Recombinant spider silk-fortified collagen produced in plants and uses thereof

Abstract

The invention described herein relates to novel, non-naturally occurring, elastic, animal-component-free recombinant fusion biopolymers produced in plants by transient expression. More specifically, the present invention describes polynucleotides encoding fusion proteins of non-human sclerostin and human collagen wherein the fusion proteins are capable of forming hydroxylated triple helix fibers. The fusion protein is especially a fusion protein of spider silk protein-like protein and human collagen. More particularly, the present invention relates to a spider silk protein I / I type collagen fusion protein capable of forming a hydroxylated triple helix fiber, or a silk fibroin III / I type collagen fusion protein capable of forming a hydroxylated triple helix fiber. The present invention has improved properties (e.g., thermal stability, Young's modulus, cell adhesion, degradability, etc.) compared to native type I collagen. The invention also describes a method of use thereof, such as as defined in the claims for use as an electrospun scaffold particularly suitable for biomedical or cosmetic applications.

Description

Technical Field

[0001] The invention described herein relates to novel non-naturally occurring elastic, animal-free recombinant fusion biopolymers produced in plants by transient expression. More specifically, the present invention describes polynucleotides encoding a fusion protein of non-human sclerostin and human collagen, wherein the fusion protein is capable of forming hydroxylated triple helical fibers. In particular, a fusion protein of a spidroin-like protein and human collagen. More particularly, a spidroin I / type I collagen fusion protein capable of forming hydroxylated triple helical fibers, or a fibroin III / type I collagen fusion protein capable of forming hydroxylated triple helical fibers. Compared to natural type I collagen, the present invention has improved properties (e.g., thermal stability, Young's modulus, cell adhesion, degradability, etc.). The present invention also describes methods of use thereof, such as as defined in the claims for use as an electrospun scaffold particularly suitable for biomedical or cosmetic applications. Background Art

[0002] The extracellular matrix (ECM) is a noncellular component present in all tissues and organs and not only provides an essential physical scaffold for cellular components but also initiates key biochemical and biomechanical cues required for tissue morphogenesis, differentiation, and homeostasis [1].

[0003] In addition, ECM is a highly dynamic structure, which is constantly remodeled by enzymatic or non-enzymatic, and its molecular components are subject to numerous post-translational modifications. Through these physical and biochemical properties, ECM produces the biochemical and mechanical properties of each organ, such as its tensile and compressive strength and elasticity, and also mediates protective effects by maintaining the buffering effect of extracellular homeostasis and water retention. In addition, ECM triggers signal transduction and regulatory gene transcription by binding growth factors (GF) and interacting with cell surface receptors, thereby guiding basic morphological organization and physiological functions. The biochemical and biomechanical, protective and organizational properties of ECM in a given tissue may vary greatly from one tissue to another (example lung and skin and bone), even in one tissue (such as renal cortex and renal medulla) and from one physiological state to another physiological state (normal and cancerous).

[0004] The ECM is composed of two major classes of macromolecules: proteoglycans (PGs) and fibrins [2, 3]. The major fibrous ECM proteins are collagen, elastin, and fibronectin [4]. PGs fill most of the extracellular space within the tissue in the form of a hydrated gel [2]. PGs have a variety of functions that reflect their unique buffering, hydration, binding, and resistance properties. For example, in the glomerular basement membrane, perlecan plays a role in glomerular filtration [5, 6]. In contrast, in ductal epithelial tissue, decorin, biglycan, and lumican associate with collagen fibers to create a molecular structure within the ECM that is essential for mechanical buffering and hydration and provides a simple, enzymatic reservoir for growth factors (GFs) by binding these factors.

[0005] Collagen is the most abundant fibrous protein within the interstitial ECM and accounts for 30% of the total protein content of multicellular animals. Collagen, which constitutes the major structural element of the ECM, provides tensile strength, regulates cell adhesion, supports chemotaxis and migration, and guides tissue development [7]. The majority of interstitial collagen is transcribed and secreted by fibroblasts that are either localized in the matrix or recruited to the matrix from adjacent tissues [8].

[0006] By applying tension to the matrix, fibroblasts are able to organize collagen fibrils into sheets and cords, thus significantly influencing the arrangement of collagen fibers.Although in a given tissue, collagen fibers are usually a heterogeneous mixture of different types, one type of collagen frequently predominates.

[0007] The structure of collagen is a triple helix, in which three polypeptide chains are coiled together in a spiral. A single polypeptide chain consists of a repeating triplet amino acid sequence called GLY-XY. X and Y can be any amino acids, and the third amino acid is glycine. The amino acids proline and hydroxyproline are present in high concentrations in collagen. The most common triplet is proline-hydroxyproline-glycine (Gly-Pro-Hyp), which accounts for approximately 10.5% of the triplets in collagen.

[0008] Collagen associates with elastin (another major ECM fiber). Elastin fibers provide recoil for tissues that undergo repeated stretching. Importantly, elastin stretching is severely limited by its tight association with collagen fibrils [9]. Secreted tropoelastin (a precursor of elastin) molecules assemble into fibers and are highly cross-linked to each other via their lysine residues by members of the lysyl oxidase (LOX) enzyme family (including LOX and LOXL)

[10] . Elastin fibers are wrapped by glycoprotein microfibrils (mainly fibrillin), which are also essential for the integrity of elastin fibers [9].

[0009] A third fibrous protein, fibronectin (FN), is intimately involved in directing the organization of the interstitial ECM and, in addition, plays a critical role in mediating cell attachment and function. FN can be stretched to several times its resting length by cellular traction forces

[11] . This force-dependent unfolding of FN exposes cryptic integrin binding sites within the molecule, leading to pleiotropic changes in cell behavior and implicating FN as an extracellular mechanorelactor.

[0010] Like FN, other ECM proteins such as tenascin exert pleiotropic effects on cell behavior, including promoting fibroblast migration during wound healing [12, 13]. In fact, the levels of tenascin C and W are elevated in the matrix of some transformed tissues, where they can inhibit the interaction between syndecan 4 and FN to promote tumor growth and metastasis

[13] .

[0011] Collagen, elastin and fibronectin are all considered to be biodegradable biopolymers, being naturally occurring biomolecules with the highest degree of biocompatibility. In other words, they are polymeric biomolecules. These also include other biopolymers based on animal proteins, such as wool, silk, gelatin and polysaccharides (such as cellulose, starch, carbohydrate polymers produced by bacteria and fungi). Biopolymers, particularly biopolymers of carbohydrate origin, have been found to be very promising in biomedical applications in various forms due to their low toxicity, low antigenicity, high bioactivity, biodegradability, stability, processability of complex shapes with appropriate porosity to support cell growth, mechanical properties and reproducibility.

[0012] Biopolymers are produced from renewable resources and are easily biodegradable due to the presence of oxygen and nitrogen atoms in their structural backbone. Biodegradation converts them into CO2, water, biomass, moisture and other natural substances. Therefore, biopolymers are naturally recycled through biological processes.

[0013] Bioactive properties such as antimicrobial, immunomodulatory, cell proliferation and angiogenic properties of polymers create a microenvironment that is conducive to the healing process and offer a multitude of applications in pharmaceutical, medical and cosmetic applications. However, one of the main advantages of biopolymers is their ability to absorb large amounts of water in the dry state and to donate water upon hydration.

[0014] Collagen and elastin are usually isolated from natural sources (such as cow, pig or poultry leather), and collagen also comes from cartilage or bone. Bone is usually dried, defatted, crushed and demineralized to extract collagen, while leather and cartilage are usually chopped and digested with proteolytic enzymes. Since collagen and elastin are resistant to most proteolytic enzymes, this process can easily remove most of the contaminating proteins coexisting with collagen or elastin.

[0015] U.S. Pat. No. 3,949,073 to Daniels et al. discloses the preparation of soluble collagen by dissolving tissue in aqueous acid followed by enzymatic digestion. The resulting atelopeptide collagen is soluble and has substantially lower immunogenicity than unmodified collagen. It can be injected into a suitable location in a subject along with a fibrillation promoter (described in the patent as a polymerization promoter) to form a fibrous collagen implant in situ to reinforce hard or soft tissue. This material is now available from Collagen Corporation (Palo Alto, Calif.) under the trademark Collagen implants.

[0016] U.S. Pat. No. 4,488,911 to Luck et al. discloses a method for preparing collagen in solution (CIS) in which native collagen is extracted from animal tissue in dilute aqueous acid and then treated with an enzyme such as pepsin, trypsin or Digestion. Enzymatic digestion removes the terminal peptide portion of the collagen molecule to provide "terminal peptide" collagen in solution. The terminal peptide CIS so produced is essentially non-immunogenic and, due to the loss of the primary cross-linked region, is also essentially non-cross-linked. The CIS can then be precipitated by dialysis in a moderate shear environment to produce collagen fibers similar to native collagen fibers. The precipitated reconstructed fibers can additionally be cross-linked using chemical reagents (e.g., aldehydes such as formaldehyde and glutaraldehyde) or using heat or radiation. Due to its biocompatibility and reduced immunogenicity, the resulting product is suitable for medical implants.

[0017] U.S. Pat. No. 4,424,208 to Wallace et al. discloses an improved collagen preparation suitable for soft tissue augmentation. Wallace's preparation comprises reconstructed fibrillar atelocollagen (e.g. The invention relates to a combination of a collagen) and a particulate cross-linked atelocollagen dispersed in an aqueous medium. The addition of the particulate cross-linked collagen improves the durability of the implant, or improves the ability to resist shrinkage after implantation.

[0018] Smestad et al., U.S. Pat. No. 4,582,640, discloses a glutaraldehyde cross-linked atelocollagen (GAX) formulation suitable for medical implants. Collagen is cross-linked under conditions that favor intrafiber bonding over interfiber bonding and provide a more durable product than non-cross-linked atelocollagen and is available from Collagen Corporation under the trademark Implant is commercially available.

[0019] US Patent No. 4,642,117 to Nguyen et al. discloses a method for reducing the viscosity of atelopepeptide CIS by mechanical shearing. The reconstituted collagen fibers are passed through a fine mesh screen until the viscosity is reduced to a level suitable for injection.

[0020] U.S. Pat. No. 4,563,350 to Nathan et al. discloses an osteoinductive bone repair composition comprising osteoinductive factors, at least 5% non-remodeled (non-fibrous) collagen and the remainder remodeled collagen and / or mineral powder (e.g., hydroxyapatite). CIS can be used for non-remodeled collagen, and Collagen implants (ZCI) preferably have reconstructed collagen components. The material is implanted into bone defects or fractures to accelerate osteoclast ingrowth and promote new bone growth.

[0021] Chu's U.S. Pat. No. 4,557,764 discloses a "secondary nucleation" collagen precipitate that exhibits desirable ductility and putty-like consistency. Collagen is provided in a solution (e.g., at a concentration of 2 mg / mL-4 mg / mL), and a "primary nucleation product" is precipitated by rapid titration and centrifugation. The remaining supernatant (containing a large amount of the original collagen) is then poured off and allowed to stand overnight. The precipitated secondary nucleation product is collected by centrifugation.

[0022] Chu's U.S. Pat. No. 4,689,399 discloses a collagen film preparation prepared by compressing and drying a collagen gel. The resulting product has high tensile strength.

[0023] JAM Ramshaw et al., Anal Biochem (1984) 141:361-65 and PCT application WO 87 / 04078 disclose precipitation of bovine collagen (types I, II and III) from aqueous PEG solutions in which there is no binding between the collagen and the PEG.

[0024] Werner, U.S. Pat. No. 4,357,274, discloses a method for increasing the durability of sclerosing proteins (e.g., meninges) by soaking the defatted tissue in H2O2 or PEG for several hours and then lyophilizing. The resulting modified whole tissue exhibits increased durability.

[0025] Hiroyoshi, U.S. Pat. No. 4,678,468, discloses the preparation of a polysiloxane polymer having an interpenetrating network of a water-soluble polymer dispersed therein. The water-soluble polymer may be a collagen derivative, and the polymer may additionally include heparin. The polymer is molded into an artificial vascular graft and is designed to prevent clotting.

[0026] Other patents disclose the use of collagen preparations with bone chips or minerals. For example, US Pat. No. 4,314,380 to Miyata et al. discloses a bone implant prepared by baking animal bone segments and soaking the baked segments in a solution of atelocollagen.

[0027] US Patent No. 4,192,02 to Deibig et al. discloses an implant material comprising powdered calcium phosphate and a biodegradable polymer, which may be collagen, in a paste formulation.

[0028] Several references in the art relate to proteins modified by covalent conjugation of polymers to alter the solubility, antigenicity, and biological clearance of the protein. For example, U.S. Pat. No. 4,261,973 discloses conjugation of several allergens with PEG or PPG (polypropylene glycol) to reduce the immunogenicity of the protein. U.S. Pat. No. 4,301,144 discloses conjugation of hemoglobin with PEG and other polymers to increase the oxygen carrying capacity of the protein.

[0029] EPO 98110 discloses coupling enzymes or interferons to polyoxyethylene-polyoxypropylene (POE-POP) block polymers to increase the half-life of proteins in serum. US Pat. No. 4,179,337 discloses conjugating hydrophilic enzymes and insulin to PEG or PPG to reduce immunogenicity.

[0030] Davis et al., Lancet (1981) 2:281-83, disclose modified uricases with long half-life and low immunogenicity by conjugation with PEG to provide uric acid metabolism in serum.

[0031] Nishida et al., J Pharm Pharmacol (1984) 36.:354-55 discloses PEG-uricase conjugates administered orally to chickens, showing a reduction in serum levels of uric acid. Inada et al., Biochem & Biophys Res Comm (1984) 122:845-50 discloses lipoprotein lipase conjugated with PEG to make it soluble in organic solvents.

[0032] M. Chvapil et al., J Biomed Mater Res (1969) 3:315-32 discloses a composition prepared from a collagen sponge and a cross-linked ethylene glycol monomethacrylate-ethylene glycol dimethacrylate hydrogel. The collagen sponge was prepared by freeze-drying an aqueous mixture of bovine collagen and methylglyoxal (tanning agent). The sponge-hydrogel composition was prepared by polymerizing ethylene glycol monomethacrylate and ethylene glycol dimethacrylate in the sponge.

[0033] U.S. Patent No. US6667051B1 to Kenton W. Gregory discloses a method for joining at least one layer of a pressed biomaterial to a tissue substrate, the method comprising: I) providing a pressed biomaterial consisting essentially of elastin or elastin raw material; II) applying a biodegradable cyanoacrylate adhesive to one of the tissue and the material; and III) bonding the pressed biomaterial to the tissue and forming a substantially watertight bond therebetween.

[0034] Patent No. AU712868B to Kenton W. Gregory et al. discloses elastin and elastin-based biomaterials and methods of using them in tissue repair and replacement. The present invention also relates to methods of fixing elastin and elastin-based biomaterials to existing tissue.

[0035] US Patent No. US11041015B2 to Nikolay Ouzounov et al. discloses recombinantly expressed non-naturally occurring full-length and truncated collagen molecules and full-length and truncated elastin molecules in bacterial cells and their production.

[0036] Patent No. EP0951537B1 to Véronique-Résidence Sainte Madeleine Gruber et al. discloses plant-produced recombinant collagen, in particular produced by stable expression, and in particular homocatalar type I collagen [α1(I)3] and uses thereof.

[0037] Klaus During's patent number CA2336064A1 discloses a method for producing fibrin, the method comprising the following steps: (a) expressing a precursor fibrin in a plant cell and (b) incubating the precursor fibrin with a protein for processing the precursor fibrin. The invention also relates to plant cells for this purpose and fibrin produced according to the method of the invention.

[0038] Patent No. US9783816B2 to Oded Shoseyov et al. discloses a method for producing at least one collagen α chain in a manner that enables the collagen α chain to accumulate in a subcellular compartment devoid of endogenous P4H activity, thereby producing collagen in plants.

[0039] Kirivikko et al. (WO 93 / 07889) describe the expression of human procollagen (type I and II). One example reports the expression of chain α-1 or α-2 procollagen I or II in mammalian cells HT1080. Another example describes the expression of the α and β chains of prolyl-4-hydroxylase in Sf9 insect cells. Suggested examples of the expression of collagen and recombinant P4H are given for S. cerevisiae and P. pastoris, but no results are demonstrated.

[0040] Although the mechanical properties of collagen, elastin and fibronectin and their very useful biological properties are unquestionably recognized, the use of such proteins is again questioned because of the risk of contamination with unconventional infectious agents. In fact, while the risks posed by bacterial contamination or viruses can be perfectly controlled, the risks associated with prion-type infectious agents are different.

[0041] These protein-like infectious agents are implicated in the development of degenerative encephalopathies in animals (scrapie in sheep, bovine spongiform encephalopathy) and in humans Creutzfeldt-Jakob disease, Gerstmann-Strauss syndrome, and Kuru. Such formal control over the period of their possible expression is difficult to achieve. These risks have actually prevented any marketing of human collagen, while the regulations established for collagen-related animals complicate the purification process and increase its cost.

[0042] In view of these difficulties, and in the face of the rapidly deteriorating profile of mammalian collagen, one solution is to produce recombinant collagen that can be easily purified in a system that is unlikely to pose a pathogenic risk to humans and is not industrially expensive. The inventors have therefore developed the present invention, for example, recombinant spider silk protein / collagen fusion proteins transiently produced in plants.

[0043] A priori, animal cells are more adapted to the expression of mammalian genes. However, the enzyme equipment for post-translational maturation is different because of different tissues, organs or species. For example, it is reported that when plasma protein is obtained from human blood or when produced by the recombinant cell-like cells of Chinese hamster ovary or in the milk of transgenic animals, the post-translational maturation of plasma protein may be different. In addition, the low-level expression obtained with mammalian cells involves a large amount of in vitro culture at high cost. Producing recombinant proteins in the milk of transgenic animals (mice, sheep and cows) reduces production costs and overcomes the problem of expression level. However, there are still problems of ethics and virus and subvirus (prion) pollution.

[0044] For these reasons, there is an urgent need to develop novel biodegradable biopolymers with broader pleiotropic anti-inflammatory effects and improved toxicity, antigenicity, bioactivity, biodegradability, stability, processability, mechanical properties and renewability. Therefore, there remains a need to produce biodegradable biopolymers with the above capabilities in a highly purified state, which are obtained from low-cost and high-yield processes, which is the inspiration for developing the present invention.

[0045] Collagen and silk constitute highly sought-after scaffold materials for regenerative and healing applications due to their important roles as mechanical support and affinity regulators in the extracellular matrix. However, substantiated concerns have arisen regarding the quality and safety of collagen extracted from animal tissues or silk derived from spiders or silkworms, particularly regarding their immunogenicity, risk of disease transmission, and overall quality and consistency. At the same time, contamination with undesirable cytokines can significantly impair their biological activity, thereby affecting cell recruitment, proliferation, and differentiation.

[0046] In general, the present invention completely overcomes the disadvantages of collagen from animal and cadaver sources due to their diversity of sources and recyclability. Summary of the invention

[0047] The present invention will be described with reference to specific embodiments and with reference to certain drawings, but the invention is not limited thereto but only to the claims. The drawings described are only schematic and not limiting. In the drawings, the size of some elements may be exaggerated and not drawn to scale for illustrative purposes. The dimensions and relative dimensions do not correspond to actual reductions in the practice of the invention.

[0048] In addition, the terms "first", "second", etc. in the specification and claims are used to distinguish similar elements and are not necessarily used to describe a sequence in time, space, ranking or any other manner. It should be understood that the terms so used are interchangeable under appropriate circumstances, and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0049] Although exemplary embodiments are described below, the present invention is limited only by the appended claims. The appended claims are hereby expressly incorporated into the Detailed Description, with each claim and each combination of claims permitted by a claim-defined dependent structure forming a separate embodiment of the present invention.

[0050] It should be noted that the term "comprises / comprising" used in the claims should not be interpreted as being limited to the means listed thereafter; it does not exclude other elements or steps. Therefore, it is interpreted as the presence of the specified features, integers, steps or components, but does not exclude the presence or addition of one or more other features, integers, steps or components or groups thereof.

[0051] In the invention described herein, various specific details are provided. Embodiments of the present invention may be performed without these specific details. In addition, for the clarity and simplicity of the present invention, well-known features, elements and / or steps are not necessarily described in detail.

[0052] References in this specification to "one embodiment" or "an embodiment" or "an embodiment" mean that the particular features, structures, or characteristics described in connection with the embodiment are included in at least one embodiment of the present invention. Thus, the phrases "in one embodiment" or "in an embodiment" appearing in various places in this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, it will be apparent to one of ordinary skill in the art that the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0053] Similarly, it should be understood that in the description of the exemplary embodiments of the present invention, in order to simplify the disclosure of the present invention and facilitate the understanding of one or more of the various creative aspects, the various features of the present invention are sometimes combined in a single embodiment, figure or its description. However, this method of disclosure should not be interpreted as reflecting the intention that the claimed invention requires more features than those explicitly stated in each claim. In contrast, as reflected in the appended claims, the creative aspects do not lie in all the features of a single aforementioned disclosed embodiment. Therefore, the claims following the specific embodiment are expressly incorporated into the present specific embodiment, and each claim exists independently as a separate embodiment of the present invention.

[0054] In addition, although some embodiments described herein include some features included in other embodiments but do not include other features, the combination of features of different embodiments is meant to be within the scope of the present invention and form different embodiments, as understood by those skilled in the art. For example, in the appended claims, any claimed embodiment can be used in any combination.

[0055] Insubstantial improvements and adjustments made to the method disclosed in the present invention should still be within the scope of protection of the present invention. At the same time, the raw materials used are not always described in detail. The raw materials can be commercial products. The process steps or preparation methods that are not always described in detail are process steps or preparation methods well known to those skilled in the art.

[0056] The starting materials used to obtain the present invention are commercially available products; process steps or preparation methods that are not always described in detail are those well known to those skilled in the art.

[0057] As used herein, the term "about" refers to ± 10%.

[0058] The term "consisting of" means "including and limited to."

[0059] The term "consisting essentially of" means that the composition, process or structure may include additional components, steps and / or parts, but only if the additional components, steps and / or parts meet the basic and novel properties of the claimed composition and / or do not materially change the basic and novel properties of the claimed composition and / or method or structure.

[0060] As used herein, the singular forms "a", "an", and "the" include plural references unless the context clearly indicates otherwise. For example, the term "a compound" or "at least one compound" may encompass a variety of compounds, including mixtures thereof, which may be used in foods, cosmetics, pharmaceuticals, industrial products, medical products, laboratory culture growth media, and many other applications.

[0061] Throughout the application, various embodiments of the present invention can be presented in range format. It should be understood that the description of the range format is only for convenience and brevity and should not be interpreted as an inflexible limitation on the open range. Therefore, it should be considered that the description of the range has specifically disclosed all possible sub-ranges and each numerical value in the range. For example, the description of a range (such as 1 to 6) should be considered to have a specifically disclosed sub-range, such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., and a single number in the range, such as 1, 2, 3, 4, 5 and 6. This application is not related to the breadth of the range.

[0062] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integer) within the indicated range. The phrases "between / a range between a first indicated numeral and a second indicated numeral" and "from / to a first indicated numeral" are used interchangeably herein and are meant to include the first and second indicated numerals and all fractional and integer numbers between them.

[0063] As used herein, the term "method" refers to manners, methods, techniques and procedures for accomplishing a given task, including but not limited to those manners, methods, techniques and procedures known to practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine or readily developable from known manners, methods, techniques and procedures.

[0064] In the description provided herein, many specific details are shown. However, it should be understood that embodiments of the present invention can be implemented without these specific details. In other cases, well-known methods, structures and techniques have not been shown in detail in order not to obscure the understanding of this description.

[0065] The object of the present invention is to provide a novel fusion biopolymer consisting of the following two parts: I) recombinant non-human sclerostin, and II) human recombinant collagen, in particular human type I collagen. The non-human sclerostin is preferably not a non-human collagen. In one embodiment, the non-human sclerostin is selected from keratin, elastin, fibrin or spider silk protein; more specifically, selected from fibrin or spider silk protein, also commonly known as spider silk protein-like protein.

[0066] In one embodiment, the present invention provides a novel fusion biopolymer consisting of the following two parts: I) recombinant silk fibroin III, and II) human recombinant type I collagen. Other combinations (such as but not limited to: type II collagen to type XXVIII collagen, any chain or any derivative thereof) are also within the scope of the present invention.

[0067] In one embodiment, the present invention provides a novel fusion biopolymer consisting of two parts: I) recombinant spider silk protein I, and II) human recombinant collagen type I. Other combinations (such as but not limited to: collagen type II to collagen type XXVIII, any chain or any derivative thereof, spider silk protein II, ampulla spider silk protein, whip spider silk protein, any type of spider silk protein or any derivative thereof) are also within the scope of the present invention.

[0068] It is an object of the present invention to provide the novel fusion biopolymers disclosed herein on a large scale, wherein the proteins can be easily manipulated to polymerize into fibers as desired.

[0069] The term "collagen" or "collagen-like" as used herein refers to a monomeric polypeptide that can form a quaternary structure with one or more collagen or collagen-like polypeptides. The quaternary structure of native collagen is a triple helix, typically composed of three polypeptides. Of the three polypeptides that form native collagen, two are typically identical and are referred to as α chains. The third polypeptide is referred to as a β chain. Thus, a typical native collagen may be referred to as AAB, where the collagen consists of two Colα1 (I) chains and one Colα2 (I) chain. The term "procollagen" as used herein refers to a polypeptide produced by a cell that can be processed into naturally occurring collagen. Preferably, the collagen chains expressed in the present invention are the α1 and 2 chains of type I collagen, although other types may also be used. Examples include other collagens that form fibrils (type II, type III, type V, and type XI), collagens that form networks (type IV, type VIII, and type X), collagens that associate with fibril surfaces (type IX, type XII, and type XIV), collagens that exist as transmembrane proteins (type XIII and type XVII), or collagens that form 11nm periodic beaded filaments (type VI). For more information, see Hulmes, 2002. The expressed collagen αI and II chains can be encoded by any polynucleotide sequence derived from any mammal. Preferably, the amino acid sequence encoding collagen αI and II chains is a human sequence, and is shown by SEQ NO:1 and 2. Its corresponding nucleotide sequence is shown by SEQ NO:3 and 4. Preferably, codons are optimized for N. benthamiana chloroplast expression.

[0070] The term "spidroin" as used herein refers to the major protein in spider silk. Different types of spider silk contain different spidroin proteins, which are all members of a single protein family. The two most ubiquitous types of spidroin proteins are the major ampullate spidroin proteins (MaSp), which are used to build dragline silk. Dragline silk fibers are composed of two types of spidroin proteins: the major ampullate spidroin protein 1 (MaSp1) and the spidroin protein 2 (MaSp2) proteins.

[0071] The term "fibroin" as used herein refers to an insoluble hardened protein comprising raw silk fibers obtained from spiders or silkworms. Preferably, the fibroin is obtained from spider species by recombinant techniques. Alternatively, fibroin may even be obtained from silkworm species, such as but not limited to Bombyx mori, other moth genera such as Antheraea, Cricula, Samia and Gonometa, from solutions containing dissolved silk or by recombinant techniques. Preferably, the spidroin and the fibroin are obtained from spider species, including but not limited to: Arachnura higginsi, Araneus circulissparsus, Araneus diadematus, Argiope picta, Argiope trifasciata, Nephila antipodiana, Beccari's Tent Spider (Cyrtophora beccarii), Celaenia excavata, Gasteracantha kuhlii, Argiope aurantia, Ordgarius furcatus, Ordgarius magnificus ... nautica), Brown-legged Spider (Neosconarufofemorata), Hooded Black-headed Spider (Zygiella calyptrata), Common Orb Spider (Parawixiadehaani), Common Orb Spider (Neoscona oxancensis), Crab-like Spiny Orb Spider (Gasteracanthacancriformis)(elipsoides), Curved Spiny Spider (Gasteracantha arcuate), Dish Clouded Spider (Cyrtophora moluccensis), Cyrtophora parnasia, Orb Spider (Dolophones conifera), Flat Spider (Dolophones turrigera), Doria's Spiny Spider (Gasteracantha doriae), Two-spot Spiny Spider (Gasteracantha mammosa),Two-tailed Tent Spider (Cyrtophora exanthematiea), Aculeperia ceropegia, Eriophora pustulosa, Flat Anepsion (Anepsion depressium), Four-spined Jewel Spider (Gasteracantha quadrispinosa), Garden Orb Weaver (Eriophora transmarina), Giant Lichen Orbweaver (Araneus bicentenarius), Golden Orb Weaver (Nephila maculate), Hasseltii (Gasteracantha hasseltii), Funnel Spider (Tegenaria atrica), Heurodes turrita, Cyclosa insulana, Jewel or Spiny Spider (Astracantha minax), Kidney Orb Weaver (Araneus mitificus), Laglaise's Garden Spider (Eriovixia laglaisei), Cyclosa bifida, Malabar Spider (Nephilengys malabarensis), Multi-Colored St. Andrew's Cross Spider (Argiope versicolor), Ornamental Tree-Trunk Spider (Herennia ornatissima), Oval St. Andrew's Cross Spider (Argiope aemula), Red Tent Spider (Cyrtophora unicolor), Russian Tent Spider (Cyrtophora hirta), St. Andrew's Cross Spider (Argiope keyserlingi), Scarlet Acusilas (Acusilas coccineus), Silver Acusilas (Argiope argentata), Spiny Garden Spider (Gasteracantha cancriformis), Spotted Garden Spider (Neosconadomiciliorum), St. Andrews Cross Spider (Argiope aetheria),St. Andrews' cross spider (Argiope keyserlingi), tree spider (Poltys illepidus), Arkys clavatus, Arkys lancearius, Poecilopachysaustralasia, Nephila, such as Nephila clavipes, Nephila senegalensis and Nephilamadagascariensis. Most preferably, the spidroin protein is derived from Nephila clavipes and the spidroin protein is derived from the cross spider. Not surprisingly, spidroin protein is also considered to be a spidroin-like analogue

[42] . The amino acid sequence of the spidroin protein I expressed in the present invention is shown in SEQ NO:5. The corresponding nucleotide sequence is shown in SEQ NO:6. Preferably, the nucleotide sequence is codon optimized for chloroplast expression in Nicotiana benthamiana. Preferably, the fibroin sequence expressed in the present invention is fibroin III, and its amino acid sequence is shown by SEQ NO:7. Its nucleotide sequence is shown by SEQ NO:8. Preferably, the nucleotide sequence is optimized for chloroplast expression in Nicotiana benthamiana. ,

[0072] According to one aspect of the invention, a method for producing collagen in a plant or isolated plant cell is defined in the claims, the method comprising expressing at least one type of collagen α chain and exogenous proline 4-hydroxylase (P4H) in a plant or isolated plant cell in a manner such that at least one type of collagen α chain and the exogenous P4H are able to accumulate in a subcellular compartment devoid of endogenous P4H activity, thereby producing collagen in the plant. In previously described techniques, attempts to produce human collagen that relies on the hydroxylation machinery naturally present in plants produce collagen that is poorly proline hydroxylated, as described by Merle et al., 2002

[14] . Such collagen denatures or loses its triple helical structure at temperatures below 30°C. Co-expression of collagen and prolyl hydroxylase produces stable hydroxylated collagen that is biologically relevant for use at body temperature

[14] . As used herein, the phrase "subcellular compartment without endogenous P4H activity" refers to any compartment region of a cell that does not include a plant P4H or an enzyme with plant-like P4H activity. Examples of such subcellular compartments include vacuoles, apoplasts and cytoplasms, as well as organelles such as chloroplasts, mitochondria, etc. Accumulation of expressed collagen chains in subcellular compartments without endogenous P4H activity can be achieved by any of several methods. For example, the expressed collagen chains can include a signal sequence for targeting the expressed protein to a subcellular compartment such as apoplasts or more preferably chloroplasts or other organelles such as mitochondria. Examples of suitable signal sequences include chloroplast transit peptides (included in Uniprot entry G5DBJ0, amino acids 1-40) and mitochondrial transit peptides (included in Uniprot entry Q9ZP06, amino acids 1-22). The Examples section below provides additional examples of suitable signal sequences, as well as guidance for using such signal sequences in the expression of collagen chains in plant cells. Alternatively, the sequence of the collagen chains can be modified in a manner that alters the cellular localization of the collagen when expressed in plants. As described above, the endoplasmic reticulum (ER) of plants includes a P4H that cannot properly hydroxylate the collagen chains. The collagen alpha chain naturally includes an ER targeting sequence that directs the expressed collagen to the ER where it undergoes post-translational modifications (including incorrect hydroxylations). Therefore, removal of the ER targeting sequence will result in cytoplasmic accumulation of collagen chains that are free of post-translational modifications (including any hydroxylations).

[0073] As described above, hydroxylation of the α chain is required for the assembly of stable type I collagen. Hydroxylation of the whole collagen proline also significantly increases the denaturation temperature by stabilizing the collagen triple helix, which is a method well known to those skilled in the art. Since the transiently expressed α chain in the wild-type plants of the present invention accumulates in compartments without endogenous P4H activity, it is usually necessary to isolate this chain from the plant, plant tissue or cell and correctly hydroxylate it using in vitro techniques, which can be achieved by the method of Torre-Blanco A, Alvizouri A.

[15] . However, this method is cumbersome and expensive to achieve the desired effect. To overcome this limitation, the method of the present invention also transiently co-expresses P4H, which is able to correctly hydroxylate the collagen α chain (i.e., hydroxylates only the proline (Y) position of the Gly-XY triplet). P4H is an enzyme composed of two subunits, α and β, and both subunits are required to form an active catalytic enzyme

[16] . Mammalian prolyl 4-hydroxylase is an α-2 / β-2 tetramer

[17] . The 59 kDa α subunit contains the substrate binding domain and the enzyme active site

[18] . There are three isoforms of the α subunit in humans and most other vertebrates, with isoform α-1 being the most common. The pair of α subunits can be any of the three isoforms

[19] . The 55-kDa β subunit is a protein disulfide isomerase (PDI), which has an additional function in the formation of collagen. As part of P4H, it retains the tetramer in the ER lumen and maintains the otherwise insoluble α subunit in an active form. In the prior art, the inventors of Patent No. EP2816117B1 (Plants producing collagen and methods for their production and use) used exogenous human P4H to produce stable transformant (e.g., transgenic) plants that are capable of producing human P4H with the goal of correctly hydroxylating only the proline (Y) position of the Gly-XY triplet. However, tetrameric human P4H is inhibited by extensin molecules through poly(L-proline), which are substrates of plant prolyl-4-hydroxylases, are rich in Ser-(Pro)4-Ser-Pro-Ser-(Pro)4 sequences, and can therefore inhibit mammalian-derived P4H. To overcome this limitation, the inventors of the present invention used an alternative approach and produced a chimeric α / β dimer that has the same specific activity as native human P4H, but does not have the inhibitory potential of poly(L-proline). The resulting chimeric P4H enzyme consists of the α subunit of Dictyostelium discoideum (slime mold) P4H (UniprotKB:Q86KR9) and the β subunit of Bostaurus, Bovine P4H (Uniprot:P05307). According to further features in the preferred embodiment, the exogenous chimeric P4H includes a signal peptide for targeting to the chloroplast and has no ER targeting or retention sequence.The amino acid sequences encoding the chimeric P4H enzymes expressed in the present invention are shown by SEQ NO: 9 for the α chain of the P4H subunit and SEQ NO: 10 for the β chain of the P4H subunit. The corresponding nucleotide sequences are shown by SEQ NO: 11 and 12. Preferably, the nucleotide sequence is optimized for chloroplast expression in Nicotiana benthamiana.

[0074] In mammals, lysyl hydroxylases, galactosyltransferases, and glucosyltransferases sequentially modify lysyl residues at specific positions to hydroxylysyl, galactosylhydroxylysyl, and glucosylgalactosylhydroxylysyl residues in collagen. However, the multifunctional enzyme lysine hydroxylase 3 (LH3), shown in Genbank No. 060568, is the only human enzyme that can convert collagen lysine to 1,2-glucosylgalactosyl-5-hydroxylysine through three consecutive reactions: hydroxylation of collagen lysine (LH activity), N-linked conjugation of galactose to hydroxylysine (GT activity), and conjugation of glucose to galactosyl-5-hydroxylysine (GGT activity). These enzymes are known to work together with prolyl hydroxylases to introduce hydroxylation of lysine and proline residues, respectively, on collagen in the endoplasmic reticulum (ER) before forming a triple helical assembly

[20] . According to further features in the described preferred embodiments, the exogenous LH3 comprises a signal peptide for targeting to chloroplasts and has no ER targeting or retention sequence. The amino acid sequence encoding the LH3 enzyme expressed in the present invention is shown by SEQ NO: 13. Its corresponding nucleotide sequence is shown by SEQ NO: 14. Preferably, the nucleotide sequence is optimized for chloroplast expression in Nicotiana benthamiana.

[0075] According to one aspect of the present invention, there is provided a method for producing a fibrous spider silk protein I / type I collagen fusion protein, the method comprising transiently co-expressing two vectors, wherein vector 1 expresses: A) spider silk protein I chain, B) type I collagen α-I chain, and C) type I collagen α-II chain, wherein the transient expression is configured so that the spider silk protein I chain and the collagen α-I and α-II chains are each capable of accumulating in a subcellular compartment devoid of endogenous P4H and LH activity. Such a compartment is preferably a chloroplast and functions by using a transport signal directed to the chloroplast. Vector 2 expresses A) the above-mentioned chimeric P4H and B) LH3, both of which are capable of accumulating in a subcellular compartment devoid of endogenous P4H and LH activity. Both vectors are preferably targeted to the chloroplast by introducing a chloroplast transit peptide at the N-terminus of the corresponding gene constructs. Such a transit peptide is preferably, but not limited to, a chloroplast protein chaperone-like protein of POR1. The individual genes assembled in the two expression vectors are separated by the introduction of a so-called "2A self-cleaving peptide", which can induce ribosome jumping during the translation of proteins in cells, thereby potentially producing multiple separate sequences expressed in a single transcript. In the present invention, this fusion protein is referred to as "SPID1COL1" from now on. The amino acid sequence encoding the SPID1COL1 fusion protein in vector 1 is shown by SEQ ID: 15. Its corresponding nucleotide sequence is shown by SEQ ID: 16. Preferably, the nucleotide sequence is optimized for chloroplast expression in Nicotiana benthamiana. The amino acid sequence encoding the P4H / LH3 protein in vector 2 is shown by SEQ ID: 17. Its corresponding nucleotide sequence is shown by SEQ ID: 18. Preferably, the nucleotide sequence is optimized for chloroplast expression in Nicotiana benthamiana.

[0076] According to one aspect of the present invention, a method for producing a fibrous silk fibroin III / type I collagen fusion protein is provided, the method comprising transiently co-expressing two vectors, wherein vector 1 expresses: A) silk fibroin III chain, B) type I collagen α-I chain, and C) type I collagen α-II chain, wherein the transient expression is configured so that the silk fibroin III chain and the collagen α-I and α-II chains are each capable of accumulating in a subcellular compartment devoid of endogenous P4H and LH activity. Such compartments are preferably chloroplasts and function by using transport signals directed to chloroplasts. Vector 2 expresses A) the above-mentioned chimeric P4H and B) LH3, both of which are capable of accumulating in subcellular compartments devoid of endogenous P4H and LH activity. Both vectors are preferably targeted to chloroplasts by introducing a chloroplast transit peptide at the N-terminus of the respective gene constructs. Such a transit peptide is preferably, but not limited to, a chloroplast protein chaperone-like protein of POR1. Each gene assembled in two expression vectors is separated by introducing a so-called "2A self-cleaving peptide", which can induce ribosome jumping during the translation of proteins in cells, thereby possibly producing multiple separate sequences expressed in a single transcript. In the present invention, this fusion protein is referred to as "FIB3COL1" from now on. The amino acid sequence encoding the FIB3COL1 fusion protein in vector 1 is shown by SEQ ID:19. Its corresponding nucleotide sequence is shown by SEQ ID:20. Preferably, the nucleotide sequence is optimized for chloroplast expression in Nicotiana benthamiana. The amino acid sequence encoding the P4H / LH3 protein in vector 2 is shown by SEQ ID:17. Their corresponding nucleotide sequences are shown by SEQ ID:18. Preferably, the nucleotide sequence is optimized for chloroplast expression in Nicotiana benthamiana.

[0077] According to further features in the preferred embodiments, the plant is selected from tobacco, maize, alfalfa, rice, potato, soybean, tomato, wheat, barley, rapeseed, sugar beet, sunflower and cotton, more preferably Nicotiana benthamiana, wherein the part is a leaf, seed, root, tuber or stem, more preferably a leaf.

[0078] Plant: Generally understood to be any unicellular or multicellular organism with photosynthetic ability or its cells, tissues, organs, parts or propagation materials (such as seeds or fruits). For the purpose of the present invention, all genera and species of higher and lower plants of the plant kingdom are included. Annual, perennial, monocotyledonous and dicotyledonous plants are preferred. The term includes mature plants, seeds, buds and seedlings and parts derived therefrom, propagation materials (such as seeds or microspores), plant organs, tissues, protoplasts, callus and other cultures, such as cell cultures and any other type of plant cells grouped to produce functional or structural units. Mature plants refer to plants at any desired developmental stage other than seedlings. Seedlings refer to young immature plants in early developmental stages. Annual, biennial, monocotyledonous and dicotyledonous plants are preferred host organisms for producing transgenic plants. In addition, the expression of genes is favorable in all ornamental plants, useful or ornamental trees, flowers, cut flowers, shrubs or lawns. The plants that can be mentioned by way of example but are not limited to are: angiosperms, bryophytes such as, for example, Hepaticae (mosses) and Musci (lichens); Pteridophytes such as true ferns, horsetails and lycophytes; Gymnosperms such as conifers, cycads, ginkgo and gnetum; Algae such as Chlorophyceae, Phaeophyceae, Rhodophyceae, Myxophyceae, Xanthophyceae, Bacillariophyceae (diatoms) and Euglenophyceae. Most preferably, plants that are not used for food or feed purposes are such as Arabidopsis thaliana or preferably cultivated tobacco (Nicotiana tabacum), or most preferably Nicotiana benthamiana. Alternatively, plants used for food or feed purposes may also be used, such as plants of the Leguminosae family, such as peas, alfalfa and soybeans; plants of the Poaceae family, such as rice, maize, wheat, barley, sorghum, millet, rye, triticale or oats; the Umbelliferae family, in particular the genus Daucus, very particularly the species carota (carrot), and the genus Apium, very particularly the species Graveolens dulce (celery) and many other species;The family Solanaceae, in particular the genus Lycopersicon, very particularly the species esculentum (tomato), and the genus Solanum, very particularly the species tuberosum (potato) and the species melongena (eggplant), and the genus Capsicum, very particularly the species annuum (pepper) and many other species; the family Leguminosae, in particular the genus Glycine, very particularly the species Glycinemax (soybean), alfalfa, pea, lucerne, bean or peanut and many other species; and the family Cruciferae (Brassicacae), in particular the genus Brassica, very particularly the species Brassica napus (oil seed rape), Brassica rapa (Brassica napus) and Brassica rapa (Brassica rapa). campestris)(beet), Brassica oleracea cv Tastie(cabbage), Brassica oleracea cv Snowball Y(cauliflower) and Brassica oleracea cv Emperor(broccoli); and Arabidopsis, very particularly the species Arabidopsis thaliana and many other species; the family Compositae, in particular the genus Lactuca, very particularly the species Lactuca oleracea. sativa) (lettuce) and many other species; Asteraceae, such as sunflower, Tagetes, lettuce or Calendula and many other species; Cucurbitaceae, such as melon, pumpkin (pumpkin / squash) or zucchini and linseed. More preferred are cotton, sugar cane, hemp, flax, chillies and various tree, nut and vine species. ;

[0079] According to further features in the described preferred embodiments, the plant is subjected to stress conditions. The stress conditions are selected from drought, salinity, wounding, cold and spraying stress-inducing compounds and / or compounds known in the art that increase endogenous ascorbate (vitamin C) levels. Since it has long been known that both P4H and LH3 enzymes are oxidatively inactivated during catalysis and require the cofactor ascorbate (vitamin C) to reactivate the enzymes by reducing the iron center from Fe(III) to Fe(II), it may be beneficial to manage vitamin C through biofortification

[21] .

[0080] According to another aspect of the present invention, a method for transiently expressing collagen α chain or an isolated plant cell is provided, wherein the isolated plant cell is capable of accumulating collagen α chain having a hydroxylation pattern identical to that produced when collagen α chain is expressed in human cells.

[0081] The term "transient expression" or "transient gene expression" used herein refers to the transient expression of a gene expressed in a short time after nucleic acid (most commonly the plasmid DNA encoding an expression cassette) is introduced into a eukaryotic cell. Transient expression should result in the limited-time use of the nucleic acid transferred, because any long-term expression is referred to as "stable expression". The transient expression used in plant cells or plants according to the present invention makes it possible to produce a high yield compatible with commercial use. In the case of transient expression, the harvest of plant biomass occurs during the peak expression of the recombinant protein, such as usually 5 days to 9 days after transfection. The transient expression of the above-mentioned recombinant fusion protein in the Nicotiana benthamiana plant can advantageously enable a high-throughput platform to produce compounds on an industrial scale and / or at low cost. However, embodiments of the present invention are not necessarily limited thereto, for example, any other suitable expression host can be used equally, including but not limited to bacteria, yeast, insects, mammals, or other plant expression systems.

[0082] The term "fluorescent protein" is a protein commonly used in genetic engineering techniques, used as a reporter gene for the expression of exogenous polynucleotides. When the protein is exposed to ultraviolet or blue light, it fluoresces and emits bright visible light. The protein that emits green light is green fluorescent protein (GFP), and the protein that emits red light is red fluorescent protein (RFP).

[0083] The term "gene" as used herein refers to a polynucleotide encoding a specific protein and may refer to the coding region alone or may include regulatory sequences preceding the coding sequence (5' non-coding sequences) and following the coding sequence (3' non-coding sequences).

[0084] The term "host cell" is a cell programmed to express an introduced exogenous polynucleotide, preferably, the host cell is a chloroplast subcellular compartment of N. benthamiana.

[0085] As used herein, the term "non-naturally occurring" refers to collagen, spider silk protein or silk fibroin that does not normally occur in nature. Non-naturally occurring collagen, spider silk protein and silk fibroin portions are recombinantly prepared. Non-naturally occurring collagen, spider silk protein or silk fibroin is recombinant collagen, recombinant spider silk protein or recombinant silk fibroin.

[0086] The term "signal peptide" refers to an amino acid sequence that recruits the cellular machinery of the host cell to transport the expressed protein to a specific location or organelle of the host cell. Preferably, the target peptide sequence is located at the C-terminus of the amino acid structure of the protein. Preferably, the signal peptide is a transit peptide that functions to target the chloroplast.

[0087] According to another aspect of the present invention, after extraction from a plant or plant cell expressing the fusion protein, the resulting spider silk protein / collagen or silk core protein / collagen fusion protein is purified. In order to facilitate its purification, the fusion protein can be fused with a tag (His6, GST, MBP, FLAG, etc.), and the tag is preferably located at the N or C terminal position of the mature protein.

[0088] The usual methods of plant growth and methods of introducing expression vectors into plant tissues can be used by those skilled in the art. The methods are varied and depend on the selected plant. Typically, the method includes a first step of culturing (aeroponic or hydroponic, preferably free-floating) plants under LED lighting. After the first step, especially after five weeks of hydroponic culture on free floaters, the plants are infiltrated with Agrobacterium under vacuum, wherein the Agrobacterium contains a DNA fragment encoding the above-mentioned spider silk protein / collagen or silk core protein / collagen fusion protein. The step of Agrobacterium infiltration can be performed by any vacuuming method. Preferably, in the method used according to the present invention, it is performed under vacuum by the Venturi effect. Agrobacterium refers to a soil-borne Gram-negative rod-shaped plant pathogen that can cause crown gall. The term "Agrobacterium" includes, but is not limited to, Agrobacterium tumefaciens strains (usually causing crown gall in infected plants) and Agrobacterium rhizogenes strains (causing hairy root disease in infected host plants). Infection of plant cells with Agrobacterium usually results in the production of opines (e.g., nopaline, agropine, octopine, etc.) by the infected cells. Therefore, Agrobacterium strains that result in the production of nopaline (e.g., strains LBA4301, C58, A208) are referred to as "nopaline-type" Agrobacterium; Agrobacterium strains that result in the production of octopine (e.g., strains LBA4404, Ach5, B6) are referred to as "octopine-type" Agrobacterium; Agrobacterium strains that result in the production of agropine (e.g., strains EHA105, EHA101, A281) are referred to as "agropine-type" Agrobacterium.

[0089] After agroinfiltration, the plants are typically further cultured for 5 to 9 days. Finally, the protein is extracted and purified using industry standard methods known in the art.

[0090] The term "expression vector" or "vector" as used herein refers to a nucleic acid assembly capable of directing transient expression of an exogenous gene. An expression vector may include a promoter operably linked to an exogenous gene, a restriction endonuclease site, a nucleic acid encoding one or more selection markers, and other nucleic acids useful in the practice of recombinant technology. Preferably, the expression vector used in step a) comprises: a prokaryotic DNA element encoding an origin of bacterial replication and an antibiotic resistance gene; at least one heterologous nucleotide sequence encoding the above-mentioned fusion protein according to the present invention is operably linked to a strong promoter, preferably a 35S promoter; an expression cassette for expressing a silencing suppressor, preferably p19; and DNA elements for controlling the processing of transcripts, such as termination / polyadenylation sequences, preferably Tnos sequences. Many plant functional expression promoters and enhancers that may be tissue-specific, developmentally specific, constitutive or inducible can be used in conjunction with the construct of the present invention. The phrase "plant promoter" or "promoter" as used herein in the specification and claims section includes promoters that can direct gene expression in plant cells (including organelles containing DNA, more specifically protoplasts). Such promoters may be derived from plants, bacteria, viruses, fungi or animal sources. Such promoters may be constitutive (i.e., capable of directing high levels of gene expression in multiple plant tissues), tissue-specific (i.e., capable of directing gene expression in one or more specific plant tissues), inducible (i.e., capable of directing gene expression under stimulation) or chimeric (i.e., formed by parts of at least two different promoters). The plant promoter used may be a constitutive promoter, a tissue-specific promoter, an inducible promoter or a chimeric promoter. Examples of constitutive plant promoters include, but are not limited to, CaMV35S and CaMV19S promoters, FMV34S promoters, sugarcane bacilliform badnavirus promoters, CsVMV promoters, Arabidopsis ACT2 / ACT8 actin promoters, Arabidopsis ubiquitin UBQ1 promoters, barley leaf-thionine BTH6 promoters and rice actin promoters. Examples of tissue-specific promoters include, but are not limited to, the bean phaseol storage protein promoter, the DLEC promoter, the PHS promoter, the zein storage protein promoter, the lupin gamma promoter from soybean, the AT2S1 gene promoter, the ACT11 actin promoter from Arabidopsis thaliana, the napA promoter from Brassica napus, and the potato patatin gene promoter.

[0091] It should be understood that constructs comprising two expressible inserts (e.g., spider silk protein chains and type I collagen αI and / or αII chains, or P4H and LH3 sequences) preferably include a single promoter for each insert, or alternatively, these constructs can express a single transcript chimera, including two inserted sequences from a single promoter. In this case, the chimeric transcript includes a "self-cleaving" 2A sequence between the two inserted sequences (e.g., a 2A sequence for expressing two proteins from a single promoter in the expression construct), so that the downstream insert can be translated from it. Preferably, T2A is used, encoding (GSG)EGRGSLLTCEDVEENPGP, and a (GSG) residue can be added to the 5' end of the peptide to improve cleavage efficiency. Other 2A sequences such as, but not limited to:

[0092] ·(GSG)ATNFSLLKQAGDVEENPGP

[0093] (GSG)QCTNYALLKLAGDVESNPGP

[0094] (GSG)VKQTLNFDLLKLAGDVESNPGP

[0095] This use of 2A sequences can circumvent the commonly known limitations of internal ribosome entry site (IRES) sequences. These elements are quite large (500-600 bp) and may take up valuable space in viral transfer vectors (which have limited packaging capacity). In addition, it may not be feasible to express more than two genes at a time using IRES elements. In addition, scientists have reported lower expression of downstream cistrons due to factors such as the experimental cell type and the specific gene cloned into the vector

[22] .

[0096] Collagen and silk are widely used in biomedicine, regenerative medicine, food and cosmetics industries. Therefore, although plant-expressed collagen and silk fiber components and modifying enzymes are useful in industrial synthesis of collagen and silk, complete collagen and / or silk production in plants is preferred for its simplicity and cost-effectiveness.

[0097] The present invention successfully addresses the shortcomings of currently known collagen configurations by providing plants capable of expressing correctly hydroxylated spider silk protein / collagen or silk fibroin / collagen fusion proteins having improved properties (e.g., thermal stability, Young's modulus, cell adhesion, etc.) compared to native human collagen. The resulting spider silk protein / collagen or silk fibroin / collagen fusion proteins thus obtained can be used in biomedical applications, cosmetics, aesthetics, but are not limited thereto.

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, this patent specification (including definitions) shall prevail. In addition, materials, methods and examples are illustrative only and are not intended to be limiting. Other objects, advantages and novel features of the present invention will become apparent to those of ordinary skill in the art upon examination of the following examples, which are not intended to limit the present invention. In addition, each of the various embodiments and aspects of the present invention as described above and as described in the claims section below finds experimental support in the following examples. Example

[0099] Large quantities of highly purified biochemically modified active recombinant photolyase fusion proteins in Nicotiana benthamiana plants can be obtained. To facilitate rapid and simple purification of the recombinant photolyase fusion protein, a 6xHis tag (also known as a polyhistidine tag, His6 tag, and / or hexahistidine tag) can be attached to the N-terminus of the protein. We have shown that the recombinant fusion protein is biologically active and exhibits improved functionality compared to native heterotrimeric type I collagen.

[0100] Nicotiana benthamiana is a bioreactor particularly suited for transient expression of recombinant proteins in a production environment. This small ornamental plant has a high leaf-to-stem ratio and is highly productive in hydroponics. Nicotiana benthamiana tolerates transfection vectors and provides maximum heterologous protein synthesis within 5-7 days after transfection. Scale-up of this bioreactor allows for the cultivation of more plants rather than reengineering the process.

[0101] Plants possess all the eukaryotic cell machinery to accurately produce human and animal proteins. Therefore, the bioreactor is a single plant. Plants are well suited to express complex proteins such as monoclonal antibodies and minimize risk by not supporting the growth of human or animal pathogens.

[0102] For all agroinfiltration experiments discussed below, 5-week-old Nicotiana benthamiana plants were used.

[0103] N. benthamiana seeds were grown in a greenhouse. Emergence and germination of N. benthamiana plants were performed under LED lighting with a 16 / 8 hour light / dark cycle, 7 days / week. Red and blue diodes (25% blue and 75% red) were selected to match the action spectrum of photosynthesis. Other wavelengths are not productive. The LEDs are focused on the plants. Plants grow to usable maturity 20% faster in this system compared to other commercial solutions. All seeds were germinated at 26.6°C using rock wool growth medium using fluctuation hydroponics well known in the art.

[0104] Example 1: Spider silk protein I / type I collagen fusion protein

[0105] As described above, two separate vectors were constructed. One vector carried the spidroin 1 / type I collagen construct and the other carried the P4H / LH3 construct. Both vectors were designed to target subcellular compartments known to have no endogenous P4H or LH activity.

[0106] The inventors constructed a basic set of Golden Gate cloning-compatible modular vectors comprising an expression cassette and an acceptor backbone (referred to as GGC-TC1 and GGC-TC2). The acceptor backbone is a binary T-DNA vector suitable for transient expression in plants and is designed to hold a geminiviral replicon system capable of producing a circular DNA replicon for high-level polyprotein expression.

[0107] 1) Spider silk protein I / type I collagen tricistronic construct:

[0108] For the biosynthesis of the genes of interest: spider silk protein 1 (Uniprot: P19837, entry version N°69, 2017-03-15), type I collagen alpha-I without signal peptide (1-22) and without N-terminal propeptide (23-161) and C-terminal propeptide (1219-1464) (Uniprot: P02452, entry version N°22, 2017-05-10), without signal peptide (1-22) and without N-terminal propeptide (23-79) and C-terminal propeptide ( 1120-1366) (Uniprot: P08123, entry version N°202, 2017-05-10), the A2 sequence GSGEGRGSLLTCEDVEENPG inserted between spidroin I, collagen type I α-I and collagen type I α-II chains, a chloroplast transit peptide inserted at the N-terminus (included in Uniprot entry G5DBJ0, amino acids 1-40, entry version N°10, 2017-11-22) and a 6x HIS tag were used as template.

[0109] 2) P4Hα-β chimeric / LH3 tricistronic vector construct:

[0110] For the biosynthesis of the gene of interest: a chimeric P4H enzyme comprising: an α subunit (Uniprot: Q86KR9, entry version N°81, 2017-06-07) and a β subunit (Uniprot: P05307, entry version N°141, 2017-05-10) sequence without its native signal peptide (1-20), an A2 sequence GSGEGRGSLLTCEDVEENPGP inserted between the α subunit and the β subunit; ... a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme The LH3 sequence (Uniprot: O60568, entry version N°165, 2017-09-27) with the signal peptide (1-24), the A2 sequence GSGEGRGSLLTCEDVEENPGP inserted between the LH3 and PH4β subunit sequences, and the chloroplast transit peptide located at the N-terminus (included in Uniprot entry G5DBJ0, amino acids 1-40, entry version N°10, 2017-11-22) were used as templates.

[0111] Using the Golden Gate cloning method, a T2A-ligated tricistronic vector in which three transgenes encoding I) spider silk protein I and II) type I collagen (called FIB1COL1) were placed in combination along the expression cassette in a binary vector. The P4Hα-β chimeric / LH3 construct also contained three genes (e.g., P4Hα subunit, P4Hβ subunit, and LH3, called P4HLH3) and were placed in combination along the expression cassette.

[0112] So-called “level 0” modular vectors containing the various parts of the expression cassette, such as the promoter (Pro), T2A signal (T2A), coding sequence (CDS), and terminator (Ter), were constructed using the assembly protocol described in the Golden Gate modular cloning system (Weber et al., 2011)

[23] . All level 0 modules were flanked by inward BsaI restriction enzyme sites and fusion sites (5 bp overhangs) to allow directional linear assembly in the orientations Pro-CDS1-T2A-CDS2-T2A-CDS3-Ter (FIB3COL1) and Pro-CDS1-T2A-CDS2-T2A-CDS3-Ter (P4HLH3), thereby generating T2A-linked tricistronic constructs. To construct the Pro, T2A and Ter modules, the sequences of the CmYLCV promoter and AtHSP 3'UTR were retrieved from the following publications in the prior art: Stavolone et al., (2003)

[24] , Nagaya et al., (2010)

[25] and Liu et al., (2017)

[26] , respectively.

[0113] Since the above-mentioned gene uses rare codons in tandem and may reduce the efficiency of translation or even break away from the translation mechanism, the codon usage preference in Nicotiana benthamiana was used by increasing the codon adoption index (CAI) from 0.70 to 0.91. GC content and unfavorable peaks have been optimized to extend the half-life of mRNA. The stem-loop structure that affects the stability of ribosome binding and mRNA is destroyed. In addition, negative cis-acting sites were screened and successfully modified. Pro, T2A, CDS and Ter modules are included in pLUG-Prime vector (iNtRON Biotechnology). Primers carrying inward BsaI site and fusion site were used to prepare codon-optimized CDS modules by PCR.

[0114] A "level 1" receptor backbone was constructed based on a modified pLSLR vector (Baltes et al., 2014)

[27] (Addgene plasmid #51493). First, the CaMV 35S promoter flanked by the UBI1 intron was inserted into BamHI and HindIII digested pLSLR with a pCAMBIA1300 backbone, and the existing bidirectional cis-acting replication elements "LIR-SIR-LIR" (LIR = long intergenic region, SIR = short intergenic region) were arranged in a "SIR-LIR-SIR" structure. The benefit of this structure (composed of sequences between the origins within the repeated SIR) and delivery mechanism is that the population of replicating viral genomes is homogeneous and predictable. The resulting vectors were named pLSLR-35SSPID1COL1 and pLSLR-35SP4HLH3, respectively. The fragment for Golden Gate cloning flanked by two BsaI sites (5′-CTATGGAGACCGAGGTCTCGTAAG-3′) was then inserted into pLSLR-35SSPID1COL1 and pLSLR-35SP4HLH3. Cloning into PpuMI and BspHI digested pLSLR-35SSPID1COL1 and pLSLR-35SP4HLH3 formed GGC-TC1 and GGC-TC2, respectively. (For the physical map of the resulting vector, see Figure 1 )

[0115] In order to construct the tricistronic (GGC-BC1 and GGC-TC1) vector of T2A connection in the direction of Pro-CDS1-T2A-CDS2-T2A-CDS3-Ter, the level 0 module was directed to be assembled into the level 1 receptor backbone using a single digestion connection procedure. The level 0 module and the level 1 receptor were mixed with BsaI (BsaI-HFv2, New England Biolabs) and T4 ligase (ThermoFisher) in an equimolar ratio. The reaction was carried out for 10 cycles, 5 minutes at 37°C and 10 minutes at 16°C, then 5 minutes at 50°C and 5 minutes at 80°C. The assembled level 1 construct was amplified in Escherichia coli DH5α, and the subsequent plasmid recovery, restriction digestion and sequencing procedures confirmed the correct vector assembly. The resulting tricistronic vectors were transformed into agrobacterium tumefaciens EHA105 and octopine agrobacterium tumefaciens LBA4404 by electroporation for Agrobacterium infiltration experiments. The transformed cells were plated on LB agar medium containing 50 mg / ml ampicillin (Sigma-Aldrich). (See the schematic diagram of "Level 0", "Receptor Skeleton" and "Level 1 Construct" for details.) Figure 2 )

[0116] Agrobacterium infiltration is used for transient expression in the Nicotiana benthamiana with Agrobacterium tumefaciens. The strain is as previously described. The frozen cell liquid of the transformed Agrobacterium of 100ml is inoculated in 5ml LB culture fluid (Thermo Fisher), and supplemented with 50μg / ml rifampicin and 50μg / ml kanamycin. Overnight, the culture is incubated at 28 ℃, with 220rpm vibration. 500ml is used to inoculate the LB culture medium of 50ml. The culture cells are incubated at 28 ℃ with 220rpm vibration, until the culture reaches OD600=0.6. By centrifuging and collecting cells at 6000rpm, and resuspended in 50ml MES buffer (10mM MES; pH5.5, 10mM MgC). This mixture is incubated at room temperature for 2.5 hours with 120mM acetosyringone, and added to the Agrobacterium suspension in the infiltration buffer (1x MS, 10mM MES, 2.5% glucose). For the influence of monosaccharide on the induction of virulence gene, different 2% is added into the Agrobacterium suspension in the infiltration buffer (1x MS, 10mM MES, 200mM acetosyringone).By immersing the tissue of being exposed to air in the Agrobacterium suspension and applying 50mbar-400mbar vacuum 45 seconds of the Nicotiana benthamiana plant, the Nicotiana benthamiana plants of 5 weeks age were infiltrated in the vacuum chamber.In case vacuum is broken, the Nicotiana benthamiana plant infiltrated is taken out from the vacuum chamber, thoroughly rinsed in water, and grown 5 days-7 days under the same growth conditions of growth before infiltration.In order to avoid any variable, each experiment has carried out Agrobacterium infiltration to the blade and the blade position, the blade of each strain plant of similar age of equal size.

[0117] Example 2: Silk fibroin III / type I collagen fusion protein

[0118] As described above, two separate vectors were constructed. One vector carried the fibroin III / type I collagen construct and the other carried the P4H / LH3 construct. Both vectors were designed to target subcellular compartments known to have no endogenous P4H or LH activity.

[0119] The inventors constructed a basic set of Golden Gate cloning compatible modular vectors comprising an expression cassette and an acceptor backbone (referred to as GGC-TC3 and GGC-TC2). The acceptor backbone is a binary T-DNA vector suitable for transient expression in plants and is designed to hold a geminivirus replicon system capable of producing a circular DNA replicon for high-level polyprotein expression.

[0120] 1) Silk fibroin III / type I collagen tricistronic construct:

[0121] For the biosynthesis of the genes of interest: fibroin 1 (Uniprot: Q16987, entry version N°44, 2017-08-30), type I collagen alpha-I (Uniprot: P02452, entry version N°22, 2017-05-10) without signal peptide (1-22) and without N-terminal propeptide (23-161) and C-terminal propeptide (1219-1464), without signal peptide (1-22) and without N-terminal propeptide (23-79) and C-terminal propeptide (1 120-1366) of type I collagen α-II (Uniprot: P08123, entry version N°202, 2017-05-10), the A2 sequence GSGEGRGSLLTCEDVEENPGP interposed between the fibroin III, type I collagen α-I and type I collagen α-II chains, a chloroplast transit peptide located at the N-terminus (included in Uniprot entry G5DBJ0, amino acids 1-40, entry version N°10, 2017-11-22), and a 6x HIS tag were used as template.

[0122] 2) P4Hα-β chimeric / LH3 tricistronic vector construct:

[0123] For the biosynthesis of the gene of interest: a chimeric P4H enzyme comprising: an α subunit (Uniprot: Q86KR9, entry version N°81, 2017-06-07) and a β subunit (Uniprot: P05307, entry version N°141, 2017-05-10) sequence without its native signal peptide (1-20), an A2 sequence GSGEGRGSLLTCEDVEENPGP inserted between the α subunit and the β subunit; ... a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme comprising: a chimeric P4H enzyme The LH3 sequence (Uniprot: O60568, entry version N°165, 2017-09-27) with the signal peptide (1-24), the A2 sequence GSGEGRGSLLTCEDVEENPGP inserted between the LH3 and PH4β subunit sequences, and the chloroplast transit peptide located at the N-terminus (included in Uniprot entry G5DBJ0, amino acids 1-40, entry version N°10, 2017-11-22) were used as templates.

[0124] Using the Golden Gate cloning method, a T2A-ligated tricistronic vector in which three transgenes encoding I) fibroin III and II) type I collagen (called FIB3COL1) were placed in combination along the expression cassette in a binary vector. The P4Hα-β chimeric / LH3 construct also contained three genes (e.g., P4Hα subunit, P4Hβ subunit, and LH3, called P4HLH3) and were placed in combination along the expression cassette.

[0125] Using the assembly protocol described in the Golden Gate modular cloning system (Weber et al., 2011)

[23] , a so-called “level 0” modular vector containing the various parts of the expression cassette (such as the promoter (Pro), T2A signal (T2A), coding sequence (CDS) and terminator (Ter)) was constructed.

[0126] All level 0 modules were flanked by inward BsaI restriction enzyme sites and fusion sites (5 bp overhangs) to allow directional linear assembly in the orientations Pro-CDS1-T2A-CDS2-T2A-CDS3-Ter (FIB3COL1) and Pro-CDS1-T2A-CDS2-T2A-CDS3-Ter (P4HLH3), resulting in T2A-linked tricistronic constructs. To construct the Pro, T2A, and Ter modules, the sequences of the CmYLCV promoter and AtHSP 3'UTR were retrieved from the following prior art publications: Stavolone et al., (2003)

[24] , Nagaya et al., (2010)

[25] , and Liu et al., (2017)

[26] , respectively.

[0127] Since the above-mentioned gene uses rare codons in tandem and may reduce the efficiency of translation or even break away from the translation mechanism, the codon usage preference in Nicotiana benthamiana was used by increasing the codon adoption index (CAI) from 0.70 to 0.91. GC content and unfavorable peaks have been optimized to extend the half-life of mRNA. The stem-loop structure that affects the stability of ribosome binding and mRNA is destroyed. In addition, negative cis-acting sites were screened and successfully modified. Pro, T2A, CDS and Ter modules are included in pLUG-Prime vector (iNtRON Biotechnology). Primers carrying inward BsaI site and fusion site were used to prepare codon-optimized CDS modules by PCR.

[0128] A "level 1" receptor backbone was constructed based on a modified pLSLR vector (Baltes et al., 2014)

[27] (Addgene plasmid #51493). First, the CaMV 35S promoter flanked by the UBI1 intron was inserted into BamHI and HindIII digested pLSLR with a pCAMBIA1300 backbone, and the existing bidirectional cis-acting replication elements "LIR-SIR-LIR" (LIR = long intergenic region, SIR = short intergenic region) were arranged in a "SIR-LIR-SIR" structure. The benefit of this structure (composed of sequences between the origins within the repeated SIR) and delivery mechanism is that the population of replicating viral genomes is homogeneous and predictable. The resulting vectors were named pLSLR-35SSPID1COL1 and pLSLR-35SP4HLH3, respectively. The fragment for Golden Gate cloning flanked by two BsaI sites (5′-CTATGGAGACCGAGGTCTCGTAAG-3′) was then inserted into pLSLR-35SSPID1COL1 and pLSLR-35SP4HLH3. Cloning into PpuMI and BspHI digested pLSLR-35SFIB3COL1 and pLSLR-35SP4HLH3 formed GGC-TC3 and GGC-TC2, respectively.

[0129] In order to construct the tricistronic (GGC-BC3 and GGC-TC2) vector of T2A connection in the direction of Pro-CDS1-T2A-CDS2-T2A-CDS3-Ter, the level 0 module was directed to be assembled into the level 1 receptor backbone using a single digestion connection procedure. The level 0 module and the level 1 receptor were mixed with BsaI (BsaI-HFv2, New England Biolabs) and T4 ligase (ThermoFisher) in an equimolar ratio. The reaction was carried out for 10 cycles, 5 minutes at 37°C and 10 minutes at 16°C, then 5 minutes at 50°C and 5 minutes at 80°C. The assembled level 1 construct was amplified in Escherichia coli DH5α, and the subsequent plasmid recovery, restriction digestion and sequencing procedures confirmed the correct vector assembly. The obtained tricistronic vector was transformed into agrobacterium tumefaciens EHA105 and octopine agrobacterium tumefaciens LBA4404 by electroporation for agroinfiltration experiments. The transformed cells were plated on LB agar medium containing 50 mg / ml ampicillin (Sigma-Aldrich).

[0130] Agrobacterium infiltration is used for transient expression in the Nicotiana benthamiana with Agrobacterium tumefaciens. The strain is as previously described. The frozen cell liquid of the transformed Agrobacterium of 100ml is inoculated in 5ml LB culture fluid (Thermo Fisher), and supplemented with 50μg / ml rifampicin and 50μg / ml kanamycin. Overnight, the culture is incubated at 28 ℃, with 220rpm vibration. 500ml is used to inoculate the LB culture medium of 50ml. The culture cells are incubated at 28 ℃ with 220rpm vibration, until the culture reaches OD600=0.6. By centrifuging and collecting cells at 6000rpm, and resuspended in 50ml MES buffer (10mM MES; pH5.5, 10mM MgC). This mixture is incubated at room temperature for 2.5 hours with 120mM acetosyringone, and added to the Agrobacterium suspension in the infiltration buffer (1x MS, 10mM MES, 2.5% glucose). For the effect of monosaccharides on the induction of virulence genes, different 2% were added to the Agrobacterium suspension in infiltration buffer (1x MS, 10 mM MES, 200 mM acetosyringone). Five-week-old Nicotiana benthamiana plants were infiltrated in a vacuum chamber by immersing air-exposed tissue of Nicotiana benthamiana plants in the Agrobacterium suspension and applying 50-400 mbar vacuum for 45 seconds.

[0131] Once the vacuum was broken, the infiltrated N. benthamiana plants were removed from the vacuum chamber, rinsed thoroughly in water, and grown for 5-7 days under the same growth conditions used for growth prior to infiltration. To avoid any variability, leaves and locations on leaves, equal-sized leaves of each plant of similar age were agroinfiltrated for each experiment.

[0132] result

[0133] Extraction and purification of SPID1COL1 and FIB3COL1 heterotrimeric fusion proteins

[0134] To extract SPID1COL1 and FIB3COL1 proteins, infiltrated Nicotiana benthamiana leaves (300 g of each protein) were harvested and ground and mixed with 2.5 g of activated charcoal and cold (4°C) extraction buffer (100 mM Tris-HCl pH 8.0, 4 mM EDTA, 600 mM NaCl, 25 mM DL-dithiothreitol (DTT), 0.5% NP40, 2% poly(vinyl polypyrrolidone) (PVPP), 10% glycerol and 2x Roche EDTA-free complete protease inhibitor cocktail (Roche Diagnostics, Germany)) at a ratio of 2 ml per gram of leaf (fresh weight). During this protocol, the temperature was maintained below 12°C. The resulting crude extract was then filtered using Whatman No. 1 filter paper and the filtered extract was then centrifuged (15000 g, 30 minutes at 5°C). The resulting supernatant was then collected and, together with 1 g / L activated charcoal, CaCl2 was added to a final concentration of 10 mM. Insoluble contaminants were then further removed by centrifugation (20000 g, 30 min at 15°C).

[0135] SPID1COL1 and FIB3COL1 in the supernatant were recovered by precipitating by gradually adding crystalline NaCl to a final concentration of 2.85M (20 minutes, stirring constantly at room temperature). The solution was incubated in a cold room for 6 hours without stirring. The precipitates containing SPID1COL1 and FIB3COL1 were collected after centrifugation (22000g, 2 hours at 5°C). The precipitate was then resuspended in 200mL of a solution of 250mM acetic acid + 2M NaCl using a magnetic stirrer for 5 minutes, followed by centrifugation (22000g, 30 minutes at 5°C). The supernatant was then discarded and the precipitate was resuspended (1 hour at room temperature) in 200mL of 0.5M acetic acid. Insoluble matter was removed by centrifugation (15000g, 30 minutes at 15°C). The resulting supernatant was passed through 3 layers of Whatman No. 1 filter paper.

[0136] Then, the obtained SPID1COL1 and FIB3COL1 proteins were precipitated by slowly adding NaCl to a final concentration of 3M at room temperature and continuously stirring for 25 minutes. The solution was incubated in a cold room at 4°C for 8 hours, and SPID1COL1 and FIB3COL1 proteins were collected after centrifugation (22000g, 2.5 hours at 5°C). All remaining trace supernatants were removed. As described above, the precipitate was redissolved and the SPID1COL1 and FIB3COL1 precipitation steps were repeated in acetic acid and NaCl solutions, respectively. After incubation and collection of the precipitate containing SPID1COL1 and containing FIB3COL1, the sample was redissolved in 50mL of 10mM HCl by vigorous pipetting mixing and vortexing at room temperature for 5 minutes. The solution was transferred to a dialysis bag (Thermo Fisher, MWCO 25000Da) and dialyzed with 5L of 10mM HCl (3 hours at 4°C). Additional dialysis was performed. By using 30mL syringe to filter through 0.2μ filter, SPID1COL1 and FIB3COL1 protein are sterilized. Then use Vivaspin PES 6mL filter tube (Sartorius, MWCO 300000) to concentrate SPID1COL1 and FIB3COL1 protein, then load it into nickel-nitrilotriacetic acid (Ni-NTA) affinity resin (Amintra). In short, wash column with 10 column volumes of washing buffer (respectively 5mM and 20mM imidazoles, 20mM Tris-HCl, 50mM NaCl, pH7.4), and elute recombinant protein with elution buffer (250mM imidazoles, 20mMTris-HCl, 50mM NaCl, pH7.4). Analyze the SPID1COL1 and FIB3COL1 protein samples of purification by SDS-PAGE, Southern blot analysis, western blot analysis, and quantify by ELISA. Total soluble protein (TSP) in crude plant extracts was estimated using the Bradford assay (Bio-Rad) following the manufacturer's instructions.

[0137] Southern blot analysis

[0138] The genomic DNA of leaves infiltrated with Agrobacterium expressing SPID1COL1, FIB3COL1 and P4HLH3 was extracted by DNeasy plant DNA kit (Qiagen), digested with EcoRI / BglII, and subjected to Southern blot analysis. The results showed that after Agrobacterium infiltration, the synthesized SPID1COL1, FIB3COL1 and P4HLH3 open reading frames (ORFs) were successfully transformed into Nicotiana benthamiana leaves. Biotin Deca-labeled DNA labeling kit (ThermoScientific) and biotin colorimetric detection kit (ThermoScientific) were used to label and detect, respectively. The presence of amplified fragments with expected sizes indicated that the gene was successfully transformed into Nicotiana benthamiana leaves by Agrobacterium infiltration. Due to the partial digestion of some DNA of the sample, higher molecular weight fragments can be seen. The digested recombinant GGC-TC1, GGC-TC2 and GGC-TC3 vectors were used as positive controls and produced bands of the same size, while the uninfiltrated leaves were used as negative controls. (The results of Southern blotting of total DNA from infiltrated tobacco leaves using the SPID1COL1 probe, FIB3COL1 probe, and P4HLH3 probe after digestion with EcoRI and BglII are shown in Figure 3 , 4 and 5)

[0139] Detection of chimeric genes by RT-PCR

[0140] Reverse transcription polymerase chain reaction (RT-PCR) was used to confirm the transcription of the corresponding spidroin I, type I collagen αI, type I collagen αII, fibroin 3, P4Hα subunit, P4Hβ subunit and LH3 genes. RT-PCR analysis was performed on RNA samples extracted from infiltrated Nicotiana benthamiana leaves using specific primers for each gene to amplify the core region of each gene. Total RNA was extracted using the Illustra RNAspin Mini Kit (GE healthcare). Oligonucleotide pairs in the core region were designed to detect the presence of the corresponding genes in the core region:

[0141] For spidroin I, TE-F: 5′-GGAGGACAAGGAGCTGGAG-3′ and TE-R: 5′-CTAGAAGCAGCAGCAGAAGC-3′;

[0142] for type I collagen α-I, TE-F: 5′-ACCTATGGGACCTCCTGGAT-3′ and TE-R: 5′-GCAGGTCCAGTTTCTCCTCT-3′;

[0143] for type I collagen αII, TE-F: 5′-AGAACCTGGATCTGCTGGAC-3′ and TE-R: 5′-CCAGGAGGTCCCATTACTCC-3′;

[0144] for the P4Hα subunit, TE-F: 5′-GCTGGAATGAATAAAGGAACTGA-3′ and TE-R: 5′-ATCTTCCTCCATTTAAATATACAGCTA-3′;

[0145] For P4Hβ subunit, TE-F: 5′-TCCTGCTTCTGCTGATAGAACT-3′, TE-R: 5′-TCAGGTTCTTCAGCTTCTTCT-3′;

[0146] For LH3, TE-F: 5'-TGTAGTACATGGAAATGGACCT-3', TE-R: 5'-GGAGGAGGTTGTCCTCCAG-3';

[0147] For fibroin III, TE-F: 5′-CTGCTGCTGGAGGATATGGA-3′ and TE-R: 5′-TCCTCCAGGTCCTTGTTGTC-3′.

[0148] According to the manufacturer's instructions, use III and One-step RT-PCR was performed using Taq DNA polymerase. These reactions produced the expected bp fragments of the core regions of the genes: spidroin I (228 bp fragment), collagen α-I (431 bp fragment), collagen α-II (386 bp fragment), P4H α subunit (214 bp fragment), P4H β subunit (226 bp fragment), LH3 (278 bp fragment), and fibroin III (239 bp fragment). The fragments amplified by RT-PCR indicated that all infiltrated leaves clearly showed transcription of the corresponding genes on days 3, 5, 7, and 10, as shown in Figure 2 . Figure 6 and Figure 7 as shown, while non-infiltrated leaves showed negative results.

[0149] Western blot analysis

[0150] Western blot was performed to confirm the production of chimeric SPID1COL1 and FIB3COL1 proteins in the tissues of plants. 500 mg of leaves were ground in 0.5 mL 50 mM Tris-HCl (pH 7.5) rich in 1 x Roche complete protease inhibitor mixture (Roche Diagnostics, Germany) without EDTA, and total soluble proteins were extracted from the infiltrated plants. The crude extract was boiled for 5 minutes in 300 μL of 4X SDS sample loading buffer (Sigma Aldrich: Tris-HCl: 0.2 M, DTT: 0.4 M, SDS: 277 mM, 8.0% (w / v), bromophenol blue: 6 mM, glycerol: 4.3 M), and centrifuged (12000 rpm, 7 minutes at room temperature). Supernatant samples (25 μL) were separated on 10% polyacrylamide gels (NuPAGE BIS-TRIS gels, Thermo Fisher) and immunodetected for proteins of interest using standard western blotting procedures. Spider silk protein 1, type I collagen α-I chain, type I collagen α-II chain, P4H α subunit, P4H β subunit, LK3, and fibroin III were detected using custom designed anti-Dictyostelium discoideum (slime mold) P4H α subunit antibody, custom designed anti-bovine P4H β subunit antibody, anti-rabbit LH antibody (LSBio), anti-rabbit MASP (MASP1) polyclonal antibody (LSBio), anti-type I collagen antibody (OriGene Technologies), and custom designed anti-rabbit fibroin III polyclonal antibody. Broad range prestained protein molecular weight markers were purchased from Thermo Fisher (PageRuler prestained protein molecular weight ladder, 30 kDa to 240 kDa). As expected, no reaction was shown with non-infiltrated plants used as negative control. (For comparison of Western blot results at 1, 3, 5, 7, 9 and 10 days after infiltration and at top, middle and bottom of leaf positions, see Figure 8 )

[0151] Thermal stability

[0152] To evaluate the thermal stability of SPID1COL1 and FIB3COL1 proteins, their sensitivity to pepsin or a trypsin / chymotrypsin mixture was determined according to the method of P. Bruckner (1981)

[28] . Using temperatures between 32°C and 42°C, the study showed that purified SPID1COL1 and FIB3COL1 were resistant to pepsin up to 39.4°C and 39.8°C (50% degradation point measured by scanning the SPID1COL1 and FIB3COL1 bands after PAGE) ( Fig. 9). In order to obtain more accurate data on the thermal stability of the resulting SPID1COL1 and FIB3COL1 proteins, circular dichroism (CD) spectroscopy was performed. CD measurements of 45 μg / mL SPID1COL1 or 45 μg / mL FIB3COL1 prepared in 10 mM HCl were performed using a Jasco J-810 circular dichroism spectrophotometer (Jasco) in a 10 mm pathlength UV fused quartz cuvette (CV10Q7A, Thorlabs). 1 mL of sample was filled into the cuvette for each measurement. CD spectra were obtained at room temperature by continuous wavelength scanning from 200 nm to 270 nm at a scanning speed of 50 nm per minute. The average of three scans for each sample was calculated. The spectrum is a typical triple helical conformation, which is consistent with the earlier work established by F. Ruggiero (2000)

[29] (data not shown). The thermal transition curves of SPID1COL1 and FIB3COL1 proteins measured by circular dichroism at 225 nm showed that T m The values ​​are 41.6°C and 43.4°C, respectively, at which 50% of the SPID1COL1 and FIB3COL1 molecules remain in a fully folded conformation. In contrast, the T values ​​of bovine heterotrimeric type I collagen shown in the prior art are m The value is 40℃( Fig.10 ). The gradual decrease in the number of SPID1COL1 and FIB3COL1 detected is due to the fact that the degree of hydroxylation can vary from one SPID1COL1 or FIB3COL1 molecule to another, thus generating populations of triple helices with different denaturation temperatures. These results show that co-expression of chimeric P4H and LH3 enzymes with SPID1COL1 and FIB3COL1 proteins proved to be crucial for conformation and stability.

[0153] Structural analysis

[0154] To visualize the fibrillar lattice network of SPID1COL1 and FIB3COL1, the resulting fusion proteins were allowed to assemble into fibrils, collected and analyzed by scanning electron microscopy (SEM) (see Fig.11). To prepare samples for SEM, fibril formation of SPID1COL1 and FIB3COL1 proteins was induced by mixing with 5 μL of fibril formation buffer (60 mM NaH2PO4, 1.4% NaCl (w / v), pH 9.5) and incubating at 37°C for 1 hour. SPID1COL1 and FIB3COL1 samples were then immersed in 0.1 M phosphate buffer (pH 7.3) and 2.5% glutaraldehyde (4°C), and the samples were then washed three times in phosphate buffer and gradually dehydrated by adding increasing concentrations of ethanol (25%-100%). The samples were washed again for 25 minutes and finally dried in a critical point dryer (Leica EM CPD300). The resulting samples were gold-coated (coating thickness 25 nm) using an EMACE600 sputter coater (Leica), and SEM images were obtained using a Camscan MX 2600FEGSEM using an accelerating voltage of 10 kV and a magnification of 500x. Long and homogeneous fibrils and lattice structures characteristic of spidroin I, fibroin III, and type I collagen were observed, indicating the correct structures of SPID1COL1 and FIB3COL1 proteins.

[0155] Biological Functionality

[0156] In culture, collagen-producing extracellular matrix proteins can bind to biological substrates and, at the same time, to cell surfaces, thereby promoting the attachment, expansion, and growth of these cells (Klebe, 1974; Pearlstein, 1976). To determine the biological functionality of the resulting SPID1COL1 and FIB3COL1 proteins, isolated endothelial cells derived from adult human umbilical vein (HUVEC) were seeded on antimicrobial plastic substrates pre-coated with SPID1COL1, FIB3COL1, or natural human skin type I collagen (GenoSkin). Human endothelial cells were isolated from normal term umbilical vein as described by Gimbrone et al. (1974)

[30] . The yields of endothelial progenitor cells (EPCs) obtained from SPID1COL1 and FIB3COL1 were 2-fold and 2.5-fold higher than those obtained with natural human tissue-derived type I collagen and several-fold higher than those obtained from uncoated substrates. Furthermore, SPID1COL1 and FIB3COL1 proteins were more effective in isolating cells from endothelial cell samples isolated from HUVECs containing very low or high endogenous levels of EPCs. Most of the cells isolated and grown on SPID1COL1 and FIB3COL1 proteins exhibited typical spherical cells, which was supported by strong interactions with the SPID1COL1 or FIB3COL1 protein matrix, while cells grown on native human skin type I collagen or uncoated substrates were mostly round. These results show that the bioactivity of SPID1COL1 and FIB3COL1 was proven to be superior to naive human tissue-derived collagen as they were able to support the attachment and proliferation of isolated endothelial cells from adult human umbilical vein. (See Fig.12 )

[0157] Amino acid composition analysis

[0158] To further verify the identity of the expressed SPID1COL1 and FIB3COL1 proteins at the amino acid composition level, the samples were digested with a thiol-specific protease (ficin) and further purified to simulate the migration of pure human skin type I collagen samples. After the purified SPID1COL1 protein was electrophoretically separated into spidroin I and type I collagen, and the FIB3COL1 protein was electrophoretically separated into fibroin III and type I collagen, the corresponding bands were subjected to protein sequence analysis using LCMS-8050 triple quadrupole LC-MS / MS (Shimadzu), and the bands were considered to correspond to spidroin I and fibroin III and type I collagen (data analyzed by Traverse MS data analysis software). The bands shown in the figure were identified as type I collagen αI (Homo sapiens; p = 1.08 × 10 -30 ), type I collagen αII (Homo sapiens; p = 1.24 × 10-14 ) and spider silk protein I (Nephila clavata; p = 1.48 × 10 -12 ). All identified peptides (sequence coverage of 80%) showed 100% identity with human collagen, spider silk protein I and fibroin III protein sequences, respectively. Amino acid analysis of the resulting SPID1COL1 and FIB3COL1 proteins showed significant identity with human extracted type I collagen heterotrimer levels [31, 32, 33] and spider silk protein I and fibroin III levels. In addition, the hydroxylysine content of SPID1COL1 and FIB3COL1 was 36- and 39-fold higher, respectively, than that detected in LH3-free Nicotiana benthamiana plants

[14] , confirming the heterologous activity of the chimeric P4H and LH3 proteins. The percentage of hydroxyproline content measured (8.24% for SPID1COL1 and 8.32% for FIB3COL1) is very similar to that reported for recombinant transgenic plant-derived collagen by Merle et al. (2002) (8.41%)

[14] and by Hanan Stein et al. (2009) (7.55%)

[34] , and the hydroxylysine content (0.86%) is similar to that of human collagen (1%), which is also consistent with the 0.74% found in the study by Hanan Stein et al. (2009)

[34] . (Amino acid analysis of SPID1COL1 and FIB3COL1 relative to human collagen heterotrimers is shown in Table 1).

[0159] Table 1

[0160]

[0161]

[0162] Example 3: SPID1COL1 and FIB3COL1 electrospun scaffolds

[0163] One of the main goals of tissue engineering is to make cell-compatible scaffolds and select (bio) materials that can interact with cells to ensure the physiological activity of the structure. There are a series of requirements for these materials, such as non-toxicity, low immunogenicity, well-defined biodegradation rate, etc. The structure of the scaffold should mimic the natural extracellular matrix structure as much as possible and exert its function to reconstruct natural conditions for cells. The inventors of the present invention studied three different scaffold structures made with SPID1COL1, FIB3COL1 or natural human type I collagen. Both spider-based spider silk proteins and silk core proteins have unique combinations of physicochemical and biological properties and can be used alone or in combination (e.g., SPID1COL1 and FIB3COL1) in different fields of tissue engineering. The main advantage of spider silk or silk fibroin compared to other cytocompatible materials such as collagen is their mechanical properties

[35] , which ensures the application of spider silk or silk fibroin as framework reinforcement components in various structures [36,37] and as composite additives for polymers (such as collagen) that are mechanically insufficient [38-40] or have weak mechanical properties under wet conditions

[41] .

[0164] The silk protein I from the spider Nephila clavata and the silk protein III from the spider Araneae spidrota (analogs of silk protein 2

[42] ) are characterized by the presence of a large number of repetitive sequences in the central part (so-called primary repeats of 25-40 amino acid residues in size) and unique sequences of 100-300 amino acid residues in the N and C domains. All the repetitive sequences contain poly-Ala (alanine) blocks of 4-8 amino acid residues, which alternate with Gly (glycine) repeats, with silk protein I alternating with GGX motifs and silk protein III (as well as silk protein II) alternating with GPGXX motifs

[43] . This alternation of hydrophobic and hydrophilic regions of the molecule ensures the amphipathic nature of the interaction with tissues. The presence of up to 15% of proline residues in the amino acid sequence of silk protein III, which is absent in silk protein I

[44] , has a significant influence on the further formation of the higher-order structure and determines various properties of these proteins. In addition, silk proteins I and silk protein III are characterized by the ability to undergo phase transitions during dehydration. This property ensures the structural stability of proteins in the structures they are based on.

[0165] The electrospinning method is one of the most promising methods for making scaffolds with defined structures. Electrospun scaffolds have a multilayer fiber structure with high porosity and high surface area / volume ratio (SA:V). Many different types of structures based on silk protein have been made using the electrospinning method [45,46]. It is well known in the art that electrospun collagen nanofibers are inherently mechanically weak and readily soluble in water [47,48,49]. Rapid degradation is not ideal for tissue engineering applications because the scaffold will disappear before the cells lay out their own ECM. Therefore, collagen fibers must be cross-linked to reduce water solubility, to increase resistance to enzymatic degradation and to enhance mechanical strength.

[0166] The present invention makes it possible to make cytocompatible scaffolds using SPID1COL1 or FIB3COL1 proteins, which combine mechanical properties and high cytocompatibility with modification potential compared to traditional animal-derived type I collagen. These properties allow to meet the requirements of tissue engineering. In addition, SPID1COL1 and FIB3COL1 are characterized by high strength and elastic modulus compared to traditional animal-derived type I collagen, which is necessary for accelerating regeneration potential and reducing surgical trauma. Therefore, in the course of this study, a comparative analysis of the structure, biological properties and regeneration potential of SPID1COL1 and FIB3COL1 electrospun scaffolds relative to commercial animal-derived type I collagen was performed, and new data on their structure and biological properties were obtained, highlighting the obvious performance superiority of SPID1COL1 and FIB3COL1.

[0167] Fabrication of SPID1COL1- and FIB3COL1-based scaffolds

[0168] Aqueous solutions of SPID1COL1, FIB3COL1, and bovine type I collagen (30% concentration) were dried in a critical point dryer (Leica EM CPD300) in a petri dish. The dried proteins were dissolved in a ternary mixture of phosphate buffered saline (PBS) / 1,1,1,3,3,3 hexafluoro-2-propanol (HFIP) / acetic acid (as solvent) at a ratio of 1:1:1 and a rate of 50 mg / mL. HFIP is a volatile solvent (boiling point of 61°C) that evaporates under normal atmospheric conditions to produce polymer fibers in a dry state

[50] . This approach has been successfully used to develop various scaffolds evaluated in in vitro and in vivo studies [51-55]. The resulting solution was centrifuged at 11500×g for 12 minutes, and each protein was then mixed separately at a volume ratio of 7:3 to a total protein concentration of 50 mg / mL. Microfibrous scaffolds were fabricated using an electrospinning method using an E-Fiber EF100 electrospinning device (SKE Research Equipment). The solution loaded into the CadenceScience tuberculin glass syringe (FisherScientific) was deposited onto a fixed collector surface (steel plate) under an electric field of 6.7kV-7kV by a standard 18G blunt needle. The solution feed rate was 0.125mL / h, and the needle-collector distance was 10cm. The support was dried in a critical point dryer (Leica EM CPD300) and then separated. In order to make a support for cell adhesion and proliferation research, the solution was deposited on a cover glass attached to the collector with similar parameters.

[0169] Morphology and characterization of electrospun nanofibers

[0170] The structures of SPID1COL1, FIB3COL1 and bovine type I collagen (ThermoFisher) scaffolds were analyzed using scanning electron microscopy (SEM). The SEM method was able to confirm the porous fiber structure of the resulting scaffolds, as well as to estimate the average thickness of their fiber compositions. In brief, the nanofibers were fixed in a mixture of 1.5% glutaraldehyde / 3% paraformaldehyde in 100mM sodium arsenate buffer (pH 7.4) containing 2.5% sucrose at room temperature for 40 minutes, and then fixed in 1% osmium tetroxide in 100mM sodium arsenate buffer (pH 7.4) at room temperature for 20 minutes. The corresponding samples were dehydrated with a graded ethanol series (50 / 75 / 85 / 95 / 100% in water) and then critically dried using a critical point dryer (Leica EM CPD300). Subsequently, the resulting samples were gold coated (coating thickness 10 nm) using an EM ACE600 sputter coater (Leica), and SEM images were obtained using a Camscan MX 2600FEGSEM. The average diameter of the electrospun fibers was analyzed from at least five different parts of the SEM images using Image J software, and the average diameter of SPID1COL1 was 630 nm ± 81 nm, the average diameter of FIB3COL1 was 564 nm ± 22 nm, and the average diameter of bovine type I collagen was 319 nm ± 29 nm (see Fig.11 ). It has been demonstrated that the fiber diameter and arrangement of SPID1COL1, FIB3COL1, and bovine type I collagen all affect NSC adhesion, proliferation, and differentiation, thereby eliminating the superiority of SPID1COL1 and FIB3COL1 compared to native bovine type I collagen (Table 2). These tests were performed on primary mouse neural stem cells. After sacrificing pregnant females, BALB / cA mouse embryos at embryonic day 13.5-14.5 (E13.5-E14.5) were isolated and placed in ice-cold Hank's Balanced Salt Solution (HBSS, Thermo Fisher) to extract neural stem cells. Spinal cords were retrieved from 1 to 2 liters of embryos at a time and washed in HBSS. After washing, the tissue was placed in NeuroCult-XF Proliferation Medium (StemCell Technologies) and mechanically dissociated by repeated gentle trituration with a wide-bore tip (Thermo Fisher). The suspension was placed in a 4% paraformaldehyde (4% dapoxetine) containing NeuroCult-XF Proliferation Medium (StemCell Technologies) and the relevant NeuroCult TMProliferation supplement (STEMCELL Technologies) and penicillin (100U) / streptomycin (125μg / mL; Thermo Fisher) in T75 TC treated Falcon cell culture flasks (Fudau). Cells were grown as free-floating clustered neurospheres at 37 ° C, 92% air and 8% CO2, and were passaged by mechanical dissociation every 5 days, and prevented from attaching every other day (gentle knocking of the culture flask). The proliferation dynamics of the culture were studied by microscopy (e.g., neurospheres were collected every 2 days and the total number of viable cells at each passage was assessed by trypan blue exclusion). For microscopy, dark dense spheres were considered unhealthy and consisted of more dead cells than light spheres, because surviving neurospheres were usually translucent. Initially, single cells proliferated to form small cell clusters that gently adhered to SPID1COL1, FIB3COL1 or bovine type I collagen scaffolds; however, as the density of the spheres increased, some of these clusters were lifted. Cells used for transplantation or in vitro differentiation were passaged 5 times. Table 2. Effect of fiber diameter and arrangement on adhesion, proliferation and differentiation. Cell counts were performed three days after cell seeding on the scaffolds.

[0171] Bracket Fiber diameter Adhesion (%) proliferation(%) Differentiation (% neurons) SPID1COL1 630nm±81nm 91 90 80 FIB3COL1 564nm±22nm 87 92 82 Natural bovine type I collagen 319nm±29nm 68 78 38

[0172] In vitro differentiation and immunocytochemistry

[0173] In vivo extracellular matrices such as collagen and laminin exhibit micro- to nanoscale fibrous morphology, which explains why electrospun matrices significantly affect stem cell adhesion, survival, proliferation, and differentiation. To gain insight into how SPID1COL1, FIB3COL1, or bovine type I collagen affect neural development, the above neurospheres were seeded on electrospun SPID1COL1, FIB3COL1, or native bovine type I collagen scaffolds to study their effects on adhesion and proliferation. Therefore, three days after the last passage, the above neurospheres were plated as pellets on poly-D-lysine (PDL, Sigma-Aldrich), laminin-coated coverslips (Thermo Fisher), or electrospun meshes in NeuroCult NS-A differentiation medium (StemCell Technologies) with penicillin (100 U) / streptomycin-125 μg / mL; Thermo Fisher). The cells were differentiated for 7 days and then fixed in 4% paraformaldehyde (PFA) for 15 minutes at room temperature. After washing in phosphate buffered saline (PBS, pH 7.2) and blocking in a blocking solution containing 5% normal goat serum and 0.25% Triton X-100 in 0.02 M PBS (PBS+), the cultures were subjected to immunocytochemistry and reacted with primary antibodies overnight at 5°C. After five washes in PBS+, the cultures were further incubated with Alexa Fluor 488- and 594-conjugated secondary antibodies (Invitrogen) in PBS+ at a ratio of 1:100 for 2.5 hours at room temperature in the absence of light (dark room). After five washes in PBS, 4',6-diamidino-2-phenylindole (DAPI, Thermo Fisher) was added for 5 minutes, followed by gentle washing in PBS and placing the coverslips on microscope slides. Negative controls lacking primary antibodies were also performed, and no positive signals were detected. Cells were also evaluated after 1, 5, 10, 21, and 28 days. The cell viability of SPID1COL1 and FIB3COL1 scaffolds at day 3 was estimated by trypan blue exclusion to be approximately 90% and 92%, respectively, while the cell viability of native bovine type I collagen scaffolds was only approximately 78%. The small clusters observed on the native bovine type I collagen scaffolds were dark and dense, indicating that the cells were unhealthy or dead. By day 5, the neurospheres on the SPID1COL1 and FIB3COL1 scaffolds were still mainly translucent and had cell viabilities of approximately 86% and 88%, respectively. Some spheres adhered to the scaffolds because individual cells were proliferating and forming small cell clusters. The neurospheres on the native bovine type I collagen scaffolds did not adhere as easily as the SPID1COL1 or FIB3COL1 scaffolds; as the dense spheres of dark, dense unhealthy or dead cells were lifted and the density of the spheres increased.By day 5, cell viability on native bovine type I collagen scaffolds was only approximately 63% (. Fig.13 ). Since the spidroin I or fibroin III portion of the SPID1COL1 or FIB3COL1 proteins both promoted more significant proliferation rates compared to native bovine type I collagen, the inventors also investigated whether it also had an effect on differentiation. To test this, three days after the sixth passage, the above neurospheres were plated as pellets on poly-D-lysine (PDL), laminin-coated coverslips or electrospun meshes in NeuroCult NS-A Differentiation Medium (StemCell Technologies). After seven days, the neurospheres readily adhered, flattened, and expanded to produce large numbers of migrating cells. The cells were stained for neuron-specific β-tubulin (Tuj1) to visualize neurons, glial fibrillary acidic protein (GFAP) for astrocytes, O4 for oligodendrocytes, and nestin to visualize intermediate microfilaments to identify neuroepithelial stem cells ( Fig.14 ). Neurons, astrocytes, and nestin-expressing cells were observed for all treatments, but no oligodendrocytes were detected. Electrospun SPID1COL1 and FIB3COL1 treatments showed the highest proportions of neurons (80% for SPID1COL1 and 82% for FIB3COL1), astrocytes (61% for SPID1COL1 and 62% for FIB3COL1), and nestin-positive cells (40% for SPID1COL1 and 41% for FIB3COL1). In contrast, treatment with native bovine type 1 collagen showed much lower proportions of neurons (38%), astrocytes (14%), and nestin-positive cells (35%). Since electrospun SPID1COL1 and FIB3COL1 scaffolds produced the highest proportions of neurons, astrocytes, and nestin-positive cells compared to native bovine type I collagen, they have the potential to become excellent scaffolds for neural tissue engineering. In this cell culture model, the biocomposite scaffolds SPID1COL1 and FIB3COL1 increased the proportion of cells that differentiated into neurons, astrocytes, and nestin-positive cells compared to laminin and native bovine type I collagen. The SPID1COL1 and FIB3COL1 scaffolds performed better than native bovine type I collagen scaffolds to mimic the native ECM, thus encouraging their use as components of therapeutic strategies to repair damaged spinal cords. Fig.14 )

[0174] Tensile Strength

[0175] Sufficient tensile strength is critical for peripheral nerve replacements because they must withstand manipulation during surgery. In addition, subsequent tissue movement associated with cardiopulmonary circulation and patient movement must be tolerated, especially as tissue begins to infiltrate the scaffold and increase axonal growth

[56] . The tensile properties of electrospun SPID1COL1, FIB3COL1, and bovine type I collagen nanofiber scaffolds were determined using a benchtop Sauter SD 500N100 tensile tester (Imlab) at a load cell capacity of 10 N. Dog-bone-shaped specimens with dimensions of 10 mm wide × 15 mm long and a thickness of 500 μm were tested at room temperature at a crosshead speed of 10 mm / min and a gauge length of 20 mm [57, 58]. A minimum of 20 specimens of a single scaffold were tested until fracture; the results obtained were then plotted to determine the stress-strain curve of the scaffold. Fig.15 The maximum stress-strain comparison of electrospun SPID1COL1, FIB3COL1, and native type I collagen nanofibers is shown. The maximum tensile strength of SPID1COL1 and FIB3COL1 scaffolds is approximately 45 times higher than that of native bovine type I collagen scaffolds, with an average tensile strength of 89.77 ± 2.18 MPa and an ultimate strain of 84% at 122.51 MPa for SPID1COL1 and an average tensile strength of 91.49 ± 3.04 MPa and an ultimate strain of 82% at 126.23 MPa for FIB3COL1, while for native bovine type I collagen scaffolds, the average tensile strength was 0.54 ± 0.68 MPa and the elongation at break was 58% at 1.32 MPa. Therefore, considering the well-known fact that the tensile strength of fresh human sciatic nerve is 11.63 ± 1.80 MPa

[59] , the tensile strength of native bovine type I collagen scaffolds is not sufficient to be used as nerve grafts alone. Both SPID1COL1 and FIB3COL1 scaffolds showed significantly improved tensile properties, making them suitable for neural tissue engineering.

[0176] degradation

[0177] To determine the degradation rate of SPID1COL1 and FIB3COL1 scaffolds, a combination of lipase (7 mg / mL) and collagenase (1 mg / mL) was dissolved in PBS (pH 7.4). For native type I collagen scaffolds, a single concentration of 1 mg / mL collagenase was used. The samples were weighed and then placed in tubes of the corresponding enzyme solution maintained at 37°C. The samples were removed, blotted with paper towels until the mass remained constant, and weighed after 2, 4, 8, and 24 hours, and then weighed every 24 hours until the sample mass remained constant. The net weight of the scaffold was calculated by subtracting the wet chamber weight from the scaffold containing the wet chamber weight. Once the initial wet pore weight is reached, a value of 0 is assigned. Fig.16As shown, bovine type I collagen scaffolds degrade faster than SPID1COL1 and FIB3COL1 scaffolds. When incubated in collagenase solution at 37 °C for 64 h, native bovine type I collagen nanofibers were resistant for up to 36 h and completely degraded at 64 h. However, SPID1COL1 and FIB3COL1 scaffolds were substantially more stable and resistant to degradation by lipase and collagenase, as complete degradation of the nanofibers required 100 h. Compared to native bovine type I collagen, SPID1COL1 and FIB3COL1 scaffolds showed resistance for up to 96 and 100 h in lipase / collagenase solution, respectively, showing their superiority against degradation.

[0178] in conclusion

[0179] It should be understood that, for the sake of clarity, certain features of the present invention described in the context of separate embodiments may also be provided in combination in a single embodiment. By contrast, for the sake of brevity, the various features of the present invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination.

[0180] Although the present invention has been described in conjunction with specific embodiments of the present invention, it is apparent that many alternatives, modifications and variations will be apparent to those skilled in the art. In addition, any reference cited or identified in this application should not be construed as admitting that such reference can be used as prior art of the present invention.

[0181] This work provides evidence that a combination of biochemical and topographical cues can influence the direction of cell differentiation and raises important questions about the mechanisms of fate specification enhanced by substrate topography. Electrospun nanofibrous scaffolds provide mechanical stability, structural guidance, and a matrix for cell integration with surrounding tissue. Collagen acts as an ECM-mimetic nanoscaffold by providing specific cell adhesion ligands to physically support cells. SPID1COL1 and FIB3COL1 proteins showed improved cell differentiation in vitro compared to native type I collagen and resembled the role of native ECM in vivo. We found that SPID1COL1 and FIB3COL1 nanofibers had increased fiber diameter and improved mechanical properties compared to native type I collagen nanofibers, allowing for control of a certain proportion of desired cell types for possible therapeutic purposes.

[0182] References

[0183] 1.Frantz C,Stewart KM,Weaver VM.The extracellular matrix at aglance.J Cell Sci.2010;123(Pt 24):4195-4200.doi:10.1242 / jcs.023820

[0184] 2. H,Sainio A,Koulu M,Wight TN,Penttinen R.Extracellularmatrix molecules:potential targets in pharmacotherapy.Pharmacol Rev.2009Jun;61(2):198-223.doi:10.1124 / pr.109.001289.PMID:19549927;PMCID:PMC2830117.

[0185] 3.Schaefer L,Schaefer RM.Proteoglycans:from structural compounds tosignaling molecules.Cell Tissue Res.2010Jan;339(1):237-46.doi:10.1007 / s00441-009-0821-y.Epub 2009Jun 10.PMID:19513755.

[0186] 4.Alberts B.,Johnson A.,Lewis J.,Raff M.,Roberts K.,Walter P.(2007).Molecular Biology of the Cell.London:Garland Science;

[0187] 5.Harvey SJ,Miner JH.Revisiting the glomerular charge barrier in themolecular era.Curr Opin Nephrol Hypertens.2008Jul;17(4):393-8.doi:10.1097 / MNH.0b013e32830464de.PMID:18660676.

[0188] 6.Morita H,Yoshimura A,Inui K,Ideura T,Watanabe H,Wang L,Soininen R,Tryggvason K.Heparan sulfate of perlecan is involved in glomerularfiltration.J Am Soc Nephrol.2005Jun;16(6):1703-10.doi:10.1681 / ASN.2004050387.Epub 2005May 4.PMID:15872080.

[0189] 7.Rozario T,DeSimone DW.The extracellular matrix in development andmorphogenesis:a dynamic view.Dev Biol.2010May 1;341(1):126-40.doi:10.1016 / j.ydbio.2009.10.026.Epub 2009Oct 23.PMID:19854168;PMCID:PMC2854274.

[0190] 8.De Wever O,Demetter P,Mareel M,Bracke M.Stromal myofibroblasts aredrivers of invasive cancer growth.Int J Cancer.2008Nov 15;123(10):2229-38.doi:10.1002 / ijc.23925.PMID:18777559.

[0191] 8.Wise SG,Weiss AS.Tropoelastin.Int J Biochem Cell Biol.2009Mar;41(3):494-7.doi:10.1016 / j.biocel.2008.03.017.Epub 2008Apr 1.PMID:18468477.

[0192] 9.Lucero HA,Kagan HM.Lysyl oxidase:an oxidative enzyme and effectorof cell function.Cell Mol Life Sci.2006Oct;63(19-20):2304-16.doi:10.1007 / s00018-006-6149-9.PMID:16909208.

[0193] 10.Smith ML,Gourdon D,Little WC,Kubow KE,Eguiluz RA,Luna-Morris S,Vogel V.Force-induced unfolding of fibronectin in the extracellular matrix ofliving cells.PLoS Biol.2007Oct 2;5(10):e268.doi:10.1371 / journal.pbio.0050268.PMID:17914904;PMCID:PMC1994993.

[0194] 11.Trebaul A,Chan EK,Midwood KS.Regulation of fibroblast migration bytenascin-C.Biochem Soc Trans.2007Aug;35(Pt 4):695-7.doi:10.1042 / BST0350695.PMID:17635125.

[0195] 12.Tucker RP,Chiquet-Ehrismann R.The regulation of tenascinexpression by tissue microenvironments.Biochim Biophys Acta.2009May;1793(5):888-92.doi:10.1016 / j.bbamcr.2008.12.012.Epub 2008Dec 31.PMID:19162090.

[0196] 13.Merle C,Perret S,Lacour T,Jonval V,Hudaverdian S,Garrone R,Ruggiero F,Theisen M.Hydroxylated human homotrimeric collagen I inAgrobacterium tumefaciens-mediated transient expression and in transgenictobacco plant.FEBS Lett.2002Mar27;515(1-3):114-8.doi:10.1016 / s0014-5793(02)02452-3.

[0197] 14.Torre-Blanco A,Alvizouri AM.In vitro hydroxylation of proline inthe collagen of the cysticercus of Taenia solium.Comp Biochem Physiol B.1987;88(4):1213-7.doi:10.1016 / 0305-0491(87)90026-5.

[0198] 15.Myllyharju J.Prolyl 4-hydroxylases,the key enzymes of collagenbiosynthesis.Matrix Biol.2003Mar;22(1):15-24.doi:10.1016 / s0945-053x(03)00006-4.

[0199] 16.Berg RA,Prockop DJ.The thermal transition of a non-hydroxylatedform of collagen.Evidence for a role for hydroxyproline in stabilizing thetriple-helix of collagen.Biochem Biophys Res Commun.1973 May 1;52(1):115-20.doi:10.1016 / 0006-291x(73)90961-3.

[0200] 17.Annunen P,Helaakoski T,Myllyharju J,Veijola J,Pihlajaniemi T andKivirikko KI(1997)Cloning of the human prolyl 4-hydroxylaseαsubunit isoformα(II)and characterization of the type II enzyme tetramer.Theα(I)andα(II)subunits do not form a mixedα(I)α(II)β2 tetramer.J Biol Chem,272,17342–17348.

[0201] 18.Gorres KL,Raines RT.Prolyl 4-hydroxylase.Crit Rev Biochem MolBiol.2010Apr;45(2):106-24.doi:10.3109 / 10409231003627991.

[0202] 19.Myllyharju,J.&Kivirikko,K.I.Collagens,modifying enzymes and theirmutations in humans,flies and worms.Trends Genet.20,33–43(2004).

[0203] 20.Guengerich FP.Introduction:Metals in Biology:α-Ketoglutarate / Iron-Dependent Dioxygenases.J Biol Chem.2015;290(34):20700-20701.doi:10.1074 / jbc.R115.675652

[0204] 21.Ibrahimi A,Vande Velde G,Reumers V,Toelen J,Thiry I,Vandeputte C,Vets S,Deroose C,Bormans G,Baekelandt V,Debyser Z,Gijsbers R.Highly efficientmulticistronic lentiviral vectors with peptide 2A sequences.Hum GeneTher.2009Aug;20(8):845-60.doi:10.1089 / hum.2008.188.

[0205] 22.Weber,E.,Engler,C.,Gruetzner,R.,Werner,S.,and Marillonnet,S.(2011).A modular cloning system for standardized assembly of multigeneconstructs.PLoS One6:e16765.doi:10.1371 / journal.pone.0016765

[0206] 23.Stavolone L,Kononova M,Pauli S,Ragozzino A,de Haan P,Milligan S,Lawton K,Hohn T.Cestrum yellow leaf curling virus(CmYLCV)promoter:a newstrong constitutive promoter for heterologous gene expression in a widevariety of crops.Plant Mol Biol.2003 Nov;53(5):663-73.doi:10.1023 / B:PLAN.0000019110.95420.bb.

[0207] 24.Nagaya S,Kawamura K,Shinmyo A,Kato K.The HSP terminator ofArabidopsis thaliana increases gene expression in plant cells.Plant Cell Physiol.2010 Feb:51(2):328-32.doi:10.1093 / pcp / pcp188.

[0208] [ PMC free article ] [ PubMed ] [ Cross Ref ] 25. Liu Z, Chen O, Wall JBJ, Zheng M, Zhou Y, Wang L, Vaseghi HR, Qian L, LiuJ 197(1):2193.doi:10.1038 / s41598-017-02460-2.

[0209] 26.Baltes NJ,Gil-Humanes J,Cermak T,Atkins PA,Voytas DF.DNA repliconsfor plant genome engineering.Plant Cell.2014 Jan:26(1):151-63.doi:10.1105 / tpc.113.119792.Epub 2014 Jan 17.PMID:24443519;PMCID:PMC3963565.

[0210] 27.P.Bruckner,DJProckop Anal.Biochem.,110(1981),pp.360-368

[0211] 28.F.Ruggiero,J.-Y.Exposito,P.Bournat,V.Gruber,S.Perret,J.Comte,B.Olagnier,R.Garrone,M.Theisen FEBS Lett.,469(2000),pp.132-136

[0212] 29.Michael A.Gimbrone,Jr.,Ramzi S.Cotran,Judah Folkman J Cell Biol(1974)60(3):673–684.https: / / doi.org / 10.1083 / jcb.60.3.673

[0213] 30.Nokelainen M,Tu H,Vuorela A,Notbohm H,Kivirikko KI,MyllyharjuJ.High-level production of human type I collagen in the yeast Pichiapastoris.Yeast.2001 Jun 30;18(9):797-806.doi:10.1002 / yea.730.

[0214] 31.Myllyharju J,Nokelainen M,Vuorela A,Kivirikko KI.Expression ofrecombinant human type I-III collagens in the yeast pichia pastoris.BiochemSoc Trans.2000;28(4):353-7.PMID:10961918.

[0215] 32.Vuorela A,Myllyharju J,Nissi R,Pihlajaniemi T,KivirikkoKI.Assembly of human prolyl 4-hydroxylase and type III collagen in the yeastpichia pastoris:formation of a stable enzyme tetramer requires coexpressionwith collagen and assembly of a stable collagen requires coexpression withprolyl 4-hydroxylase.EMBO J.1997 Nov17;16(22):6702-12.doi:10.1093 / emboj / 16.22.6702.

[0216] 33.Stein H,Wilensky M,Tsafrir Y,Rosenthal M,Amir R,Avraham T,Ofir K,Dgany O,Yayon A,Shoseyov O.Production of bioactive,post-translationallymodified,heterotrimeric,human recombinant type-I collagen in transgenictobacco.Biomacromolecules.2009 Sep 14;10(9):2640-5.doi:10.1021 / bm900571b.

[0217] 34.Stoppato,M.;Stevens,H.Y.;Carletti,E.;Migliaresi,C.;Motta,A.;Guldberg,R.E.Effects of silk fibroin fiber incorporation on mechanicalproperties,endothelial cell colonization and vascularization of PDLLAscaffolds.Biomaterials 2013,34,4573–4581

[0218] 35.Mobini,S.;Hoyer,B.;Solati-Hashjin,M.;Lode,A.;Nosoudi,N.;Samadikuchaksaraei,A.;Gelinsky,M.Fabrication and characterization ofregenerated silk scaffolds reinforced with natural silkfibers.J.Biomed.Mater.Res.A 2013,101,2392–2404.

[0219] 36.Park,S.;Edwards,S.;Hou,S.;Boudreau,R.;Yee,R.;Jeong,K.J.Multi-interpenetrating network(ipn)hydrogel by gelatin and silkfibroin.Biomater.Sci.2019,7,1276–1280.

[0220] 37.Panas-Perez,E.;Gatt,C.J.;Dunn,M.G.Development of a silk andcollagen fiber scaffold for anterior cruciate ligament reconstruction.J.Mater.Sci.Mater.Med.2013,24,257–265

[0221] 38.Ghezzi,C.E.;Marelli,B.;Muja,N.;Hirota,N.;Martin,J.G.;Barralet,J.E.;Alessandrino,A.;Freddi,G.;Nazhat,S.N.Mesenchymal stem cell-seededmultilayered dense collagen-silk fibroin hybrid for tissue engineeringapplications.Biotechnol.J.2011,6,1198–1207

[0222] 39.Vasconcelos,A.;Gomes,A.C.;Cavaco-Paulo,A.Novel silk fibroin / elastin wound dressing.Acta Biomater.2012,8,3049–3060

[0223] 40.Shunji Yunoki,Toshiyuki Ikoma,Junzo Tanaka.Development of collagencondensation method to improve mechanical strength of tissue engineeringscaffolds,Material Characterization,Volume 61,Issue 9,2010,Pages 907-911,ISSN1044-5803,https: / / doi.org / 10.1016 / j.matchar.2010.05.010.

[0224] 41.Gatesy,J.;Hayashi,C.;Motriuk,D.;Woods,J.;Lewis,R.Extremediversity,conservation,and

[0225] 42.convergence of spider silk fibroin sequences.Science 2001,291,2603-2605

[0226] 43. L,Scheibel T.The elaborate structure of spider silk:structure and function of a natural high performance fiber.Prion.2008;2(4):154-161.doi:10.4161 / pri.2.4.7490

[0227] 44.Hayashi,C.Y.;Shipley,N.H.;Lewis,R.V.Hypotheses that correlate thesequence,structure,and mechanical properties of spider silk proteins.Int.J.Biol.Macromol.1999,24,271–275

[0228] 45.Zhao,L.;Chen,D.;Yao,Q.;Li,M.Studies on the use of recombinantspider silk protein / polyvinyl alcohol electrospinning membrane as wounddressing.Int.J.Nanomed.2017,12,8103–8114

[0229] 46.Meng,Z.X.;Wang,Y.S.;Ma,C.;Zheng,W.;Li,L.;Zheng,Y.F.Electrospinningof PLGA / gelatin randomly oriented and aligned nanofibers as potentialscaffold in tissue engineering.Mater.Sci.Eng.2010,30,1204–1210

[0230] 47.Telemeco T,Ayres C,Bowlin G,Wnek G,Boland E,Cohen N,etal.Regulation of cellular infiltration into tissue engineering scaffoldscomposed of submicron diameter fibrils produced by electrospinning.ActaBiomater.2005;1:377–385.doi:10.1016 / j.actbio.2005.04.006

[0231] 48.Rho KS,Jeong L,Lee G,Seo BM,Park YJ,Hong SD,et al.Electrospinningof collagen nanofibers:effects on the behavior of normal human keratinocytesand early-stage wound healing.Biomaterials.2006;27:1452–1461.doi:10.1016 / j.biomaterials.2005.08.004.

[0232] 49.Buttafoco L,Kolkman N,Engbers-Buijtenhuijs P,Poot A,Dijkstra P,Vermes I,et al.Electrospinning of collagen and elastin for tissue engineeringapplications.Biomaterials.2006;27:724–734.doi:10.1016 / j.biomaterials.2005.06.024.

[0233] 50.Matthews JA,Wnek GE,Simpson DG,Bowlin GL.Electrospinning ofcollagen nanofibers.Biomacromolecules 2002;3:232-238.

[0234] 51.Zhang X,Reagan MR,Kaplan DL.Electrospun silk biomaterial scaffoldsfor regenerative medicine.Adv Drug Deliv Rev 2009;61:988-1006.

[0235] 52.Boland ED,Matthews JA,Pawlowski KJ,Simpson DG,Wnek GE,BowlinGL.Electrospinning collagen and elastin:preliminary vascular tissueengineering.Front Biosci2004;9:1422-1432.

[0236] 53.Rho KS,Jeong L,Lee G,Seo B,Park YJ,Hong S,Roh S,Cho JJ,Park WH,MinB.Electrospinning of collagen nanofibers:Effects on the behavior of normalhuman keratinocytes and early-stage wound healing.Biomaterials 2006;27:1452-1461.

[0237] 54.Zhang X,Baughman CB,Kaplan DL.In vitro evaluation of electrospunsilk fibroin scaffolds for vascular cell growth.Biomaterials 2008;29:2217-2227.

[0238] 55.Noh HK,Lee SW,Kim J,Oh J,Kim K,Chung C,Choi S,Park WH,MinB.Electrospinning of chitin nanofibers:Degradation behavior and cellularresponse to normal human keratinocytes and fibroblasts.Biomaterials 2006;27:3934-3944.

[0239] 56.Ma,M.;Wei,P.;Wei,T.;Ransohoff,R.M.;Jakeman,L.B.Enhanced axonalgrowth into a spinal cord contusion injury site in a strain of mouse(129X1 / SvJ)with a diminished inflammatory response.J.Comp.Neurol.2004,474,469-486.

[0240] 57.Prabhakaran,M.P.;Venugopal,J.;Chan,C.K.;Ramakrishna,S.Surfacemodified electrospun nanofibrous scaffolds for nerve tissueengineering.Nanotechnology 2008,19,455102–455109.

[0241] 58.Mobarakeh,L.G.;Prabhakaran,M.P.;Morshed,M.;Esfahani,M.H.N.;Ramakrishna,S.Electropsun PCL / gelatin nanofibrous scaffolds for nerve tissueengineering.Biomaterials 2008,29,4532–4539.

[0242] 59.Borschel GH,Kia KF,Kuzon WM Jr,Dennis RG.Mechanical properties ofacellular peripheral nerve.J Surg Res.2003 Oct;114(2):133-9.doi:10.1016 / s0022-4804(03)00255-5.

[0243] 60.Perona R.Cell signalling:growth factors and tyrosine kinasereceptors.Clin Transl Oncol.2006;8(2):77–82.doi:10.1007 / s12094-006-0162-1

[0244] 61.Richardson,S.M.Tissue engineering today,nottomorrow.Regen.Med.2007,2,91-94

[0245] 62.Sahoo,S.;Ang,L.T.;Goh,J.C.H.;Toh,S.L.Growth factor deliverythrough electrospun nanofibers in scaffolds for tissue engineering applications.J.Biomed.Mater.Res.Part A 2009,4,1539-1550.

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

Claims

1. A method for producing a fusion protein of non-human sclerostin and human collagen, wherein the fusion protein is capable of forming hydroxylated triple helical fibers in plants or isolated plant cells, hereinafter also referred to as self-fibrotic heterotrimeric collagen containing the fusion protein, the method include: (a) targeting to and accumulating in chloroplasts of plants or isolated plant cells the nucleotide sequence encoding non-human sclerostin, the nucleotide sequence encoding human type I collagen α-I chain, and the nucleotide sequence encoding human type I collagen α-II chain as shown in SEQ ID 16, including a signal peptide sequence for targeting to chloroplasts, all of which have no ER retention sequence, (b) targeting to the chloroplasts of the plant or isolated plant cells and accumulating therein a nucleotide sequence encoding an exogenous non-human chimeric prolyl 4-hydroxylase (P4H), which is capable of specifically hydroxylating the Y position of the Gly-XY triplet of the type I collagen α-I chain and the type I collagen α-II chain, and (c) co-expressing the genes of (a) and (b) in the chloroplasts of the plant or isolated plant cells to obtain a fusion protein of non-human sclerostin and human collagen.

2. The method according to claim 1, wherein the co-expression of the genes of (a) and (b) is achieved in two separate vectors and by a tricistronic expression vector with A2 activity, wherein the tricistronic expression vector with A2 activity is capable of inducing ribosome jumping during protein translation in cells, thereby enabling the gene of (a) and the gene of (b) to be expressed simultaneously in a single plant; in particular, by a tricistronic expression vector with A2 activity having an A2 sequence as shown in SEQ ID 18 and 20.

3. The method according to claim 1, wherein the exogenous non-human chimeric P4H comprises: A) a non-human P4H α subunit sequence shown in SEQ ID 11 and 18, and B) a non-human P4H β subunit sequence shown in SEQ ID 12 and 18, an exogenous human lysine hydroxylase 3 (LH3) sequence, wherein the exogenous human lysine hydroxylase 3 is capable of specifically hydroxylating collagen lysine to 1,2-glucosylgalactosyl-5-hydroxylysine of the type I collagen α-I chain and the type I collagen α-II chain.

4. The method according to claim 3, wherein the exogenous human LH3 is represented by SEQ ID 14 and 18, including a signal peptide sequence for targeting to chloroplasts, as represented by SEQ ID 18, and all of the sequences are free of ER retention sequences.

5. A method according to any one of the preceding claims, wherein the method comprises avoiding the co-expression of C-terminal and / or N-terminal collagen propeptides necessary for the assembly of collagen molecules into fibrils, thereby enabling the formation of a triple helical fibril structure.

6. The method according to any one of the preceding claims, wherein the non-human sclerostin is selected from spider silk protein I or silk fibroin III.

7. The method according to claim 6, wherein the spider silk protein I is encoded by the nucleotide sequences shown in SEQ ID 6 and 16. 8 . The method according to claim 6 , wherein the fibroin III is encoded by the nucleotide sequences shown in SEQ ID 8 and 20.

9. The method of claim 1, wherein the plant is a Nicotiana benthamiana or Nicotiana tabacum plant.

10. The method according to claim 1, further comprising filtering and / or purifying the extracted fusion protein of non-human sclerostin and human collagen.

11. The method of claim 10, wherein the filtering and / or purifying comprises a chromatography step.

12. The method of claim 1, wherein the plant is transiently transformed.

13. The method of claim 12, comprising introducing a nucleotide sequence into at least one Agrobacterium tumefaciens strain using the viral vector of any one of claims (a) to (c).

14. A fusion protein of non-human sclerostin and human collagen obtained using the method according to any one of the preceding claims.

15. Use of the fusion protein according to claim 14 for producing nanofibers.

Images