μl of a dope solution (10 mg / ml C ₁₆ , 1.0% (w / v) PAA). A needle made of colorless glass (5 cm long and 1 mm in diameter) was brought into contact with a spinning solution, and then it was retracted by hand from a microscope slide at a speed of approximately 10 cm / sec. This method as a result led to the drawing of fibers from the spinning solution, including granules from PS. The granules were embedded in the fiber and distributed fairly evenly (Figure 2). Atomic force microscopy demonstrated the coating of granules with fiber material and their tight connection with the fiber (Figure 3).

Since this experiment can be carried out under conditions corresponding to living cells in temperature, pressure, and solvent, it demonstrates the possibility of including cells instead of the used granules from PS, which had a diameter similar to the diameters of many cells, for example fibroblasts.

Example 5: Zein Fibers

Zein is a reserve protein from corn and has been used as a material for decades. It is available with reasonable purity in large quantities at low cost (Tsai 1979). 300 mg / ml zein in 85% ethanol was mixed with 2% (w / v) PAA, with a final concentration of PAA of 0.5% (w / v). 100 μl of this dope was placed on a microscope slide. A needle made of colorless glass (5 cm long and 1 mm in diameter) was brought into contact with a spinning solution, and then it was retracted by hand from a microscope slide at a speed of approximately 10 cm / sec. This method as a result led to the drawing of zein fibers from the spinning solution (Figure 4). Such zein fibers were insoluble in water and had a smooth surface. They apparently showed good mechanical stability, but were quite fragile and unyielding. Zein fibers could be made more pliable in a formaldehyde bath.

Example 6: Incorporation of crystallites into fibers

Dyes (Sudan red (Sigma 20161-8) or Nile red (Fluka 72485)) were added to a spinning solution (10 mg / ml C ₁₆ , 1.0% (w / v) PAA) in suspension in the form of a fine powder. Both dyes were embedded in the fibers. Both dyes are hydrophobic and adhere firmly to the hydrophobic proteins of spider silk.

Example 7: Enzyme Incorporation into Fiber

Fluorescent proteins such as green fluorescent protein (GFP) were included in the fibers (10 mg / ml C ₁₆ , 1.0% (w / v) PAA). After switching on, bright fluorescence was observed. This demonstrates the still intact protein folding (GFP) in the fibers and therefore the protein itself.

Β-galactosidase was included in the fibers (10 mg / ml C ₁₆ , 1.0% (w / v) PAA). As a result of enzymatic hydrolysis, hydrolysis of o-nitrophenyl-β-D-galactopyranoside (ONPH) occurs with the formation of a yellow o-nitrophenol dye. The concentration of o-nitrophenol is established to quantify the activity of β-galactosidase in the fiber. Enabling enzymes can be used for any application that requires enzymes that are large enough to be trapped in the fiber while allowing diffusion of the substances in and out.

Claims (24)

1. A method of spinning fibers from a spinning solution, comprising the steps of:
(a) preparing a spinning solution containing (i) a polymer that can be added to the aqueous solution at a concentration of at least 0.15 mg / ml, (ii) polyacrylamide (PAA) and (iii) a solvent; and
(b) drawing the fiber from the dope, or combining extrusion and drawing the fiber from the dope, resulting in a fiber. 2. The method according to claim 1, where the polymer is a polypeptide, preferably a silk polypeptide comprising at least two identical repeating units, bovine serum albumin (BSA), zein or casein. 3. The method according to claim 2, where the silk polypeptide includes at least two identical repeating units, each of which includes at least one consensus sequence selected from the group consisting of:
i) GPGXX (SEQ ID NO: 3), where X is any amino acid, preferably in each case independently selected from A, S, G, Y, P, N and Q;
ii) GGX, where X is any amino acid, preferably in each case independently selected from Y, P, R, S, A, T, N and Q; and
iii) A _x , where x is an integer in the range from 5 to 10. 4. The method according to claim 3, where the repeating units are independently selected from module A (SEQ ID NO: 20), module C (SEQ ID NO: 21), module Q (SEQ ID NO: 22), module K (SEQ ID NO : 23), module sp (SEQ ID NO: 24), module S (SEQ ID NO: 25), module R (SEQ ID NO: 26), module X (SEQ ID NO: 27), or module Y (SEQ ID NO: : 28) or their variants. 5. The method according to claim 2, where the silk polypeptide further comprises at least one non-repeating (NR) unit. 6. The method according to claim 5, where the NR unit is NR3 (SEQ ID NO: 41) or its variants or NR4 (SEQ ID NO: 42) or its variants. 7. The method according to claim 1, where the concentration of the polymer is in the range from 0.15 mg / ml to 350 mg / ml, preferably in the range from 0.5 mg / ml to 50 mg / ml, and more preferably in the range from 1 mg / ml to 20 mg / ml. 8. The method according to claim 1, where the polyacrylamide (PAA) is covalently attached to the polymer. 9. The method according to claim 1, where the concentration of polyacrylamide (PAA) is in the range from 0.1% (w / v) to 5% (w / v). 10. The method according to claim 1, where the solvent is selected from the group consisting of a polar solvent, preferably water, aqueous buffer or alcohol; preferably isopropanol, hexafluoroisopropanol, glycerol, ethanol (EtOH), propanol, butanol, octanol, a non-polar solvent, preferably hexane, dodecane, oil, acetone or organic solvents, preferably ethyl acetate; and mixtures thereof. 11. The method according to claim 1, where the drawing of the fiber is initiated as a result of introducing the air / liquid interface at the solution into contact with the drawing tool and drawing the drawing tool away from said interface. 12. The method according to claim 1, where the hood is carried out at speeds of at least 0.1 cm / s, preferably 10 cm / s, and more preferably 10 m / s. 13. The method according to claim 1, where the spinning solution further comprises a compound that increases Newtonian viscosity, preferably monosaccharides, polysaccharides or polyols. 14. The method according to claim 1, where the dope solution further comprises a dispersed dispersed material dispersed therein. 15. The method according to 14, where the dispersed material is selected from the group consisting of cells, granules, metals, conductive electricity, crystallites, protein / protein complexes and enzymes or growth factors. 16. The method according to 14, where the dispersed material in the spinning solution is present in an amount in the range from 0.01% (wt./vol.) To 10% (wt./vol.), Preferably from 0.5% (mass ./vol.) up to 1% (mass./vol.). 17. The method according to claim 1, where the method after step (b) comprises the step of (c) curing the fiber in the curing solution or crosslinking the fiber. 18. A spinning solution for implementing the method according to claims 1-17, containing (i) a polymer that can be introduced into an aqueous solution with a concentration of at least 0.15 mg / ml, (ii) polyacrylamide (PAA) and ( iii) a solvent. 19. The fiber obtained by the method according to claims 1-17. 20. A thread comprising a fiber according to claim 19. 21. A woven or non-woven material comprising fiber according to claim 19. 22. The use of a fiber according to claim 19 or a thread according to claim 20, comprising one or more cells, for preserving said cells, where said cells are included in them. 23. The use of claim 22, wherein one or more cells is used (i) in a screening study of cells to detect one or more cellular functions;
(ii) as a framework material for tissue engineering and the cultivation of artificial organs; and / or (iii) for wound healing. 24. The use according to claim 23, wherein the analysis of cell function is carried out before, during and / or after exposure to one or more cells with a test compound or stimulus, preferably a mechanical, thermal and / or electrical stimulus.

Images