Recombinant spider silk protein

Abstract

The recombinant spider silk protein comprises an NT domain, a REP domain and a CT domain. The REP domain has the formula pA 1 -pG-pA 2 pG stands for glycine-rich domain, pA 1 and pA 2 represents the alanine-rich domain. 1 and pA 2 One of them is a polyalanine domain, pA 1 and pA 2 The other is a polyalanine domain in which every third or fourth alanine residue is replaced by an isoleucine or valine residue.Spider silk spun from the recombinant spider silk protein of the invention has improved mechanical properties.

Description

[Technical field]

[0001] The present invention relates generally to recombinant spider silk proteins, and in particular to recombinant spider silk proteins capable of producing silk fibres with improved mechanical properties. [Background technology]

[0002] Spiders can make seven types of silk, each with its own mechanical properties, produced in different glands (major ampullate, minor ampullate, flagellate, tubular, grape-like, collecting, and pear-like glands). These silks are made of silk proteins named according to their major expression glands: major ampullate (MaSp), minor ampullate (MiSp), flagellate (FlSp), tubular (TuSp), grape-like (AcSp), collecting (AgSp), and pear-like (PiSp). All spider silk proteins, also called spidroins in the art, have an N-terminal (NT) domain, an extensive repeat region (REP), and a C-terminal (CT) domain. The mechanical properties are thought to be determined by the REP domain (Guerette et al., Science 11:112-115 (1996)). Flagellate silk, the most extensible fiber, is made mainly from spider silk protein (FlSp) with a Pro-rich REP domain predicted to form a spring-like structure. Large ampullate silk, the strongest fiber, also called drag silk, is composed mainly of spider silk protein (MaSp) with a repeat domain of Gly-rich and poly-Ala repeats. The tensile strength of large ampullate silk comes from MaSp poly-Ala blocks that form β-sheet crystals in the silk fiber, while the Gly-rich portion mediates fiber extensibility (Bratzel et al., J Mech Behav Biomed Mater. 7:30-40 (2012); Liu et al., Adv Funct Mater. 26:5534-5541 (2016); Keten et al., Nat Mater. 9:359-367 (2010)). MaSp silk is the strongest natural fiber known (~150 MJ / m 3 ) (Gosline et al., J Exp Biol 202:3295-3303(1999), Blackledge et al., J Exp Biol 209:2452-2461(2006)).

[0003] A recombinant spider silk protein with improved solubility in water, thereby enabling large-scale production with high yields, is known in the art as NT2RepCT (WO2018 / 002216; Andersson et al., Nat Chem Biol 11:309-315(2017)). NT2RepCT contains a His6 tag, an NT domain from Euprosthenops australis MaSp1, two Gly-rich and polyAla tandem repeats (2Rep) from E. australis, and a CT domain from Araneus ventricosus MiSp.

[0004] Johansson and Rising, ACS Nano 15:1952-1959 (2021) disclose a structural biology-based approach to engineer artificial spidroins to produce biomimetic yarn fibers with improved mechanical properties.

[0005] However, there remains a need for recombinant spider proteins that can produce silk fibres with improved mechanical properties. Summary of the Invention

[0006] It is a general object to provide recombinant spider silk proteins capable of producing silk fibres with improved mechanical properties.

[0007] This and other objects are achieved by embodiments of the present invention.

[0008] The invention is defined in the independent claims. Further embodiments of the invention are defined in the dependent claims.

[0009] One aspect of the present invention relates to a recombinant spider silk protein comprising an N-terminal (NT) domain, a repeat region (REP) domain, and a C-terminal (CT) domain. The REP domain comprises a set of domains according to the formula pA1-pG-pA2, where pG represents a glycine-rich domain and pA1 and pA2 represent alanine-rich domains. One of pA1 and pA2 is a polyalanine domain, and the other of pA1 and pA2 is a polyalanine domain in which every third or fourth alanine residue is replaced by an isoleucine or valine residue.

[0010] Further aspects of the invention relate to silk fibres made from said recombinant spider silk proteins, synthetic materials comprising said silk fibres, nucleic acid molecules encoding said recombinant spider silk proteins, expression vectors comprising said nucleic acid molecules, and host cells comprising said expression vectors.

[0011] An additional aspect of the invention relates to a method for producing silk fibers, the method comprising extruding a spin dope containing the recombinant spider silk protein described above into an aqueous buffer having an acidic pH to induce polymerization of the recombinant spider silk protein into silk fibers, the method also comprising isolating the silk fibers from the aqueous buffer.

[0012] The recombinant spider silk proteins of the invention can be spun into silk fibers with very high tensile strength and breaking strain, and toughness comparable to that of natural drag silk. The recombinant spider silk proteins of the invention can also be produced in high yields and concentrated to high concentrations to produce a spin dope suitable for the production of silk fibers.

[0013] The embodiments, together with further objects and advantages thereof, may best be understood by reference to the following description taken together with the accompanying drawings, in which: [Brief description of the drawings]

[0014] [Figure 1A]Schematic diagram of the designed construct. NT2RepCT(A15-A14) consists of an N-terminal domain (NT, PDB:4FBS), a repeat region containing two polyAla blocks, and a C-terminal domain (CT, PDB 3LR2). Both subunits of the soluble NT2RepCT dimer are shown (one is shaded). [Figure 1B] Schematic diagram of the designed constructs. Protein sequence alignment of the repeat region of A15-A14 and its engineered constructs. Note that all constructs contain NT, a repeat portion, and a CT. Substitutions of polyAla blocks are indicated. The sequences presented in B are found in SEQ ID NOs: 1-16. [Figure 2A] Rosetta energy profiles of A15-A14 and (A3I)3-A14 (profiles for all designed proteins are shown in Table 1). Bars indicate the Rosetta energy for transferring hexapeptides (shown at the first residue of each hexapeptide), dark grey bars indicate Rosetta energies below -23 kcal / mol (dashed line). Light grey bars indicate Rosetta energies above the threshold, making it unlikely to form a steric zipper (https: / / services.mbi.ucla.edu / zipperdb / ). [Figure 2B] The bars indicate the Rosette energy of the hexapeptide with the lowest predicted energy among A15-A14 and the engineered minispidroins (all hexapeptides are shown in Table 1). [Figure 2C] A hypothetical zipper structure of two β-sheets composed of hexapeptides AAAAAA (SEQ ID NO: 17) derived from A15-A14 and AIAAAAI (SEQ ID NO: 24) derived from (A3I)3-A14, respectively. [Figure 3A] CD spectroscopy of purified engineered minispidroins. Initial spectrum at 20 °C. [Figure 3B] CD spectroscopy of purified engineered minispidroins. Molar ellipticities measured at 222 nm from 20 °C to 90 °C were converted to percent natively folded and then normalized. [Figure 3C]CD spectroscopy of purified engineered minispidroins. CD spectroscopy of different constructs upon heating to 90°C. [Figure 3D] CD spectroscopy of purified engineered minispidroins.CD spectroscopy of different constructs after cooling to 20°C. [Figure 4A] Mechanical properties of spinnable engineered minispidroins compared to A15-A14. A) Photograph of spun fibers, B) strength, C) breaking strain, D) toughness modulus (dashed line shows toughness modulus of native drag yarn), E) representative stress-strain curves. Note that (A3T)3-(A3T)3 and (A3V)3-(A3V)3 have very low strains. Enlarged graphs showing these are shown in Figure 7A. Whiskers show standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4B] Mechanical properties of spinnable engineered minispidroins compared to A15-A14. A) Photograph of spun fibers, B) strength, C) breaking strain, D) toughness modulus (dashed line shows toughness modulus of native drag yarn), E) representative stress-strain curves. Note that (A3T)3-(A3T)3 and (A3V)3-(A3V)3 have very low strains. Enlarged graphs showing these are shown in Figure 7A. Whiskers show standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4C] Mechanical properties of spinnable engineered minispidroins compared to A15-A14. A) Photograph of spun fibers, B) strength, C) breaking strain, D) toughness modulus (dashed line shows toughness modulus of native drag yarn), E) representative stress-strain curves. Note that (A3T)3-(A3T)3 and (A3V)3-(A3V)3 have very low strains. Enlarged graphs showing these are shown in Figure 7A. Whiskers show standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4D]Mechanical properties of spinnable engineered minispidroins compared to A15-A14. A) Photograph of spun fibers, B) strength, C) breaking strain, D) toughness modulus (dashed line shows toughness modulus of native drag yarn), E) representative stress-strain curves. Note that (A3T)3-(A3T)3 and (A3V)3-(A3V)3 have very low strains. Enlarged graphs showing these are shown in Figure 7A. Whiskers show standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Figure 4E] Mechanical properties of spinnable engineered minispidroins compared to A15-A14. A) Photograph of spun fibers, B) strength, C) breaking strain, D) toughness modulus (dashed line shows toughness modulus of native drag yarn), E) representative stress-strain curves. Note that (A3T)3-(A3T)3 and (A3V)3-(A3V)3 have very low strains. Enlarged graphs showing these are shown in Figure 7A. Whiskers show standard deviation. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. [Diagram 5] FTIR spectroscopy of engineered fibers. Normalized and baseline subtracted absorbance spectra in the amide I region of A) A15-A14, (A3V)3-(A3V)3, (A3V)3-A14, (A3T)3-(A3T)3, and B) A15-A14, (A3I)3-A14, A15-(A3I)3, and (A2I)4-A14. C) Percent secondary structure content determined by co-fitting the absorbance spectra with the second derivative. The horizontal line indicates the β-sheet content of A15-A14. [Figure 6] Solid-state NMR 13C-13C correlation spectra (aliphatic region) of the A15-A14 fiber (dark grey) and (A3I)3-A14 fiber (light grey). Cα / Cβ correlations of Ala and Ile in α-helical and β-sheet conformations are shown. [Figure 7A] Continuation of mechanical characterization of spinnable constructs. Zoomed-in representative stress-strain curves. Full stress-strain curves are presented in Figure 4E. [Figure 7B] Mechanical properties of spinnable constructs continued. Young's modulus of fibers. [Figure 7C] Mechanical properties of spinnable constructs cont. Fiber diameter. [Figure 8] Extrusion of A15-A14 or (A3I)3-A14 at 17 or 35 μl / min through a tapered metal nozzle with an orifice diameter of 150 μm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] The present invention relates generally to recombinant spider silk proteins, and in particular to recombinant spider silk proteins capable of producing silk fibres with improved mechanical properties.

[0016] The spider silk protein of the present invention is a recombinant or engineered spider silk protein, i.e. an artificial and non-naturally occurring spider silk protein. The recombinant spider silk protein is preferably in the form of an isolated recombinant spider silk protein. The recombinant spider silk protein of the present invention can produce silk fibers with improved mechanical properties compared to NT2RepCT (WO2018 / 002216; Andersson et al., Nat Chem Biol 11:309-315(2017)). More specifically, the silk fibers produced from the recombinant spider silk protein have significantly higher strength, breaking strain, and toughness modulus compared to NT2RepCT.

[0017] Another important advantage of the recombinant spider silk proteins of the present invention is that they can be produced in high yields and concentrations, which are advantageous when preparing spin dopes used to spin the silk fibers. Generally, such spin dopes should contain very high concentrations of spider silk proteins to promote the production of silk fibers.

[0018] Thus, one aspect of the invention relates to a recombinant spider silk protein comprising an N-terminal (NT) domain, a repeat region (REP) domain and a C-terminal (CT) domain. According to the invention, the REP domain comprises a set of domains according to the formula pA1-pG-pA2, where pG stands for a glycine-rich (G-rich or Gly-rich) domain and pA1 and pA2 stand for an alanine-rich (A-rich or Ala-rich) domain. According to the invention, one of pA1 and pA2 is a polyalanine domain and the other of pA1 and pA2 is a polyalanine domain in which every third or fourth alanine (A or Ala) residue is replaced by an isoleucine (I or Ile) or valine (V or Val) residue.

[0019] Spider silk proteins, also called spidroins, are composed of NT, REP, and CT domains. The terminal domain is important for the solubility of spider silk proteins during storage and regulates their assembly into solid fibers. The REP domain of most large ampullate spidroins (MaSps) contains up to 100 tandem repeats of polyalanine blocks and glycine-rich motifs. In soluble dopes, spider silk proteins are mostly in random coil and helical conformations, but solid silk fibers contain nano-sized crystals composed of stacked antiparallel β-sheets embedded in an amorphous structure. This heterogeneous structure of the silk fibers is important because the β-sheet crystals impart strength and the amorphous structure imparts extensibility to the fibers. The amorphous matrix contains β-turns and ordered structures with conformations similar to collagen and polyproline helices, with glycine-rich regions dominating. The β-sheets formed by the polyalanine blocks are oriented with the β-strands parallel to the fiber axis, and the alanine side chains of a given β-strand fill the space close to the α-carbons in adjacent β-strands, resembling a tightly packed steric zipper.

[0020] There are two main strategies to generate artificial silk fibers. One is the expression of insoluble spider silk proteins followed by solubilization and fiber processing using organic solvents, and the other is a biomimetic approach that reproduces the molecular mechanisms and triggers of fiber formation using only aqueous solutions throughout the purification and spinning process. The first approach allows the expression of large spider silk proteins that can be spun into high tensile strength silk fibers, but the protein yields are far from those required for industrial production (Bowen et al., Biomacromolecules 19:3853 (2018); Edlund et al., New Biotechnology 42:12 (2018)). Using the second approach, small spider silk proteins, often called minispidroins, have been developed, consisting of an NT domain, a short REP domain, generally consisting of two polyalanine / glycine-rich domains, and a CT domain. Such minispidroins are highly water-soluble and can be spun into silk fibers using a biomimetic spinning mechanism. Moreover, one of these minispidroins, NT2RepCT, can be produced in bioreactor culture at a yield of 14.5 g / L, ensuring economically feasible mass production (Edlund et al., New Biotechnology 42:12 (2018); Schmuck et al., Materials Today 50:16 (2021)). Although thread fibers spun from NT2RepCT are superior to previously published as-spun thread fibers, they still only reach about 15% of the tensile strength of native thread fibers (Gosline et al., Journal of Experimental Biology 202:3295 (1999); Andersson et al., Nature Chemical Biology 13:262 (2017)). NMR spectroscopy revealed that the two polyalanine domains of minispidroins are in an α-helical conformation in the soluble state and convert to a β-sheet conformation in the as-spun wet fibers.However, upon drying of the yarn fibers, the polyalanine domains revert to an α-helical conformation (Otikovs et al., Angew Chem Int Ed Engl 56:12571 (2017)), which may explain the inferior mechanical properties of dried NT2RepCT fibers compared to native yarn fibers.

[0021] The recombinant spider silk protein of the present invention improves the mechanical properties of silk fibers by increasing the β-strand tendency and β-sheet-beta-sheet interactions of the polyalanine domains. Notably, alanine residues have a low tendency to form β-strands, while more hydrophobic residues such as valine, cysteine, isoleucine, and phenylalanine show a high β-strand tendency and therefore may be considered better candidates for forming stable β-sheets in silk fibers. However, spider silk proteins are secreted proteins and therefore need to pass through a translocon when produced by glandular epithelia. If the nascent polypeptide chain contains a segment rich in valine, isoleucine, cysteine, or phenylalanine, the translocon mediates insertion into the endoplasmic reticulum (ER) membrane, and thus any spidroin segment rich in these amino acid residues is captured by the cell. In fact, alanine is the most hydrophobic residue that allows passage through the translocon, suggesting that spider silk proteins have evolved to optimize the hydrophobicity in their β-sheet-forming segments to the extent possible as secreted proteins. Intracellular expression in prokaryotes circumvents the constraints of the secretory pathway that native spider silk proteins must follow, since translation and accumulation of the target protein occurs in the cytosol.

[0022] The tensile strength of the silk fibers is imparted by polyalanine stretches that are zipped together by the tight packing of side chains in β-sheet crystals. Spider silk proteins are secreted, so they should be free of long stretches of hydrophobic residues, because such segments would be inserted into the ER membrane. At the same time, hydrophobic residues have a high β-strand propensity and can mediate interactions between tight β-sheets, which are attractive features for the generation of strong artificial silks. The recombinant spider silk proteins of the present invention are predicted to form stronger β-sheets more avidly than the wild-type protein by selectively replacing alanine residues with isoleucine or valine residues in one of the polyalanine domains.

[0023] As further shown in the Examples section, replacement of alanine residues in the alanine-rich domain with threonine (T) results in silk fibres that are poorly expressed or have significantly lower extensibility (breaking strain) and toughness compared to the spider silk proteins of the invention, despite the fact that threonine is branched at the β carbon and should therefore favour a β-strand conformation in the silk protein.

[0024] Furthermore, the experimental data presented herein show that replacement of more than every third or fourth alanine residue, such as every second alanine residue or indeed every alanine residue, with an isoleucine or valine residue resulted in insoluble spider silk protein, or even when performed with only one of the two alanine-rich domains, resulted in very small amounts of soluble spider silk protein. Similarly, replacement of every seventh alanine residue caused the protein to aggregate, such that it could not be spun into silk fibers.

[0025] The experimental data herein further shows that substituting alanine residues in both alanine-rich domains, i.e., both pA1 and pA2, resulted in yarn fibers that were very weak or had poor mechanical properties in terms of low breaking strain and low toughness.

[0026] Thus, improved mechanical properties of the yarn fiber are obtained when one of the alanine-rich domains of the REP domain is a polyalanine domain and the other of the alanine-rich domains of the REP domain is a polyalanine domain in which every third or fourth alanine residue is substituted with an isoleucine residue or a valine residue.

[0027] Thus, an alanine-rich domain that is a polyalanine domain comprises a plurality of alanine residues that are not sandwiched by any other amino acid residues, and thus preferably consists only of alanine residues.

[0028] In one embodiment, one of pA1 and pA2 is selected from the group consisting of the amino acid sequence A m In this embodiment, m is an integer selected within the range of 7-18, preferably within the range of 10-17, more preferably within the range of 14-16.

[0029] Thus, in this embodiment, one of the alanine-rich domains of the REP domain comprises or preferably consists of a sequence of alanine residues, more particularly m such consecutive alanine residues. The length of this polyalanine domain is 7 to 18 alanine residues, preferably 10 to 17 alanine residues. In a particularly preferred embodiment, the polyalanine domain has a length of 14 to 16 alanine residues, for example 14 alanine residues, 15 alanine residues or 16 alanine residues, more preferably 14 or 15 alanine residues.

[0030] In one embodiment, the length of the other alanine-rich domain, i.e., the polyalanine domain having alanine residues substituted by isoleucine or valine residues, is preferably 8 to 18 amino acid residues, preferably 10 to 17 amino acid residues, more preferably 14 to 16 amino acid residues, for example, 14 amino acid residues, 15 amino acid residues or 16 amino acid residues, more preferably 14 or 15 amino acid residues.

[0031] In one embodiment, the other of pA1 and pA2 is a polyalanine domain in which every third or fourth alanine residue is replaced by an isoleucine residue, or a polyalanine domain in which every third or fourth alanine residue is replaced by a valine residue. Thus, in this embodiment, the other of pA1 and pA2 comprises, or preferably consists of, alanine and isoleucine residues, or comprises, or preferably consists of alanine and valine residues.

[0032] However, it is also possible to have a polyalanine domain that comprises, or preferably consists of, alanine, isoleucine, and valine residues, in such an embodiment, at least one of every third or fourth alanine residues of the polyalanine domain is replaced by an isoleucine residue and at least one of every third or fourth alanine residues of the polyalanine domain is replaced by a valine residue.

[0033] In one embodiment, the other of pA1 and pA2 is a polyalanine domain in which every fourth alanine residue is replaced by an isoleucine or valine residue. The experimental data presented herein show that recombinant spider silk proteins having one of the alanine-rich domains in the form of a polyalanine domain in which every fourth alanine residue is replaced by an isoleucine or valine residue produced silk fibers with improved mechanical properties in terms of strength, breaking strain and toughness compared to recombinant spider silk proteins having one of the alanine-rich domains in the form of a polyalanine domain in which every third alanine residue is replaced by an isoleucine or valine residue.

[0034] In certain embodiments, the other of pA1 and pA2 is a polyalanine domain in which every fourth alanine residue is replaced with an isoleucine residue. Experimental data show that recombinant spider silk proteins having an alanine-rich domain in which the alanine residues are replaced with isoleucine residues produced silk fibers with increased strength, breaking strain, and toughness compared to corresponding recombinant spider silk proteins having an alanine-rich domain in which the alanine residues are replaced with valine residues.

[0035] Thus, in one embodiment, the other of pA1 and pA2 is a polyalanine domain in which every third or fourth alanine residue is replaced with an isoleucine residue, preferably in which every fourth alanine residue is replaced with an isoleucine residue.

[0036] In one embodiment, the other of pA1 and pA2 is (A3I) n A p , A p (IA3) n , (A3V) n A p and A p (VA3) n In this embodiment, n is an integer selected from the range of 2 to 4, and p=mn, where m is an integer selected from the range of 8 to 18.

[0037] In certain embodiments, n is 3.

[0038] In a particular embodiment, m is an integer selected within the range of 10 to 18, preferably within the range of 14 to 16; more preferably, m is 14 or 15.

[0039] Currently, other preferred amino acid sequences of pA1 and pA2 include, or preferably consist of, AAAAIAAAIAAAIAA (SEQ ID NO:43), AAAAIAAAIAAAIAAA (SEQ ID NO:44), AAIAAAIAAAIAAA (SEQ ID NO:45), AAAVAAAVAAAVAA (SEQ ID NO:46), AAAVAAAVAAAVAAA (SEQ ID NO:47), and AAVAAAVAAAVAAA (SEQ ID NO:48).

[0040] In certain embodiments, the other of pA1 and pA2 is (A3I) n A p and A p (IA3) n Presently preferred amino acid sequences according to this particular embodiment are AAAIAAAIAAAIAA (SEQ ID NO: 43), AAAIAAAIAAAIAAA (SEQ ID NO: 44), and AAIAAAIAAAIAAA (SEQ ID NO: 45).

[0041] In a preferred embodiment, the other of pA1 and pA2 is (A3I) n A p Presently preferred amino acid sequences according to this embodiment are AAAIAAAIAAAIAA (SEQ ID NO: 43) and AAAIAAAIAAAIAAA (SEQ ID NO: 44).

[0042] The REP domain comprises alternating glycine-rich domain(s) and alanine-rich domain(s). In one embodiment, the REP domain comprises a set of domains according to the formula pA1-pG-pA2, pA1-pG1-pA2-pG2, pG1-pA1-pG2-pA2 or pG1-pA1-pG2-pA2-pG3. In this embodiment, pG, pG1, pG2 and pG3 represent glycine-rich domains. Thus, in this embodiment, the REP domain comprises or preferably consists of two alanine-rich domains and one, two or three glycine-rich domains. Furthermore, the alanine-rich domain and the glycine-rich domain(s) are alternating domains within the REP domain.

[0043] In certain embodiments, the REP domain comprises, or preferably consists of, two alanine-rich domains and one or more, preferably one to three, more preferably two or three, even more preferably three glycine-rich domains.

[0044] In a particular embodiment, the REP domain comprises a set of domains according to the formula pA1-pG1-pA2-pG2, pG1-pA1-pG2-pA2 or pG1-pA1-pG2-pA2-pG3. In this particular embodiment, the REP domain comprises, or preferably consists of, two alanine-rich domains and two or three glycine-rich domains. In a preferred embodiment, the REP domain comprises a set of domains according to the formula pG1-pA1-pG2-pA2-pG3.

[0045] In one embodiment, the REP domain consists of pA1-pG-pA2, pA1-pG1-pA2-pG2, pG1-pA1-pG2-pA2, or pG1-pA1-pG2-pA2-pG3. In a particular embodiment, the REP domain consists of pA1-pG1-pA2-pG2, pG1-pA1-pG2-pA2, or pG1-pA1-pG2-pA2-pG3. In a preferred embodiment, the REP domain consists of pG1-pA1-pG2-pA2-pG3.

[0046] In one embodiment, the REP domain is (A3I)3A3-pG-A 14 , A 15 -pG-(A3I)3A2, (A3V)3A3-pG-A 14 , A 15 -pG-(A3V)3A2, (A3I)3A3-pG1-A 14 -pG2, A 15 -pG1-(A3I)3A2-pG2, pG1-(A3I)3A3-pG2-A 14 , pG1-A 15 -pG2-(A3I)3A2, pG1-(A3I)3A3-pG2-A 14 -pG3, pG1-A 15 -pG2-(A3I)3A2-pG3, (A3V)3A3-pG1-A 14 -pG2, A 15 -pG1-(A3V)3A2-pG2, pG1-(A3V)3A3-pG2-A 14 , pG1-A 15 -pG2-(A3V)3A2, pG1-(A3V)3A3-pG2-A 14 -pG3 and pG1-A 15 -pG2-(A3V)3A2-pG3, preferably (A3I)3A3-pG1-A 14 -pG2, A 15 -pG1-(A3I)3A2-pG2, pG1-(A3I)3A3-pG2-A 14 , pG1-A 15 -pG2-(A3I)3A2, pG1-(A3I)3A3-pG2-A 14 -pG3, pG1-A 15 -pG2-(A3I)3A2-pG3, (A3V)3A3-pG1-A 14 -pG2, A 15 -pG1-(A3V)3A2-pG2, pG1-(A3V)3A3-pG2-A 14 , pG1-A 15 -pG2-(A3V)3A2, pG1-(A3V)3A3-pG2-A 14 -pG3 and pG1-A 15 -pG2-(A3V)3A2-pG3.

[0047] In certain embodiments, the REP domain is (A3I)3A3-pG1-A 14 -pG2, A 15 -pG1-(A3I)3A2-pG2, pG1-(A3I)3A3-pG2-A 14 , pG1-A 15 -pG2-(A3I)3A2, pG1-(A3I)3A3-pG2-A 14 -pG3 and pG1-A 15 -pG2-(A3I)3A2-pG3.

[0048] In a preferred embodiment, the REP domain is pG1-(A3I)3A3-pG2-A 14 -pG3 and pG1-A 15 In a currently preferred embodiment, the REP domain comprises a set of domains according to a formula selected from the group consisting of pG1-(A3I)3A3-pG2-A 14 -pG3.

[0049] In one embodiment, pG, pG1, pG2, and pG3 each comprise, or preferably consist of, an amino acid sequence selected from the group consisting of GRGQGGYGQGSGGN (SEQ ID NO: 49), GQGGQGGYGRQSQGAGS (SEQ ID NO: 50), and GSGQGGYGGQGQGGYGQS (SEQ ID NO: 51).

[0050] In a particular embodiment, pG1 comprises or preferably consists of an amino acid sequence according to SEQ ID NO: 49, pG2 comprises or preferably consists of an amino acid sequence according to SEQ ID NO: 50, and pG3 comprises or preferably consists of an amino acid sequence according to SEQ ID NO: 51.

[0051] Examples of REP domains of recombinant spider silk proteins of the invention are provided below. (A3I)3-A 14 , SEQ ID NO:13 GRGQGGYGQGSGGNAAAIAAAIAAAIAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAGSGQGGYGGQGQGGYGQS A 15 -(A3I)3, SEQ ID NO: 14 GRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAIAAAIAAAIAAGSGQGGYGGQGQGGYGQS (A3V)3-A 14 , SEQ ID NO:8 GRGQGGYGQGSGGNAAAVAAAVAAAVAAAGQGGQGGYGRQSQGAGSAAAAAAAAAAAAAAGSGQGGYGGQGQGGYGQS A 15 -(A3V)3, SEQ ID NO:52 GRGQGGYGQGSGGNAAAAAAAAAAAAAAAGQGGQGGYGRQSQGAGSAAAVAAAVAAAVAAGSGQGGYGGQGQGGYGQS

[0052] The recombinant spider silk protein preferably comprises a REP domain located between the NT and CT domains, and thus preferably has the general formula NT-REP-CT.

[0053] As further described herein, the recombinant spider silk protein may also comprise other amino acid sequences than the NT, REP and CT domains, including optional N-terminal and / or C-terminal tags and / or optional linkers. Thus, in one embodiment, the recombinant spider silk protein has the general formula (X)-NT-(L1)-REP-(L2)-CT-(Y), where X represents an optional N-terminal tag, Y represents an optional C-terminal tag, L1 represents an optional first linker and L2 represents an optional second linker.

[0054] As mentioned above, the recombinant spider silk protein of the invention may contain, in addition to the NT, REP and CT domains, additional amino acid sequences or domains, which may then preferably be linked to the N-terminus of the NT domain of the recombinant spider silk protein and / or to the C-terminus of the CT domain of the recombinant spider silk protein (i.e. X-NT-REP-CT, NT-REP-CT-Y or X-NT-REP-CT-Y) and / or be provided between the NT and REP domains and / or between the REP and CT domains (i.e. NT-L1-REP-CT, NT-REP-L2-CT or NT-L1-REP-L2-CT). It is also possible to combine X, Y of the N-terminal and / or C-terminal tag with a linker (e.g., X-NT-L1-REP-CT, X-NT-REP-L2-CT, X-NT-L1-REP-L2-CT, NT-L1-REP-CT-Y, NT-REP-L2-CT-Y, NT-L1-REP-L2-CT-Y, X-NT-L1-REP-CT-Y, X-NT-REP-L2-CT-Y or X-NT-L1-REP-L2-CT-Y).

[0055] Illustrative, but non-limiting examples of such additional domains X, Y are affinity tags, solubilization tags, chromatography tags, epitope tags, fluorescent tags, signal peptides or sequences, etc.

[0056] Examples of domains that facilitate purification include various affinity tags, such as chitin-binding protein (CBP), maltose-binding protein (MBP), hemagglutinin tags, Strep tags, glutathione-S-transferase (GST), and poly(His) tags, such as the His6 tag, solubilization tags, such as thioredoxin (TRX) and poly(NANP), chromatography tags, such as the FLAG tag, epitope tags, such as the ALFA tag, V5 tag, Myc tag, HA tag, Spot tag, T7 tag, and NE tag, and fluorescent tags, such as GFP.

[0057] Illustrative examples of linkers that can be used between the NT and REP domains and / or between the REP and CT domains are various GS or GNS linkers and other peptide linkers. Such linkers can be beneficial to shorten the distance between the NT and REP domains and / or between the REP and CT domains, thereby reducing the risk of steric hindrance between the linked domains. Optional linkers can be very short, such as GS or GNS, or up to several tens of amino acids, preferably 20 amino acids or less, more preferably 15 amino acids or less.

[0058] The NT domain of spider silk proteins is thought to improve the solubility of spider silk proteins, thereby allowing very high protein concentrations in the spinning dope. Furthermore, the pH-dependent dimerization of the NT domain is a key factor enabling rapid polymerization of the spinning dope.

[0059] Although some of the CT domains of spider silk proteins do not exhibit pH-sensitive solubility (Hedhammar et al., Biochemistry 47(11):3407-3417(2008)), in general, most CT domains that have several charged amino acid residues are in fact highly soluble and have pH-dependent solubility (Andersson et al., PLoS Biology 12(8):e1001921(2014)).

[0060] The recombinant spider silk proteins of the present invention may use various combinations of NT and CT domains together with the REP domain to form recombinant spider silk proteins that can be spun into silk fibres.

[0061] Illustrative, but non-limiting examples of NT domains that may be used in accordance with the present invention are listed in Table 2 of US2019 / 0248847, the teachings of which regarding NT domains are incorporated herein by reference.

[0062] In a preferred embodiment, the NT domain of the recombinant spider silk protein is derived from the NT domain of Euprosthenops australis MaSp1.

[0063] In a particular embodiment, the NT domain comprises, or preferably consists of, SEQ ID NO:53.

[0064] Illustrative, but non-limiting examples of CT domains that may be used in accordance with the present invention are listed in Table 1 of US2019 / 0248847, the teachings of which regarding CT domains are incorporated herein by reference.

[0065] In a preferred embodiment, the CT domain of the recombinant spider silk protein is derived from the CT domain of Araneus ventricosus MiSp.

[0066] In a particular embodiment, the CT domain comprises, or preferably consists of, SEQ ID NO:54.

[0067] In one embodiment the recombinant spider silk protein comprises, or preferably consists of, an NT domain comprising, or preferably consisting of, SEQ ID NO:53, an REP domain comprising, or preferably consisting of, an amino acid sequence selected from the group consisting of SEQ ID NOs:8, 13, 14 and 52, and a CT domain comprising, or preferably consisting of, SEQ ID NO:54.

[0068] In one embodiment, the recombinant spider silk protein comprises, or preferably consists of, an NT domain which comprises, or preferably consists of, SEQ ID NO: 53, a REP domain which comprises, or preferably consists of, SEQ ID NO: 13, and a CT domain which comprises, or preferably consists of, SEQ ID NO: 54. Such a recombinant spider silk protein is shown in SEQ ID NO: 55, with linkers between the NT and REP domains and between the REP and CT domains in SEQ ID NO: 56, with an N-terminal His tag in SEQ ID NO: 57, and with a His tag and linker in SEQ ID NO: 39.

[0069] Another embodiment of a recombinant spider silk protein of the invention comprises, or preferably consists of, an NT domain which comprises, or preferably consists of, SEQ ID NO: 53, a REP domain which comprises, or preferably consists of, SEQ ID NO: 14, and a CT domain which comprises, or preferably consists of, SEQ ID NO: 54. Such a recombinant spider silk protein is shown in SEQ ID NO: 58, with linkers between the NT and REP domains and between the REP and CT domains in SEQ ID NO: 59, with an N-terminal His tag in SEQ ID NO: 60, and with a His tag and linker in SEQ ID NO: 40.

[0070] A further embodiment of a recombinant spider silk protein of the invention comprises, or preferably consists of, an NT domain which comprises, or preferably consists of, SEQ ID NO: 53, a REP domain which comprises, or preferably consists of, SEQ ID NO: 8, and a CT domain which comprises, or preferably consists of, SEQ ID NO: 54. Such a recombinant spider silk protein is shown in SEQ ID NO: 61, with linkers between the NT and REP domains and between the REP and CT domains in SEQ ID NO: 62, with an N-terminal His tag in SEQ ID NO: 63, and with a His tag and linker in SEQ ID NO: 34.

[0071] Yet another embodiment of a recombinant spider silk protein of the invention comprises, or preferably consists of, an NT domain which comprises, or preferably consists of, SEQ ID NO: 53, a REP domain which comprises, or preferably consists of, SEQ ID NO: 52, and a CT domain which comprises, or preferably consists of, SEQ ID NO: 54. Such a recombinant spider silk protein is shown in SEQ ID NO: 64, with linkers between the NT and REP domains and between the REP and CT domains in SEQ ID NO: 65, with an N-terminal His tag in SEQ ID NO: 66, and with a His tag and linker in SEQ ID NO: 67.

[0072] One embodiment relates to a recombinant spider silk protein comprising an NT domain, a REP domain and a CT domain. According to the invention, the REP domain comprises a set of domains according to the formula pA1-pG-pA2, where pG stands for a glycine-rich domain and pA1 and pA2 stand for an alanine-rich domain. According to the invention, one of pA1 and pA2 is a polyalanine domain and the other of pA1 and pA2 is a polyalanine domain with 2-4, preferably 3, alanine residues substituted with respective amino acid residues selected from the group consisting of isoleucine and valine. Thus, in this embodiment, the recombinant spider silk protein has one polyalanine domain, preferably consisting of alanine residues, and one polyalanine domain comprising, in addition to the alanine residues, 2-4, preferably 3 amino acid residues individually selected from isoleucine and valine. In this embodiment, the isoleucine and / or valine residue does not necessarily have to be every third or fourth residue in the other of pA1 and pA2.

[0073] Another aspect of the invention relates to silk fibres made from recombinant spider silk proteins according to the invention. This aspect therefore relates to silk fibres, sometimes called silk polymers, comprising recombinant spider silk proteins according to the invention. The silk fibres are then obtained by spinning a so-called spin dope comprising recombinant spider silk proteins according to the invention into silk fibres, which are further described herein.

[0074] The strength, breaking strain, tenacity modulus, diameter, and other mechanical properties referred to herein relate to average values ​​of the mechanical properties determined when testing multiple yarn fibers.

[0075] In one embodiment, the yarn fibers of the present invention have an average strength of at least 50 MPa, preferably at least 60 MPa, more preferably at least 70 MPa, for example at least 80 MPa.

[0076] In one embodiment, the yarn fibers of the present invention have an average breaking strain of at least 60%, preferably at least 70%, more preferably at least 100%, for example at least 125%.

[0077] In one embodiment, the yarn fibers of the present invention have a thermal conductivity of at least 25 MJ / m 3 , preferably at least 35 MJ / m 3 , more preferably at least 45 MJ / m 3 , e.g., at least 75 MJ / m 3 The average toughness coefficient is

[0078] The thread fibers may have an average diameter of 1 or several μm to several tens of μm. For example, the average diameter of the thread fibers is 1 μm to 100 μm, preferably 5 μm to 50 μm, and more preferably 7.5 to 15 μm.

[0079] The present invention also relates to synthetic materials comprising the yarn fibers of the present invention.

[0080] Examples of synthetic materials that include or are made from the yarn fibers of the present invention include textile materials such as filaments, yarns, ropes, and woven fabrics. Such textile materials can benefit from the high tensile strength of the yarn fibers. Other examples of synthetic materials include flexible energy absorbing materials such as armor and bumpers. The yarn fibers of the present invention can also be used in medical applications such as sutures, compression bandages, etc. Additionally, the yarn fibers can be used in scaffolds and materials in tissue engineering, implants, and other cell scaffold-based materials.

[0081] The present invention also relates to a nucleic acid molecule encoding a recombinant spider silk protein according to the invention.

[0082] As used herein, a nucleic acid molecule includes polynucleotides, oligonucleotides, and nucleic acid sequences, and generally refers to a polymer of DNA or RNA, which may be single- or double-stranded, may contain natural, non-natural, or modified nucleotides, and may contain natural, non-natural, or modified internucleotide linkages, such as phosphoramidate or phosphorothioate linkages in place of the phosphodiesters found between nucleotides in unmodified oligonucleotides. Nucleic acid molecules also include complementary DNA (cDNA) and messenger RNA (mRNA).

[0083] Illustrative, but non-limiting examples of such nucleic acid molecules are set out in SEQ ID NO: 68 for the recombinant spider silk protein of SEQ ID NO: 39, in SEQ ID NO: 69 for the recombinant spider silk protein of SEQ ID NO: 40 and in SEQ ID NO: 70 for the recombinant spider silk protein of SEQ ID NO: 34.

[0084] In one embodiment, the nucleic acid molecule is an isolated nucleic acid molecule.

[0085] A further aspect of the present invention relates to an expression vector comprising a nucleic acid molecule according to the invention.

[0086] An expression vector comprises at least one nucleic acid molecule comprising a coding sequence that can be expressed (e.g., transcribed and translated) in a cell (often referred to as a host cell) that contains the expression vector. The expression vector, in one embodiment, is selected from a DNA molecule, an RNA molecule, a plasmid, an episomal plasmid, and a viral vector.

[0087] The expression vector then comprises a nucleic acid molecule operably linked to a promoter to allow its transcription in the host cell. The promoter can be any promoter that is constitutively active or inducibly active in the host cell. An illustrative but non-limiting example of a promoter that can be used in Escherichia coli host cells is the T7 promoter. Protein production can then be induced in the host cell by the addition of isopropyl β-D-1-thiogalactoside.

[0088] In one embodiment, the expression vector is an isolated expression vector.

[0089] Yet another aspect of the present invention relates to a host cell comprising an expression vector according to the invention.

[0090] The nucleic acid molecule or expression vector can then be transcribed in the host cell so as to produce recombinant spider silk protein within the host cell.

[0091] A variety of such host cells may be used in accordance with the present invention, including, but not limited to, bacteria, yeast, mammalian cells, plant cells, and insect cells. It is presently preferred to produce the recombinant spider silk proteins of the present invention in prokaryotic cells, preferably bacteria, such as E. coli.

[0092] The recombinant spider silk protein may then be produced by the host cell, for example by culturing the host cell according to the invention under conditions allowing the production of the recombinant spider silk protein and isolating the spider silk protein from the culture. In a particular embodiment, the spider silk protein is isolated from the cytosol of the host cell.

[0093] The present invention also relates to a method for producing silk fibers, the method comprising extruding a spin dope comprising a recombinant spider silk protein of the present invention into an aqueous buffer having an acidic pH to induce polymerization of the recombinant spider silk protein into silk fibers, the method also comprising isolating the silk fibers from the aqueous buffer.

[0094] In one embodiment the spin dope comprises at least 100 mg / ml recombinant spider silk protein, preferably at least 150 mg / ml, more preferably at least 200 mg / ml recombinant spider silk protein.

[0095] In one embodiment, the aqueous buffer is an acetate buffer having a pH of 6 or less, preferably 5.5 or less. In certain embodiments, the aqueous buffer also has a pH of preferably 4 or more, preferably 4.5 or more. In a preferred embodiment, the aqueous buffer has a pH of about 5.

[0096] Recombinant spider silk proteins advantageously allow the production of silk fibers in industrially compatible equipment using metal nozzles, as disclosed in Example 2. Moreover, the silk fibers thus produced can be drawn after spinning. EXAMPLES

[0097] Example 1 - Engineered spider silk proteins for biomimetic spinning of fibers This example utilized protein engineering to generate mini-spidroins produced in high yields in prokaryotic hosts and used them to generate strong biomimetic artificial spider silk fibers. Using the Zipper database (Goldschmidt et al., Proceedings of the National Academy of Sciences of the United States of America 107:3487 (2010)), a large panel of mini-spidroins with designed modifications of poly-Ala blocks was screened, and candidates with low Rosette Energy were selected for heterologous expression. Soluble target proteins were identified, biochemically characterized, and fabricated into fibers using a biomimetic spinning device. The mechanical performance of the fibers revealed that it was possible to engineer the repeat domains of mini-spidroins, resulting in fibers with increased tensile strength.

[0098] result Thirteen different constructs were designed with substitutions in the polyAla blocks of the original NT2RepCT sequence, selecting Ile (I) and Val (V) based on the ratio of β-strand / α-helical propensity of the amino acid residues and their hydrophobicity (A to reflect the composition of the two polyAla blocks). 15 -A 14 (Fig. 1). In addition, the less hydrophobic residue Thr (T) was used because it is branched at the β-carbon and therefore favors a β-strand conformation (Koehl and Levitt, Proceedings of the National Academy of Sciences of the United States of America 96:12524 (1999); Chou and Fasman, Biochemistry 13:211 (1974)).

[0099] Figure 1B shows A 15 -A 14 The amino acid sequences of the repeat regions and engineered constructs with the indicated substitutions are shown. Substitutions were primarily introduced at every second position, resulting in beta strands with mutated side chains on the same side. Mutations were either introduced in both (e.g., (AV)7-(AV)7) or in only one of the polyAla blocks (e.g., (AV)7-A 14 The number of substitutions is 15, where all Ala is replaced by Val in the first polyAla block (e.g., V 15 -A 14 ) and three containing Val substitutions at every fourth position in the first polyAla block (e.g., (A3V)3-A 14 ). Several additional constructs were designed to analyze the effect of the position of the substituted residue: for example, (A3I)3-A 14 , A 15 -(A3I)3, and IA6IA6I-A 14 (All have three Ile substitutions but at different positions).

[0100] The packing of β-sheets into amyloid-like fibrils involves steric zippers, which are also seen in spider silk β-sheet crystals. Steric zippers are formed by tightly associated β-strands with high complementarity of the involved side chains. The Zipper database predicts the stability and propensity of a hexapeptide in a given amino acid sequence to form a steric zipper by calculating the energy of the interstrand interactions. An energy of -23 kcal / mol or less suggests a high propensity to form a steric zipper (Goldschmidt et al., Proceedings of the National Academy of Sciences of the United States of America 107:3487 (2010)).

[0101] FIG. 2A shows construct A. 15 -A 14 and (A3I)3-A 14 The estimated Rosette energies for all engineered minispidroins are shown in Table 1 and Figure 2B. 15 -A 14 The hexapeptide within the polyAla region of the constructs has a low Rosette energy (-24.6 kcal / mol) and should therefore be able to form a steric zipper (Figure 2C). 15 -A 14 Include at least one hexapeptide with a Rosette Energy (in the range of -24.9 to -29.4 kcal / mol) lower than those in Table 1. In general, the impact on the Rosette Energy increases with increasing number of hydrophobic substitutions in the polyAla region.

[0102] Table 1: Hexapeptides with lowest Rosette energy and hydropathy among engineered minispidroins. [Table 1]

[0103] Of the 15 designed proteins, seven were overexpressed and six were highly overexpressed in Escherichia coli BL21 cells (Table 2). Constructs with Val substitutions expressed lower levels than the corresponding constructs with Ile substitutions, but the number and hydrophobicity of substitutions had no overall effect on expression levels. The (AT)7-(AT)7 construct did not express well, which may be due to the fact that this repeat was designed to resemble a "CAT tail," which is known to cause aggregation and proteasomal degradation of nascent polypeptide chains (Shen et al., Science 347:75 (2015)).

[0104] A 15 -A 14 In addition, seven of the constructs were found primarily in the soluble fraction after cell lysis in 20 mM Tris-HCl, and four constructs were found in both the soluble and insoluble fractions (Table 2). Increased hydrophobicity, number of substitutions, and low Rosette energy correlated with decreased solubility after cell lysis. Nine of the 15 designed constructs and the control A 15 -A 14 Both methods yielded sufficient soluble protein for purification. Non-denaturing immobilized metal affinity chromatography yielded 4-243 mg of pure target protein per L of shake flask culture (average of 10 x 1 L cultures). Notably, six of the engineered minispidroins showed very high yields (>100 mg / L, Table 2). (AV)7-(AV)7, (AV)7-A 14 and V 15 -A 14 showed high expression but was insoluble after lysis, which may be due to the high hydrophobicity of the engineered segment. 15 Expression and purification of the -(AI)7 and (AIA2)3-(AIA2)3 constructs did not yield sufficient soluble protein for further characterization. Constructs that showed moderate to high levels of expression but were insoluble after cell lysis were treated with 8 M urea but were unable to solubilize to the extent necessary to allow purification of sufficient protein for fiber spinning (not shown).

[0105] The position of the Ile substitutions within one Ala block affected protein yields, but it did not matter whether they were located in the first or second polyAla block. 14 and A 15 Both (A3I)3-A and (A3I)3-B had three Ile substitutions in the first and second polyAla blocks, respectively, and showed comparable yields. 14 and IA6IA6I-A 14 have the same number of Ile substitutions in the first block, but at different positions, and in different yields ((A3I)3-A 14 and IA6IA6I-A 14 207 vs. 139 mg / L culture, respectively).

[0106] Table 2. Summary of engineered protein substitution numbers, expression levels, solubility after cell lysis, protein yield, and spinnability into fibers. Expression levels, solubility after cell lysis, and spinnability into fibers are rated as very high (+++), moderate (++), low (+), and none (0). Evaluation of expression levels and solubility after cell lysis was estimated by the appearance of target bands by SDS-PAGE. (-) indicates not tested. 1 ) indicates degradation during expression. * ) indicates purification using a gravity column instead of FPLC. [Table 2]

[0107] The secondary structure content and thermal stability of the purified constructs were then investigated by circular dichroism (CD) spectroscopy (Figure 3). All constructs were found to have an overall α-helical secondary structure (Figure 3A), indicating that the amino acid substitutions do not have any major effect on the secondary structure of the soluble proteins. Heating to 90 °C led to a decrease in the signal of all constructs accompanied by a transition to a β-sheet dominated secondary structure (Figure 3C). The thermally induced conformational changes were irreversible upon cooling of the samples (Figure 3D). The melting curves of all constructs showed that the proteins unfolded around 46-50 °C, implying that the substitutions had only a minor effect on the thermal stability of the proteins (Figure 3B). Of the nine engineered minispidroins successfully purified (A 15 -A 14 Of the eight constructs, eight could be concentrated to at least 200 mg / mL and generate spinnable dopes, except for (AI)7-(AI)7, which gave too little protein yield (Table 2). Dopes made from the eight constructs were transferred to syringes and extruded through thin glass capillaries into low pH aqueous buffers, following a previously described biomimetic spinning procedure (Greco et al., Molecules 25:3248 (2020); Andersson et al., Nature Chemical Biology 13:262 (2017)). Seven engineered minispidroins could be spun into fibers, including IA6, IA6I-A, and IA6I-B. 14 Only the protein aggregated prematurely in the syringe. One of the minispidroins, (A3I)3-(A3I)3, formed fibers but was too fragile to be recovered. The reason for the poor integrity of the (A3I)3-(A3I)3 fibers is unclear, but was not related to premature aggregation in the dope. The other six engineered fiber types and A 15 -A 14 The fibers were successfully collected on a motorized wheel at the edge of the spinning bath. There was no difference in the appearance of the spun fibers (Figure 4A), and the diameters of the different fiber types, determined by optical microscopy, varied from 4 to 19 µm (Figure 7C, Table 3).

[0108] Table 3. Mechanical properties of spinnable constructs and their standard deviations. [Table 3] a) Protein expressed in a bioreactor

[0109] The tensile strength of all fibers spun from the engineered proteins was (A3V)3-A 14 and (A2I)4-A 14 Except for A 15 -A 14 The two similar fiber types (A3I) and 3-A were significantly increased compared to those (Fig. 4B and Table 3). 14 and A 15 -(A3I)3 shows the highest increase in strength, the former reaching 131 MPa, which is 15 -A 14 This is almost three times higher than that of the control (Figure 4B). This indicates that rational protein engineering of spidroin polyAla blocks can indeed result in an increase in fiber tensile strength. Unexpectedly, the introduced amino acid substitutions also had a significant effect on the fiber extensibility, with the breaking strains varying widely from 3.3 to 203.5% (Figure 4C and Table 3).

[0110] The two strongest fiber types ((A3I)3-A 14 and A 15 -(A3I)3) showed a very large increase in strain (160 and 204%, respectively), while (A3V)3-A 14 , (A2I)4-A 14 Fiber is A 15 -A 14 (A3T)3-(A3T)3 and (A3V)3-(A3V)3 fibers were the least extensible (3.3 and 8.3%, respectively). Clearly, the mechanical properties of artificial spider silk fibers can be significantly improved by introducing Ile into every fourth position of the first or second polyAla block. These two minispidroins, (A3I)3-A 14 and A 15-(A3I)3 formed fibers with toughness moduli comparable to those of natural drag yarns (146 and 125 MJ / m, respectively). 3 , whereas the natural dragline from Argiope argentata, Blackledge and Hayashi, Journal of Experimental Biology 209:2452 (2006) had a yield of 136 MJ / m 3 ) (Figure 4D). (A3V)3-A 14 and (A2I)4-A 14 The fibers formed by A 15 -A 14 significantly higher toughness factors were achieved (50 and 37 MJ / m 3 For 18MJ / m 3 ).

[0111] To investigate the relationship between the secondary structure content and mechanical properties of the fibers, attenuated total reflectance Fourier transform infrared (ATR-FTIR) spectroscopy was used. The results shown in Figure 5 show that no significant differences in secondary structure content are detected between the fibers, but (A3V)3-A 14 , (A3I)3-A 14 and A 15 -(A3I)3 is A 15 -A 14 Compared to the fibers, (A3V)3-(A3V)3 and (A2I)4-A showed a slight increase in β-sheet content and a decrease in α-helix / random coil content. 14 In fiber, A 15 -A 14 No significant differences in secondary structure content were detected by ATR-FTIR spectroscopy of the different fiber types. Therefore, the unmodified fibers (A 15 -A 14 ) and the best performing engineered fiber (A3I)3-A 14 We also used solid-state NMR spectroscopy to investigate the 15 -A 14 Compared to fibers, (A3I)3-A 14In the β-sheet conformation, more Ala residues were found (Figure 6).

[0112] The changes in the mechanical properties of fibers made from engineered spidroins indicate that the intermolecular interactions of spidroins are affected. In natural dragline fibers, pulling on the fiber first leads to a reversible deformation of the amorphous regions up to the yield point, followed by a softening of the material due to the breaking of hydrogen bonds in the amorphous regions. As the amorphous protein chains stretch, the load is transferred to the β-sheet crystals, leading to stiffening of the fiber. Further increases in load cause the β-sheet crystals to undergo stick-slip deformation and the fiber to break. The increase in tensile strength of fibers made from engineered proteins suggests that our strategy of increasing the propensity of β-strands and the interactions between β-sheets may indeed result in stronger fibers, although some of the engineered fibers simultaneously showed a reduction in strain. Theoretically, increased intermolecular interactions in β-sheet formation and stacked β-sheets may not only increase the strength of the fiber, but also increase extensibility by allowing the amorphous regions to fully stretch before the load is transferred to the crystalline regions. A 15 -A 14 In the absence of poly-Ala β-sheet crystals, as in fibers, the intermolecular contacts may be too weak to allow complete extension of the amorphous protein chains before fiber fracture. At the same time, stacking all β-sheets in the crystal may be unfavorable, since only about 40% of Ala residues in native dragline are found in this conformation, the rest forming disordered β-sheets. In this study, introducing substitutions in both poly-Ala blocks resulted in fibers with dramatically reduced strain, suggesting a suboptimal packing of the protein in the fiber.

[0113] (A3I)3-A 14 The fibers exhibited better mechanical properties, making these fibers attractive candidates for large-scale production. 15 -A 14has been shown to be expressed at very high levels (~21 g / L) in a bioreactor-based E. coli fed-batch culture (Schmuck et al., Materials Today 50:16 (2021)). Following the same protocol, (A3I)3-A 14 The expression level of (A3I)3-A reached 13 g / L, and the final yield after purification using an automated purification protocol was 8.9 g / L. To our knowledge, these yields are the second highest reported for any recombinant spidroin produced in E. coli and meet the requirements for economically viable large-scale production. After purification, (A3I)3-A 14 The recombinant silk protein was concentrated to 300 mg / mL and could be easily spun into fibers. Remarkably, 8.9 g of the protein was enough to produce a fiber approximately 18 km long.

[0114] Using biological principles, we used protein engineering to design minispidroins predicted to have increased β-sheet propensity and increased β-sheet-to-β-sheet bond strength. Prokaryotic expression, protein purification, and biomimetic fiber spinning led to four different types of fibers with significantly improved tensile strength compared to the original minispidroins. Using this strategy, we successfully produced the first biomimetic fibers with durability values ​​comparable to natural dragline fibers. Finally, we show that these fibers can be produced in bioreactors with very high yields, ensuring viable large-scale production.

[0115] Experimental Section Designed mini spidroin All expressed proteins were composed of a 6xHis tag (MGHHHHHH, SEQ ID NO: 71), NT from Euprosthenops australis MaSp1 (SEQ ID NO: 53), and CT from Araneus ventricosus ampullate spidroin (MiSp) (SEQ ID NO: 54). Between NT and CT, a repeat portion containing two polyAla and three glycine-rich repeats from E. australis MaSp1 (NT2RepCT) was inserted as previously described (Andersson et al., Nature Chemical Biology 13:262 (2017)). Engineered variants containing amino acid residue substitutions in the polyAla block of the repeat region were designed as described in the results section. These constructs are named corresponding to their substitutions in the polyAla block, but also contain NT, CT, and glycine-rich regions, e.g., NT2RepCT is a repeat region containing two polyAla and three glycine-rich repeats from A. australis MaSp1 (NT2RepCT). 15 -A 14 The amino acid sequence corresponding to the designed repeat region was converted to a gene sequence, codon-optimized for expression in E. coli (Geneious) ordered from Eurofins Genomics, Germany, and subcloned (using EcoRI and BamHI restriction sites) between NT and CT of the existing NT2RepCT plasmid (Andersson et al., Nature Chemical Biology 13:262 (2017)).

[0116] The complete sequence below shows the basic layout [Table 4] has. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0117] Fibrosis tendency and hydrophobicity Since threads have been proposed to form β-sheets that pack into crystals, the Zipper database (Goldschmidt et al., Proceedings of the National Academy of Sciences of the United States of America 107:3487 (2010)) was used to estimate the fiberization propensity and Rosette energy of the engineered constructs (only the repeat regions). The Zipper database calculates the Rosette energy (Kuhlman and Baker, Proceedings of the National Academy of Sciences of the United States of America 97:10383 (2000)) and evaluates the self-complementary binding of the moving hexapeptides (Nelson et al., Nature 435:773 (2005); Sawaya et al., Nature 447:453 (2007)). The Rosetta energy combines several free energy functions to model and analyze a given protein structure, and an energy below -23 kcal / mol indicates a high tendency for fibrillation (Goldschmidt et al., Proceedings of the National Academy of Sciences of the United States of America 107:3487(2010)). Lower energy means higher stability of the two β-strands in a zipper conformation. Hydrophobicity was calculated by https: / / web.expasy.org / protparam / (Wilkins et al, In 2-D Proteome Analysis Protocols, Humana Press, New Jersey, pp.531-552(1967); Gasteiger et al., The Proteomics Protocols Handbook,571(2005); Kyte and Doolittle, Journal of Molecular Biology 157:105(1982)).

[0118] Protein expression using shake flask cultures Protein expression was performed as previously described (Greco et al., Molecules 25:3248 (2020)). Briefly, constructs were transformed into BL21(DE3) E. coli cells and grown at OD 200-300 rpm in Luria broth (Miller, VWR, USA) in shake flasks containing kanamycin at 30°C and 110 rpm. 600 The cells were grown until the β-d-1-thiogalactopyranoside reached 0.9. To induce recombinant protein expression, 0.15 mM isopropyl β-d-1-thiogalactopyranoside (final concentration, VWR, USA) was added and the temperature was reduced to 20°C. Expression was carried out overnight, after which the cells were harvested and stored at -20°C.

[0119] Protein purification and concentration Cell lysis was performed using a high pressure cell disrupter (TS Series Machine, Constant Systems Limited). After centrifugation, the supernatant was purified by Ni-immobilized metal affinity column (IMAC) (Akta start, GE Healthcare, USA or manually). After loading the supernatant onto a HisPrep™ FF16 / 10 or manually packed column (GE Healthcare, USA), the column was washed with 4–5 column volumes (CV) of 20 mM Tris-HCl, followed by 4–5 CV of 20 mM Tris-HCl, pH 8, containing 2 mM imidazole. Proteins were eluted with 20 mM Tris-HCl, pH 8, containing 200 mM imidazole. After dialysis against 20 mM Tris-HCl, pH 8, proteins were analyzed by SDS-PAGE for quality control. Depending on the solubility of the construct, the protein was concentrated to 200-400 mg / mL using a centrifugal concentrator (Vivaspin 20, 10 kDa MWCO, GE Healthcare, USA) and then frozen at -20 °C until further use.

[0120] CD spectroscopy Protein concentrations of 10 μM in 20 mM phosphate buffer were measured in a 300 μl cuvette with a path length of 1 mm using a J-1500 CD spectrometer (JASCO, USA). Temperature scans were performed at 1°C min -1 Heating rates of 100-200 nm were performed between 20 and 90 °C, and spectra were recorded from 260 to 190 nm. After heating, the samples were cooled to 20 °C in 15 min to observe the reversibility of the conformational changes. Five scans per temperature were taken as averages, smoothed, and converted to molar residue ellipticities. Thermal unfolding curves were plotted by taking the molar residue ellipticities at 222 nm, and the fraction naturally folded was calculated as (CD 測定 -CD 終了 ) / (CD 開始 -CD 終了 ) and then normalized.

[0121] Biomimetic fiber spinning Artificial fiber spinning was performed similarly to that previously described (Greco et al., Molecules 25:3248 (2020)). A round glass capillary (G1, Narishige, UK, inner diameter 0.6 mm) was pulled to a diameter of 25-78 μm with a Micro Electrode Puller (Stoelting co. 51217). A 1 mL syringe with a Luer Lok tip (BD, USA) was filled with concentrated protein and connected to a 27 G steel needle (Braun, Germany). The needle was connected to the pulled glass capillary via polyethylene tubing. Proteins were injected at a flow rate of 17 μl / min (neMESYS low pressure syringe pump, Cetoni, Germany) into an 80 cm long bath containing spinning buffer (750 mM acetate buffer, 150 mM NaCl, pH 5.0) and reeled up on a collection frame in air with minimal stretching of the fibers. Each construct was spun at least twice on different occasions.

[0122] Mechanical Testing of Textiles The fibers were attached with tape to a paper frame with a square window (1 cm x 1 cm), and the fiber diameter was measured using an optical microscope (Nikon, Japan) at 10 points along each fiber, and the average diameter was calculated. The frame was placed in a tensile tester (5943-Instron, USA with a 5N load cell), cut, and the fibers were pulled at a strain rate of 6 mm / min. All tests were performed at a relative humidity of less than 35% to avoid affecting the mechanical properties of the yarn. The number and type of fibers tested were A 15 -A 14 n=33, (A3I)3-A 14 n=30, (A3T)3-(A3T)3n=60, (A3V)3-(A3V)3n=38, (A3V)3-A 14 n=15, A 15 -(A3I)3n=13, (A2I)4-A 14 n=15. Engineering strength was calculated by dividing the measured force by the area of ​​the cross section (calculated from nominal / maximum diameter assuming a circular cross section). Engineering strain was calculated by dividing the displacement by the gauge length. Toughness coefficient was determined by calculating the area under the stress-strain curve and Young's modulus was determined from the slope of the initial linear elastic phase of the stress-strain curve.

[0123] FTIR spectroscopy FTIR spectra of the fiber bundles were recorded on a Vertex 70 instrument equipped with a Diamond ATR unit (Platinum-ATR, Bruker, Germany) and a mercury cadmium telluride detector (Bruker, Germany). The instrument was continuously purged with dry air and the spectra confirmed that no correction for water vapor was necessary. Resolution was 2 cm. -1 1000 scans were recorded at 1000 x 1000. Prior to each sample spectrum measurement, a background spectrum without sample was recorded and used to calculate the absorbance spectrum. For each sample, six spectra were acquired by pressing the fiber bundles onto the ATR crystal, with three fiber bundles oriented perpendicular to the beam and three fiber bundles oriented parallel to the beam.

[0124] The spectra were processed using the "Kinetics" software written by Erik Goormaghtigh (Universite Libre de Bruxelles, Belgium). Six spectra for each sample were averaged and the amide I band (1705–1595 cm) was extracted. -1 ) to baseline (baseline points: 1740, 1730, 1580, and 1578 cm -1 A polynomial baseline with σ = 1 was subtracted. The second derivative was calculated from the absorbance spectra, smoothed with a 15-point Savitzky-Golay algorithm, and scaled to match the absorbance values ​​(factor = 600). The absorbance and second derivative spectra were simultaneously co-fitted to analyze secondary structure content (Baldassarre et al., Molecules 20, 12599 (2015)). Eight component bands were fitted (first peak positions: 1695, 1680, 1669, 1651, 1633, 1622, 1613, 1599 cm). -1 ), the band is located at ±5 cm from its initial central peak position. -1 Each component band was assigned to a secondary structure according to the literature (Goormaghtigh et al., Sub-cellular biochemistry 23:363 (1994); Jackson and Mantsch, Critical Reviews in Biochemistry and Molecular Biology 30:95 (1995); Venyaminov and Kalnin, Biopolymers 30:1259 (1990); Barth, Biochimica et Biophysica Acta-Bioenergetics 1767:1073 (2007); Barth and Zscherp, Quarterly Reviews of Biophysics 35:369 (2002)). -1 The component bands that fit the central peak position of ca. 1651 cm were assigned to antiparallel β-sheets. -1 The component bands that matched with the α-helical / random structure were assigned to the α-helical / random structure. -1The bands at 1633 cm were assigned to different types of β-sheets according to the study of Bombyx mori silk fibers (Carissimi et al., Polymers 12:1 (2020)): -1 The bands at approximately 1622 and 1613 cm likely correspond to distorted or twisted β-sheets. -1 The bands at ca. 1680 and ca. 1669 cm have been assigned to more planar sheets and have previously been proposed to represent different orientations of methyl groups within B. mori silk fibers (Carissimi et al., Polymers 12:1 (2020); Asakura et al., Macromolecules 48:28 (2015)). These assignments follow the known relationship between band position and planarity of β-sheets (Kubelka and Keiderling, Journal of the American Chemical Society 123:12048 (2001)). -1 The band at 1599 cm was assigned to another secondary structure. -1 The bands in A and B are assigned to side chains (Barth, Progress in Biophysics and Molecular Biology 74:141 (2000)). 15 -A 14 (and all other constructs) contain 2.3% Glu and 1.4% Arg. Relative secondary structure content was calculated by dividing the area of ​​the component bands by the total fitted area of ​​all bands assigned to amide I vibrations (excluding side chain bands).

[0125] NMR spectroscopy Uniformly 13 C. 15 N-labeled A 15 -A 14 and (A3I)3-A 14 Solid-state NMR spectra of the fibers were obtained using a 3.2 mm 1 H / 13 C / 15The NMR spectra were recorded on a Bruker Avance III HD NMR spectrometer equipped with a NE-free magic angle spinning (MAS) probe. The sample temperature was set at 277 K. The MAS frequency was 12.5 kHz. 1 H- 13 C cross polarization (CP) and 2D dipole assisted rotational resonance (DARR) experiments 1 H linear slope from 49.0 to 61.2 kHz, constant at 80.5 kHz 13 C radio frequency field amplitude, and high power heteronuclear decoupling at 83.3 kHz during acquisition. 1 From H 13 Acquisition was performed using forward and reverse CP up to C. The CP contact time was 1 ms and the acquisition time was 10 ms. 13 C chemical shifts were externally referenced to adamantane (38.48 ppm relative to TMS). Spectra were processed with Bruker Topspin 4.0.

[0126] Protein expression using bioreactors (A3I)3-A 14 Fed-batch culture of E. coli for expression of A 15 -A 14 was performed as previously described (Schmuck et al., Materials Today 50:16(2021)). Briefly, (A3I)3-A 14 Precultures of BL21(DE3) E. coli transformed to overexpress were grown in LB medium (50 μg / mL kanamycin) at 37° C. OD 600Once the OD reached approximately 5, the preculture was used to inoculate (100-fold dilution) 250 mL of fresh medium (50 μg / mL kanamycin, 0.01% antifoam 204) as specified by da Silva and coworkers (da Silva et al., SpringerPlus 2:1 (2013)). The pH was adjusted to 7 with 3 M H3PO4 and 25% NH3 using a Multifors 2 (Infors) equipped with a 0.5 L glass vessel. The stirring speed was automatically adjusted from 200 to 1200 rpm to obtain a dissolved relative oxygen level (pO2) of 30%. Initially, the OD 600 The temperature was set at 28 °C until pO2 reached 50 (22 h after inoculation). The temperature was then lowered to 20 °C before the culture was induced with IPTG at 150 μM. Nutrient feeding was started automatically 25 h after inoculation using a culture medium containing 40% glycerol, indicated by a sharp increase in pO2, μ = 0.1 h. -1 An exponential feeding profile was followed, assuming a growth rate of 100 μg / ml. Therefore, the flow rate was varied between 2.8 and 20 mL / h until 125 mL of feed stock solution was consumed. After 20 h of induction, the cultures were harvested by centrifugation at 4,000 x g, the supernatant was discarded, and the cell pellet was resuspended in 20 mM Tris, pH 8 (20 mL / 10 g wet cell pellet) and stored at -20 °C.

[0127] statistics Data were analyzed in GraphPad Prism using one-way ANOVA or multivariate analysis (correlation matrix with Pearson correlation coefficients) as appropriate. Statistical significance is indicated by asterisks: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0128] Example 2 - Industrial scale wet spinning In industrial-scale wet spinning processes, the polymer solution is extruded into a coagulation bath through a metal spinneret in which a hundred or hundreds of orifices are arranged. Therefore, to make the spinning process described in Example 1 suitable for scale-up, it is necessary to consider spinnability using extrusion equipment other than glass capillaries. The reason is that glass capillaries are brittle, easily damaged, and it is not possible to ensure the integrity of the capillary and reuse it. Moreover, they are time-consuming to manufacture and difficult to make in reproducible shapes. To enable extrusion with equipment other than glass capillaries, the spidroin needs to be compatible with devices with orifices of various diameters, but preferably from 30 to at least 200 μm. Now, compatibility with metal extrusion equipment exists if the tip is not blocked by premature solidification of the polymer and if the spidroin solution can be continuously extruded as fibers.

[0129] result As described in the experimental section of Example 1, glass capillaries with orifice diameters of 25 to 78 μm were used to extrude concentrated spidroin preparations. However, when glass capillaries with diameters of more than 150 μm were used, the concentrated A 15 -A 14 Extrusion of the recombinant spider silk protein (A3I)3-A (300 mg / ml) was not possible. Instead of fibers, the capillary was probably clogged due to premature solidification, or the spinning dope would leave the capillary in the form of small globules. In either case, no continuous fibers were obtained. Surprisingly, this was due to the fact that the recombinant spider silk protein (A3I)3-A 14 The spinning dope was (A3I)3-A 14 (300 mg / ml) and extruded through glass capillaries of 150 μm or larger, continuous fibers were obtained without spontaneous clogging of the tip or formation of nodules.

[0130] This experiment was then repeated using a metal nozzle with an orifice diameter of 150 μm. Extrusion was tested at flow rates of 17 μl / min and 35 μl / min. Again, A15 -A 14 When extruded into the spinning buffer, a clogged tip or a small lump occurred (Figure 8, left image), and the concentrated (A3I)3-A 14 In the extrusion of the solution, the silk fibers were successfully extruded (Figure 8, right side). Therefore, the recombinant spider silk protein (A3I)3-A of the present invention 14 The results are consistent with those of the previous study, not only in terms of the mechanical properties shown in Example 1, but also in terms of the feasibility of an industrial-scale spinning process using metal nozzles and spinnerets for extrusion. 15 -A 14 Moreover, the silk fibers produced from the recombinant spider silk proteins of the present invention can be drawn following spinning.

[0131] Experimental Section Extrusion using glass capillaries or metal nozzles of 150 μm or more A concentrated spider silk protein preparation (300 mg / ml) was loaded into a 1 mL syringe equipped with a Luer Lok tip (BD, USA) and connected to a glass capillary with a diameter of 150 μm or more, as described in Example 1. The spinning dope was extruded into 750 mM acetate buffer (pH 5.0) at a rate of 17 μl / min, and the extrusion behavior was observed. As a next step, the syringe was fitted with a Micro-Mate® female luer hose end for tubing with an inner diameter (ID) of 1 / 16 inch (1.5875 mm) to 3 / 32 inch (2.38125 mm) (Cadence Science, Cranston, USA) and connected to a male luer lock hose end for tubing with an ID of 1 / 16 to 3 / 32 inch (Cadence Science, Cranston, USA) via approximately 5 cm of silicone tubing with an ID of 1.6 mm (667-8441, RS Pro, Gothenburg, Sweden). Finally, the male luer lock was connected to an Arque metal nozzle (Tecdia, Campbell, USA) with a tip length of 0.3 mm (A-150250000SA-B278) and an inner diameter at the tip of 150 μm. (A3I)3-A 14 (300mg / ml) and A 15 -A 14(300 mg / ml) was extruded through a metal nozzle at 17 or 35 μl / min while observing the extrusion behavior.

[0132] The above-described embodiments should be understood as some illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes can be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in different embodiments can be combined in other configurations, if technically possible.

Claims

1. A recombinant spider silk protein comprising an N-terminal (NT) domain, a repeat region (REP) domain and a C-terminal (CT) domain, The REP domain has the formula pA １ -pG-pA ２ includes a set of domains by pG stands for glycine-rich domain, pA １ and pA ２ represents the alanine-rich domain, pA １ and pA ２ one of which is a polyalanine domain, pA １ and pA ２ the other is a polyalanine domain in which every third or fourth alanine residue is substituted with an isoleucine residue or a valine residue.

2. The pA １ and pA ２ One of the amino acid sequences is A ｍ 2. The recombinant spider silk protein according to claim 1, comprising, or preferably consisting of, m, an integer selected within the range of 7 to 18, preferably within the range of 10 to 17, more preferably within the range of 14 to 16.

3. The pA １ and pA ２ 3. A recombinant spider silk protein according to claim 1 or 2, wherein the other is a polyalanine domain in which every fourth alanine residue is substituted with an isoleucine residue or a valine residue, preferably a polyalanine domain in which every fourth alanine residue is substituted with an isoleucine residue.

4. The pA １ and pA ２ The other is (A ３ I) ｎ A ｐ , A ｐ (IA ３ ) ｎ , (A ３ V) ｎ A ｐ and A ｐ (VA ３ ) ｎ an amino acid sequence selected from the group consisting of: ３ I) ｎ A ｐ and A ｐ (IA ３ ) ｎ and more preferably an amino acid sequence selected from the group consisting of (A ３ I) ｎ A ｐ wherein n is an integer selected within the range of 2 to 4, preferably 3, and p=m-n, and m is an integer selected within the range of 8 to 18, preferably within the range of 10 to 17, more preferably within the range of 14 to 16.

5. The REP domain is pA １ -pG １ -pA ２ -pG ２ , pG １ -pA １ -pG ２ -pA ２ and pG １ -pA １ -pG ２ -pA ２ -pG ３ and preferably, pG １ -pA １ -pG ２ -pA ２ -pG ３ pG １ , pG ２ and pG ３ A recombinant spider silk protein according to claim 1 or 2, wherein represents a glycine-rich domain.

6. The REP domain is (A ３ I) ３ A ３ -pG １ -A １４ -pG ２ A １５ -pG １ -(A ３ I) ３ A ２ -pG ２ pG １ -(A ３ I) ３ A ３ -pG ２ -A １４ pG １ -A １５ -pG<00OO078>-(A ３ I) ３ A ２ pG １ -(A ３ I) ３ A ３ -pG ２ -A １４ -pG ３ pG １ -A １５ -pG ２ -(A ３ I) ３ A ２ -pG ３ (A ３ V) ３ A ３ -pG １ -A １４ -pG ２ A １５ -pG １ -(A ３ V) ３ A ２ -pG ２ pG １ -(A ３ V) ３ A ３ -pG ２ -A １４ pG １ -A １５ -pG ２ -(A ３ V) ３ A ２ pG [[ID=1t25]] １ -(A ３ V) ３ A ３ -pG ２ -A １４ -pG ３ and pG １ -A １５ -pG ２ - (A ３ V) ３ A ２ -pG ３ Formula selected from the group consisting of, preferably (A ３ I) ３ A ３ -pG １ -A １４ -pG ２ , A １５ -pG １ - (A ３ I) ３ A ２ -pG ２ , pG １ - (A ３ I) ３ A ３ -pG ２ -A １４ , pG １ -A １５ -pG ２ - (A ３ I) ３ A ２ , pG １ - (A ３ I) ３ A ３ -pG ２ -A １４ -pG ３ and pG １ -A １５ -pG ２ - (A ３ I) ３ A ２ -pG ３ and more preferably pG １ - (A ３ I) ３ A ３ -pG ２ -A １４ -pG ３ and pG １ -A １５ -pG ２ - (A ３ I) ３ A ２ -pG ３ and even more preferably pG １ - (A ３ I) ３ A ３ -pG ２ -A １４ -pG ３ 6. The recombinant spider silk protein of claim 5, comprising a set of domains according to the formula:

7. p.g. １ , pG ２ and pG ３ each of which comprises, or preferably consists of, an amino acid sequence selected from the group consisting of SEQ ID NOs: 49-51, and preferably １ comprises or preferably consists of the amino acid sequence according to SEQ ID NO: 49, ２ comprises or preferably consists of an amino acid sequence according to SEQ ID NO: 50, and pG ３ 6. A recombinant spider silk protein according to claim 5, comprising, or preferably consisting of, the amino acid sequence according to SEQ ID NO:

51.

8. The REP domain is pA １ -pG １ -pA ２ -pG ２ , pG １ -pA １ -pG ２ -pA ２ and pG １ -pA １ -pG ２ -pA ２ -pG ３ a formula selected from the group consisting of: １ -pA １ -pG ２ -pA ２ -pG ３ 6. The recombinant spider silk protein of claim 5, which consists of a set of domains according to the formula:

9. 3. The recombinant spider silk protein according to claim 1, wherein the REP domain is located between the NT domain and the CT domain in the recombinant spider silk protein.

10. 10. The recombinant spider silk protein of claim 9, wherein the recombinant spider silk protein has the general formula (X)-NT-(L1)-REP-(L2)-CT-(Y), where X represents an optional N-terminal tag, Y represents an optional C-terminal tag, L1 represents an optional first linker, and L2 represents an optional second linker.

11. 3. The recombinant spider silk protein according to claim 1 or 2, wherein the NT domain is derived from the NT domain of Euprosthenops australis MaSp1, preferably wherein the NT domain comprises, or preferably consists of, SEQ ID NO:

53.

12. 3. The recombinant spider silk protein according to claim 1 or 2, wherein the CT domain is derived from the CT domain of Araneus ventricosus MiSp, preferably wherein the CT domain comprises, or preferably consists of, SEQ ID NO:

54.

13. A silk fiber made from the recombinant spider silk protein of claim 1 or 2.

14. A synthetic material comprising the yarn fiber of claim 13.

15. A nucleic acid molecule encoding the recombinant spider silk protein of claim 1 or 2.

16. An expression vector comprising the nucleic acid molecule of claim 15.

17. A host cell comprising the expression vector of claim 16.

18. 1. A method for producing a yarn fiber, comprising: extruding a spinning dope containing the recombinant spider silk protein of claim 1 or 2 into an aqueous buffer solution having an acidic pH to induce polymerization of the recombinant spider silk protein into silk fibers; and isolating the yarn fibers from the aqueous buffer solution.

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