Recombinant proteins based on fibrinogen

Abstract

The present invention relates to an isolated recombinant polypeptide comprising a fibrinogen gamma chain and a His tag. The present invention also relates to a nucleic acid encoding said polypeptide, a vector comprising said nucleic acid, a host cell, preferably a bacterial host cell, comprising said nucleic acid or vector, a biological hybrid material comprising a conjugate comprising said polypeptide, uses of the above, and a method of manufacturing said polypeptide.

Description

[0001] This invention relates to isolated recombinant polypeptides comprising a γ-chain of fibrinogen and a His tag. The invention also relates to nucleic acids encoding said polypeptides, vectors containing said nucleic acids, host cells (preferably bacterial host cells) containing said nucleic acids or vectors, biohybrid materials containing conjugates of said polypeptides, the above-described uses, and methods for manufacturing said polypeptides.

[0002] Proteins derived from the extracellular matrix (ECM) are frequently used in cell culture applications and are valued for their ability to provide cells with key biochemical factors required for cell attachment, growth, and proliferation. Fibrinogen, a blood protein, is a precursor to fibrin and plays a crucial role in wound healing, hemostasis, and angiogenesis. Following blood clot formation, fibrin serves as a novel, temporary ECM for tissue regeneration. Fibrinogen is a hexamer composed of a collection of bialpha, beta, and gamma chains linked together via intra- and inter-chain disulfide bridges. The natural polymerization of fibrinogen with fibrin is a multi-step enzymatic process catalyzed by thrombin after vascular injury. Thrombin cleaves two short amino acid sequences at the amino terminals of the Aα and Bβ chains of the fibrinogen precursor. This exposes polymerization sites within the fibrin molecule, leading to the polymerization of individual fibrinogen molecules into a 3D fibrin network. Further stabilization of this network is achieved through covalent cross-linking of the gamma chains catalyzed by factor XIIIa.

[0003] The most widespread medical uses of fibrin are as a hemostatic agent and tissue sealant. The first fibrin glue to receive FDA approval on the market was Baxter's. However, many other products have subsequently been launched. These formulations are used as hemostatic agents, occlusive agents, and tissue adhesives in various clinical settings. They are all derived from homologous fibrinogen and thrombin from combined blood donations. Because fibrin used in biomedical and cell culture applications is derived from human or animal blood, there is always a residual risk of pathogenic transmission. Furthermore, due to the source of the materials, they are subject to batch-to-batch natural variations, making it difficult to standardize fibrin materials in biomedical research. These variations may hinder GMP (Good Manufacturing Practice) requirements for specific newly developed fibrin-based products.

[0004] A safer approach to obtaining pure proteins of consistent quality without the risk of pathogenic transmission is recombinant production. Due to the complex structural and post-translational modifications involved, most heterologous expression systems used to produce intact fibrinogen molecules are based on mammalian cells. Accordingly, recombinant fibrinogen has been expressed in juvenile hamster cells, primarily to study its subunit interactions and assembly. Furthermore, it has been expressed in PER... Fully assembled fibrinogen was produced in the cell line. CHO cells have also been used for the production of recombinant fibrinogen, with yields increasing over the years. Another method to obtain fully assembled fibrinogen is to produce it in the milk of transgenic animals. This has been demonstrated, for example, in cows and mice.

[0005] The applications of fibrinogen are based on the material's attractive properties, particularly its tendency to bind growth factors and its interactions with platelets, leukocytes, fibroblasts, and endothelial cells. Therefore, fibrinogen is a widely used biopolymer in tissue engineering for repairing damaged tissues / organs, as a coating or 3D matrix in cell culture, and also as a cell and drug delivery agent, especially for growth factor delivery. Fibrinogen binds to a variety of growth factors with well-studied affinities, particularly fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF), as well as heparin. The latter has been used as a conjugate for growth factors that do not bind to fibrinogen (nerve growth factor (NGF), neurotrophin-3) in their native state. Such growth factor immobilization constructs can be used to deliver growth factors for therapeutic or cell culture applications. For example, in the field of cell culture coatings, fibrinogen has been very successfully used for solid growth factor delivery, improving the survival and proliferation of MG-63 cells in contact with FGF-2 modalities, which are immobilized by fibrinogen's native affinity for this growth factor. Numerous reports have indicated that the binding of FGF-2 to fibrinogen or fibrin can protect growth factors from degradation, thereby increasing their long-term effects in cell culture. Similarly, fibrinogen-FGF-2 appears to enhance the proliferative capacity of growth factors compared to using fibrinogen or FGF-2 alone.

[0006] Fibrinogen possesses multiple RGD, AGDV (SEQ ID NO:7), and integrin binding sites, enabling it to interact with a wide range of cell types. This bioactivity makes it an attractive matrix for stem cell differentiation, cell delivery / enrichment, and tissue engineering. In cell culture applications, fibrinogen coatings have proven to be a viable alternative to laminin (Geltrex) in induced pluripotent stem cell (iPSC) culture. iPSCs cultured on a fibrinogen coating retain their pluripotency and ability to differentiate into different germ layers. Therefore, fibrinogen represents an interesting regulatory-compliant material for developing cell therapy applications. Another interesting application in the field of cell isolation / enrichment is the use of insoluble fibrinogen particles to create cell affinity columns, which can be used to obtain and culture mesenchymal cells (fibroblasts, endothelial cells) from various tissues, such as bone marrow, without the use of trypsinization. This technique can also be used to capture cancer cells from blood suspensions.

[0007] While fibrinogen-based materials possess interesting biological activities, they often exhibit poor mechanical properties and rapid degradation. This sometimes necessitates modification of the materials with synthetic polymers to create biohybrids with superior properties. These semi-synthetic materials combine the inherent biological activity of fibrinogen with custom-tunable properties derived from the synthetic component.

[0008] PEG is an attractive polymer for modification due to its bioinertness and the ability to precisely tune mechanical properties by controlling chain length and crosslinking degree. Amine-reactive PEG has been conjugated to fibrinogen-producing hydrogels for 3D cell culture, which can be loaded with growth factors (PDGF, TGF-β) and support MSC viability and angiogenesis. A different approach has been employed to generate PEGylated precursors by crosslinking denatured fibrinogen to PEG-DA. Photopolymerization of these PEGylated precursors leads to the formation of scaffolds that facilitate migration, cell diffusion, and controlled proteolytic degradation. Another promising research approach is based on fibrin and... F127 ( F127) copolymer-based thermosensitive hydrogel. The F127 copolymer undergoes reverse thermogelation in response to temperature changes. Fibrinogen and The conjugation of F127 can control physical properties while maintaining the complete cellular compatibility of the hybrid hydrogel.

[0009] As mentioned above, recombinant fibrinogen-based materials represent a cost-effective and regulated source of fibrinogen, opening the door to a variety of cell culture and biomedical applications.

[0010] Therefore, the technical problem of the present invention is to provide fibrinogen-derived materials and corresponding methods for producing such materials, which can be recombined and produced in a rapid, simple and cost-effective manner, while avoiding the use of human or animal blood and mammalian cell cultures.

[0011] The solution to the above-mentioned technical problem is achieved through the embodiments characterized in the claims.

[0012] Specifically, in a first aspect, the present invention relates to isolated recombinant polypeptides comprising:

[0013] (i) the γ chain of fibrinogen, and

[0014] (ii) His tag.

[0015] As used herein, the term “recombinant polypeptide” refers to an artificially produced polypeptide, that is, a polypeptide produced by expressing recombinant, i.e., artificially produced DNA in living cells.

[0016] The isolated recombinant polypeptide of the present invention comprises a γ chain of fibrinogen, preferably a γ chain of human fibrinogen. In a preferred embodiment, the γ chain of said fibrinogen contains binding sites targeting integrins, leukocytes, platelets, fibroblasts, endothelial cells, and / or fibroblast growth factor 2 (FGF-2). Specifically, the γ chain of said fibrinogen may contain one or more or all of the following specific binding sites:

[0017] - Platelet binding site: FGG 427-438 The above is used to bind integrin α IIb γC-dodecapeptide of β3 (HHLGGAKQAGDV; SEQ ID NO: 8)

[0018] -FGG 404–422 Leukocytes, neutrophils, monocytes, integrin Mac-1, or α at (YSMKKTTMKIIPFNRLTIG; SEQ ID NO:9) M β2 binding site

[0019] -FGG 217–229 (WTVFQKRLDGSV; SEQ ID NO:10) and FGG 373–385 Integrin α at (GVYYQGGTYSKAS; SEQ ID NO: 11) is responsible for the binding of endothelial cells, fibroblasts, and tumor cells. V β3 binding site

[0020] -C-terminal region FGG: Binding site for growth factor FGF-2 and interleukin-β

[0021] - Factor XIII crosslinking site, FGG lysine at 433 and FGG glutamine at 425 / 426

[0022] More preferably, the fibrinogen γ-chain comprises or consists of: (i) the amino acid sequence according to SEQ ID NO:1, or (ii) an amino acid sequence having sequence identity with SEQ ID NO:1 and having fibrinogen γ-chain activity of: at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.25%, at least 95.5%, at least 95.75%, at least 96%, at least 96.25%. %, at least 96.5%, at least 96.75%, at least 97%, at least 97.25%, at least 97.5%, at least 97.75%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%. In this context, the term "having fibrinogen γ-chain activity" refers to having functional binding sites for integrins, leukocytes, platelets, fibroblasts, endothelial cells, and / or FGF-2.

[0023] Furthermore, the recombinant polypeptide of the present invention comprises a His tag, i.e., a polyhistidine tag, which contains an amino acid motif containing at least six histidine (His) residues. Preferably, the His tag comprises or consists of: (i) the amino acid sequence according to SEQ ID NO:2, or (ii) an amino acid sequence having the following sequence identity with SEQ ID NO:2 and having His tag functionality: at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.25%, at least 95.5%, at least 95.75%, at least 96%, at least 96.25%. %, at least 96.5%, at least 96.75%, at least 97%, at least 97.25%, at least 97.5%, at least 97.75%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%. In this context, the term "having His tag functionality" refers to having an amino acid motif comprising at least six (preferably consecutive) histidine (His) residues.

[0024] In a preferred embodiment, the isolated recombinant polypeptide of the present invention comprises: (i) the amino acid sequence according to SEQ ID NO:3, or (ii) an amino acid sequence having the following sequence identity with SEQ ID NO:3 and having fibrinogen activity and His tag functionality: at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.25%, at least 95.5%, at least 95.75%, at least 96%, at least 9 6.25%, at least 96.5%, at least 96.75%, at least 97%, at least 97.25%, at least 97.5%, at least 97.75%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%. In this context, the protein composed of the amino acid sequence according to SEQ ID NO:3 has an amino acid composition of 13.01% acidic amino acids, 34.47% neutral amino acids, 15.3% basic amino acids, and 37.21% hydrophobic amino acids, a molecular weight of 49.7, and a pI of 5.95. Furthermore, the amino acid sequence according to SEQ ID NO:3 includes a factor Xa protease cleavage site to allow for the removal of the His tag from the FGG when necessary.

[0025] In a particular embodiment, the isolated recombinant polypeptide of the present invention is expressed in bacterial host cells, preferably in Escherichia coli (E. coli) host cells.

[0026] In a second aspect, the present invention relates to isolated nucleic acids (preferably DNA) encoding polypeptides according to the first aspect of the invention.

[0027] In a preferred embodiment, the isolated nucleic acid of the present invention comprises: (i) the nucleotide sequence according to SEQ ID NO:4, or (ii) the nucleotide sequence having sequence identity with SEQ ID NO:4 and encoding a polypeptide having fibrinogen activity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.25%, at least 95.5%, at least 95.75%, at least 96%, at least 96.25%, at least 96.5%, at least 96.5%, at least 9 6.75%, at least 97%, at least 97.25%, at least 97.5%, at least 97.75%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, or (iii) a nucleotide sequence complementary to the nucleotide sequence of (i) or (ii).

[0028] In other preferred embodiments, the isolated nucleic acid of the present invention comprises: (i) the nucleotide sequence according to SEQ ID NO:6, or (ii) a nucleotide sequence having sequence identity with SEQ ID NO:6 and encoding a polypeptide having fibrinogen activity and His tag functionality of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 90.5%, at least 91%, at least 91.5%, at least 92%, at least 92.5%, at least 93%, at least 93.5%, at least 94%, at least 94.5%, at least 95%, at least 95.25%, at least 95.5%, at least 95.75%, at least 96%, at least 96.25%, and at least 96.5%. At least 96.75%, at least 97%, at least 97.25%, at least 97.5%, at least 97.75%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9%, or (iii) a nucleotide sequence complementary to the nucleotide sequence of (i) or (ii).

[0029] The nucleic acid of the present invention may also contain suitable upstream and downstream regulatory sequences, including promoter sequences, enhancer sequences, stop codons and other sequences known in the art.

[0030] In a third aspect, the present invention relates to vectors comprising the nucleic acids described herein. In this context, the term "vector" includes plasmid vectors, granular vectors, viral vectors, and artificial chromosomes known in the art. In a specific embodiment, the vector is a plasmid vector, for example, an expression vector suitable for expressing proteins in bacterial host cells (particularly *E. coli* host cells). Various vectors are known in the art.

[0031] In a fourth aspect, the present invention relates to host cells comprising the nucleic acid or the vector of the present invention. In a preferred embodiment, the host cell is a bacterial host cell, more preferably an *E. coli* host cell. In this context, the term "*E. coli* host cell" includes any suitable *E. coli* strain known in the art.

[0032] In a fifth aspect, the present invention relates to biohybrid materials comprising the polypeptides of the present invention and conjugates selected from one or more of the following: polyethylene glycol (PEG), PEG derivatives (such as PEG diacrylate (PEG-DA) and poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA)), poly(N-(2-hydroxypropyl)-methacrylamide) (PHPMA) and HPMA copolymers, poly(vinylpyrrolidone) (PVP), poly(ethyleneimine) (PEI), poly(acryloylmorpholine) (PAcM), poly(2-ethyl-2-oxazoline) (PEOZ), divinyl ether maleic anhydride / acid copolymer (DIVEMA), poly(styrene) -Copolymer-maleic anhydride)(SMA), poly(vinyl alcohol))(PVA), and temperature-sensitive polymers including poloxamer (Planic), poly(N-isopropylacrylamide))(PNIPAM, poly(N,N-diethylacrylamide))(PDEAM, poly(methyl vinyl ether))(PMVE), poly(N-vinylcaprolactam))(PNVCl, and mixtures thereof. In this context, the term "poloxam" refers to a nonionic triblock copolymer consisting of a central hydrophobic chain of polyoxypropylene (poly(propylene oxide)) flanked by two hydrophilic chains of polyoxyethylene (poly(ethylene oxide)). In a particular embodiment, poloxamer is, according to a trade name... F-127 is a known compound, that is, a compound with the molecular formula EO known in the art. 100 PO 65 EO 100 A polyoxyethylene-polyoxypropylene block copolymer with CAS number 9003-11-6.

[0033] As used herein, the term "conjugate" refers to the fact that the polypeptide of the present invention and one or more of the above-described portions are covalently linked to each other.

[0034] In a sixth aspect, the present invention relates to the in vitro use of the polypeptides or biohybrid materials of the present invention, i.e., uses not performed on human or animal bodies. Specifically, the present invention relates to the use of the isolated recombinant polypeptides or biohybrid materials of the present invention as coatings for cell culture plates, as 3D cell culture substrates, as in vitro tissue / organ models, as in vitro drug testing platforms, as in vitro cell and / or drug delivery platforms, as coatings for cell enrichment and / or isolation, as coatings for the enrichment and / or isolation of growth factor (FGF-2), as bioinks for bioprinting, or in enzyme-free cell isolation.

[0035] In a related seventh aspect, the present invention relates to the application of the recombinant polypeptide or the biohybrid material of the present invention in the human or animal body, i.e., for wound healing, in vivo tissue engineering, in vivo tissue / organ repair, or in vivo cell and / or drug delivery.

[0036] In an eighth aspect, the present invention relates to a method for generating the polypeptide of the present invention, comprising the following steps:

[0037] (a) Providing the host cell of the present invention,

[0038] (b) The host cells are cultured under conditions that allow expression of the polypeptide, and

[0039] (c) Isolate the polypeptide from the host cell and / or culture medium.

[0040] In this respect, the host cell is preferably a bacterial host cell, and more preferably an Escherichia coli host cell.

[0041] Methods for culturing the host cells under conditions that allow expression of the polypeptide, and methods for isolating the polypeptide from the host cells and / or culture medium, are not particularly limited and are known in the art.

[0042] In this invention, a partial sequence of human fibrinogen was generated in *E. coli*, consisting of a fibrinogen γ-chain (FGG) and a His tag for purification. Furthermore, studies have shown that FGG, as a coating for cell culture plates, is indistinguishable from human fibrinogen. Research also indicates that modification with synthetic polymers can produce the biohybrid material FGG-PEG, which is photoactive and can be photocrosslinked. Both forms of this material, the basic biopolymer FGG and its biohybrid form, can be used in a variety of cell culture and laboratory solution applications.

[0043] The biological characteristics of FGG derive from its parent molecule, fibrinogen. Fibrinogen is a blood protein and a precursor of fibrin, which physiologically serves as (a) a temporary cellular matrix, (b) hemostatic properties (for wound healing applications), (c) enhancing stem cell differentiation / delivery, and (d) inducing angiogenesis. FGG consists of the total mature protein sequence of the fibrinogen γ chain plus a His tag and contains binding sites for integrins, leukocytes, platelets, fibroblasts, endothelial cells, and fibroblast growth factor 2 (FGF-2).

[0044] Fibrinogen is widely used in biomedical applications (especially as a tissue sealant) and is typically derived from blood. While blood represents a widely available resource, there are several drawbacks to its use. Specifically, there is always a residual risk of pathogenic transmission, and the resulting fibrinogen raw material exhibits considerable batch-to-batch variability. To address these drawbacks, the present invention provides a recombinant partial fibrinogen sequence produced in *E. coli*. The advantage of using a simple host microorganism, in contrast to mammalian cell culture, lies in the absence of a virus source and more cost-effective production. In fact, the vast majority of recombinant human fibrinogen is produced in mammalian cell lines. The advantages of recombinant human FGG from *E. coli* include (i) virus-free production, (ii) cost-effective production via large-scale microbial fermentation, (iii) uniform properties (e.g., in terms of molecular weight (MW) and / or pI), and (iv) a well-defined structure for further chemical modification if desired.

[0045] It is noteworthy that the isolated recombinant peptides of the present invention surprisingly and advantageously employ a functionally active conformation (e.g., in relation to the active binding sites of integrins, leukocytes, platelets, fibroblasts, endothelial cells, and FGF-2) spontaneously, for example, after changing the buffer from a buffer containing a large amount of dissociating agent (such as, for example, 5M urea), or even directly from the corresponding solution containing a large amount of dissociating agent. Furthermore, the inclusion bodies (IBs) of the peptides can even be used directly, for example, for coating plates, without any need for dissolution. This is even more surprising, as the fibrinogen γ chain itself, i.e., in the absence of other fibrinogen chains (α and β chains), has no native conformation. However, the peptides of the present invention spontaneously employ a conformation indistinguishable from native fibrinogen, for example, when used as a coating for cell culture plates. Therefore, no complex and time-consuming refolding procedures are required using the peptides of the present invention. Moreover, the advantage of not requiring chromatographic separation / isolation makes the product even more cost-effective.

[0046] Studies have shown that when used as a cell culture coating, FGG's performance is indistinguishable from that of natural fibrinogen. The primary application of FGG is as a cell plate coating for mammalian cell culture. This can be further distinguished from other coatings by its tendency to bind and stabilize FGF-2 (solid growth factor delivery) and many specific cell types. FGG, in the form of insoluble inclusion bodies, can be used as a biodegradable matrix for harvesting anchorage-dependent cells from tissues or blood. Similarly, it can be used in this form to modify disposable bags or chromatography membranes, all for the purpose of providing specific cell and growth factor affinity surfaces.

[0047] The advantages of the basic material FGG according to the invention over existing solutions all relate to the fact that it does not require the use of homologous fibrinogen from human or animal blood, i.e., it is a certified material with reproducible material properties and no risk of pathogenic transmission. Other advantages stem from the fact that the production is carried out in E. coli, not in mammalian cell culture, thus providing an animal-free process, virus-free production, and considerable cost-effectiveness.

[0048] To broaden the scope of applications of FGG according to the present invention, the base material may also be modified to create biohybrid materials that provide additional functionality. These biohybrid FGG biopolymers can be used in 3D mammalian cell culture, or even more specifically, as photoactive bioinks in bioprinting, or as temperature-responsive materials in the field of enzyme-free cell separation.

[0049] The advantages of such modified biohybrids according to the invention over existing solutions again involve obtaining the biological component FGG from recombinant technology rather than from natural sources. Therefore, the modified biohybrids will possess more reproducible mechanical and biological properties. Furthermore, the creation of biohybrids from recombinant fibrinogen produced in mammalian cell cultures will certainly be limited by high costs.

[0050] The attached image shows:

[0051] Figure 1 ：

[0052] The sequence of FGG, including the target binding site, disulfide bonds, and connections to other chains, as well as a 3D FGG structural model, are shown.

[0053] Figure 2 ：

[0054] After IPTG induction for 0 and 3 hours, SDS-PAGE (A) and Western blot (B) of soluble and insoluble fractions of cell lysates, M: molecular weight marker (expressed as kDa), +: positive control as fibrinogen from human plasma, the location of the γ chain is highlighted in red.

[0055] Figure 3 ：

[0056] Purification Procedure Overview. Two-step purification can be used for FGG purification. Step 1: Chromatogram of dissolved inclusion bodies run on an IMAC column (HisTrap, 5 mL, GE Healthcare). The gray area represents a sharp elution peak containing the labeled FGG protein. Step 2: Size exclusion chromatography (SEC, Superdex 75 pg, GE Healthcare) of the peak eluted from the affinity column resulted in a peak highlighted in gray. Further analysis of the peak by SDS-PAGE showed that FGG was separated at approximately 55 kDa.

[0057] Figure 4:

[0058] (A) Polymerization assay of recombinant FGG with FXIII.0 (control), with time above lane showing increased reaction duration and FGG dimer formation intensity at 100 kDa. (B) FGG in cell culture medium resulted in a dose-dependent increase in survival compared to control (cell culture medium only).

[0059] Figure 5 ：

[0060] (A) CTB assay, showing AD-MSC proliferation on different types of cell culture coatings during culture days 1, 3, and 7; (B) Growth of AD-MSCs in FGG, fibrinogen, BSA, and uncoated cell culture wells, shown as confluence as determined by Fiji. Cell concentration: 15,000 cells / well; using Cytation 5 TM System imaging (4× magnification).

[0061] Figure 6 ：

[0062] After 1, 3, and 7 days of culture, AD-MSCs grew in FGG, fibrinogen, BSA, and uncoated cell culture wells at 15,000 cells / well; using Cytation 5 TM The system was used for fluorescence imaging (4× magnification), and cells were stained with calcein-AM. Cells grew on BSA without coating and formed aggregates.

[0063] Figure 7 ：

[0064] Cell morphology of AD-MSCs after 24 h of growth in FGG, fibrinogen, BSA, and uncoated cell culture wells; cell concentration: 15,000 cells / well in a 48-well plate; using Cytation 5 TMThe system was used for fluorescence imaging (left: 4×, right: 20× magnification), live cell imaging (calcein-AM, green), nuclear staining (DAPI, blue), dead cell imaging (propidium iodide, red), FGG is a coating of purified soluble protein, and FGG2 coating is directly derived from unpurified soluble IB.

[0065] Figure 8 ：

[0066] Time-scan oscillation analysis of FGG-PEGDA (FGG biohybrid biopolymer modified with photoactive PEG-DA) showed the formation of a strong hydrogel during in-situ UV light irradiation. Rheological parameters were: 1% amplitude, 1 Hz, 37 °C, FGG-PEGDA 19 mg / ml.

[0067] This invention relates to the following amino acid and nucleotide sequences:

[0068] SEQ ID NO:1

[0069] Fibrinogen γ chain (FGG)

[0070] MYVATRDNCCILDERFGSYCPTTCGIADFLSTYQTKVDKDLQSLEDILHQVENKTSEVKQLIKAIQLTYNPDESSKPNMIDAATLKSRKMLEEIMKYEASILT HDSSIRYLQEIYNSNNQKIVNLKEKVAQLEAQCQEPCKDTVQIHDITGKDCQDIANKGAKQSGLYFIKPLKANQQFLVYCEIDGSGNGWTVFQKRLDGSVDFK KNWIQYKEGFGHLSPTGTTEFWLGNEKIHLISTQSAIPYALRVELEDWNGRTSTADYAMFKVGPEADKYRLTYAYFAGGDAGDAFDGFDFGDDPSDKFFTSHN GMQFSTWDNDNDKFEGNCAEQDGSGWWMNKCHAGHLNGVYYQGGTYSKASTPNGYDNGIIWATWKTRWYSMKKTTMKIIPFNRLTIGEGQQHHLGGAKQAGDV

[0071] SEQ ID NO:2

[0072] His tags

[0073] MGHHHHHHHHHHSSGHIEGRHMLEDI

[0074] SEQ ID NO 3

[0075] His Tag - FGG

[0076] MGHHHHHHHHHHSSGHIEGRHMLEDIMYVATRDNCCILDERFGSYCPTTCGIADFLSTYQTKVDKDLQSLEDILHQVENKTSEVKQLIKAIQLTYNPDESSKPNMIDAATLKSRKMLEEIMKYEASILTHDSSIRYLQEIYNSNNQKIVNLKEKVAQLEAQCQEPCKDTVQIHDITGKDCQDIANKGAKQSGLYFIKPLKANQQFLVYCEIDGSGNGWTVFQKRLDGSVDFKKNWIQYKEGFGHLSPTGTTEFWLGNEKIHLISTQSAIPYALRVELEDWNGRTSTADYAMFKVGPEADKYRLTYAYFAGGDAGDAFDGFDFGDDPSDKFFTSHNGMQFSTWDNDNDKFEGNCAEQDGSGWWMNKCHAGHLNGVYYQGGTYSKASTPNGYDNGIIWATWKTRWYSMKKTTMKIIPFNRLTIGEGQQHHLGGAKQAGDV

[0077] SEQ ID NO:4

[0078] FGG encoded sequence

[0079]

[0080] SEQ ID NO:5

[0081] Nucleic acid for FGG expression in Escherichia coli

[0082]

[0083] SEQ ID NO:6

[0084] His tag-FGG encoded sequence

[0085]

[0086] SEQ ID NO:7

[0087] AGDV binding site

[0088] AGDV

[0089] SEQ ID NO:8

[0090] FGG 427-438 Platelet binding sites on

[0091] HHLGGAKQAGDV

[0092] SEQ ID NO:9

[0093] FGG 404–422 Mac-1 or αMβ2 binding sites at the location

[0094] YSMKKTTMKIIPFNRLTIG

[0095] SEQ ID NO:10

[0096] FGG 217–229 The binding site of integrin αVβ3 at the location

[0097] WTVFQKRLDGSV

[0098] SEQ ID NO:11

[0099] FGG 373–385 The binding site of integrin αVβ3 at the location

[0100] GVYYQGGTYSKAS

[0101] The present invention will be further illustrated by the following embodiments, but is not limited thereto. Example

[0102] Example 1

[0103] Design and production strains of the construct Tuner TM Creation of (DE3)-FGG

[0104] The gene sequence used for recombinant expression of the human γA chain in Escherichia coli corresponds to the mature protein (amino acids 27-437, gene accession number C9JC84). Figure 1 The gene was generated via a gene synthesis service (Life Technologies). The expression vector pET-16b (Merck Millipore, USA) was selected to generate FGG using the *E. coli* expression system. The FGG gene sequence was cloned in-frame along with the N-terminal polyhistidine affinity tag (His tag) sequence. Sequencing of the entire fragment in both directions confirmed the integrity of the coding sequence. The confirmed plasmid was then used to transform competent cells via heat shock transformation. TM(DE3) Host cell, producing the production strain Tuner TM (DE3)-FGG. After induction with 0.58 mM isopropyl-β-D-thiogalactopyranoside (IPTG), the recombinant biopolymer was expressed as inclusion bodies (IB) in the intracellular fraction at 35°C. Figure 2 ).

[0105] Example 2

[0106] Production and purification of FGG

[0107] FGG is produced in 10L fed-batch cultures, which can be scaled up or down as needed. It is produced at pH 6.8 in minimal DNB medium containing 50 mg / ml carbenicillin, using a tuner... TM (DE3)-FGG preculture was used for inoculation, having been cultured for 10–12 h (OD600 = 0.1). The main culture was performed in a stainless steel benchtop bioreactor (Sartorius) equipped with all necessary sensors for process control. The process was carried out in minimal DNB medium containing 50 mg / ml carbenicillin. Operating parameters were set and controlled at pH 6.8, 35°C, an aeration rate of 4 L / min, and a dissolved oxygen level of 30% air saturation. Glucose addition (0.8 g / min) was initiated after glucose depletion in the main medium. Typical fermentation lasted 18 h, after which the medium was removed from the biomass by centrifugation.

[0108] Cell pellets were washed, resuspended in lysis buffer (50 mM MOPS, 150 mM NaCl, 1 mM EDTA), and lysed in a Freund's crusher at 9 psi through multiple channels (8×). The resulting mixture was homogenized and centrifuged at 10,000×g for 60 min at 4 °C. Pellet pellets containing IB were washed and centrifuged multiple times (3×) to remove residual impurities in a buffer of 50 mM Tris HCl, 100 mM NaCl, 1 M urea, 1 mM EDTA, and 1% Triton. The typical total yield of this process is 350 g wet weight of FGG-containing IB (70% total FGG protein in 1 g IB).

[0109] Further processing of FGG depends on its final application. Since this protein is not present in the native environment of the intact fibrinogen molecule, it is not expected to possess a "native" conformation. Its biological activity is primarily sequence-dependent. Results showed that a simple dissolution of FGG in (50 mM Tris HCl, 150 mM NaCl, 5 M urea, pH 8, frozen overnight, thawed, and then centrifuged at 15,000 × g and 4 °C for 30 min to remove aggregates) can be used for coating cell culture plates with excellent results (see [link to relevant documentation]). Figure 7 Additionally, IB can be used for direct coating of plates without the need for dissolution. For applications requiring high purity (>90%) of soluble FGG protein, a two-step chromatographic purification process has been developed, consisting of ion metal affinity chromatography (IMAC) and size exclusion chromatography (SEC).

[0110] Details of the purification procedure

[0111] use A purification system (GE Healthcare, USA) was used to apply soluble FGG to a HisTrap HP column (GE Healthcare, USA, column volume, CV = 5 ml) previously equilibrated with 5CV equilibration buffer (5 M urea, 50 mM Tris-HCl, 150 mM NaCl, 20 mM imidazole, pH 8). The column was then washed with 5CV equilibration buffer to remove impurities. Elution was performed with a buffer containing 5 M urea, 50 mM Tris-HCl, 150 mM NaCl, 200 mM imidazole, pH 8. The flow rate was maintained at 5 ml / min. The eluted IMAC fraction (5 ml) was further purified by size exclusion chromatography using a HiLoad Superdex 75 pg (GE Healthcare, USA, CV = 120 ml). The SEC column was equilibrated with 2CV equilibration buffer (10 mM Tris-HCl). The sample was then applied and eluted from the column using 1.5 CV run buffer (50 mM Tris HCl, 150 mM NaCl, 2 M urea, pH 8). The flow rate in the SEC was maintained at 1 ml / min. After purification, the typical purity of the 55 kDa FGG protein was 64.5% after IMAC and 91.3% after SEC. After SEC, the sample was further purified using… 20 (Sartorius, MWCO = 30 kDa) was used to buffer and exchange PBS at pH 7.4, while simultaneously removing urea. The final FGG product was filtered through a 0.22 μm PES sterile filter (Sartorius, Germany) and frozen before further use. Figure 3 ).

[0112] Example 3

[0113] Characterization and application of FGG

[0114] One approach to characterizing recombinant FGG produced in *E. coli* is to investigate whether its cross-linking sites for factor XIII (Gln398 and Lys406) can be obtained in a manner similar to that of natural fibrinogen. Purified soluble recombinant FGG (170 μg / mL) was polymerized with factor XIII (0.5 mg / mL, 1.1 U / mL) with the addition of human α-thrombin (0.1 U / mL). The reaction was run at 37 °C in 50 mmol / L TBS buffer at pH 7.4 and terminated by adding 1% SDS and 2% 2-mercaptoethanol at selected intervals. A control sample (labeled 0) was prepared by adding SDS and 2-mercaptoethanol to fibrinogen prior to thrombin and factor XIII. SDS-PAGE analysis demonstrated that the recombinant FGG could be cross-linked with factor XIII, producing dimers (approximately 100 kDa) and higher molecular weight FGG structures after 30 min (Figure 4).

[0115] The bioactivity of the generated FGG was tested in mammalian cell cultures. Mammalian cells, such as adipose-derived mesenchymal stem cells (AD-MSCs) used in this experiment, require the ability to adhere to the extracellular matrix for growth, communication, and other cell-to-cell interactions. Cell culture plates were coated with recombinant FGG, along with human fibrinogen and BSA. Uncoated plates were used as a control group. All proteins were coated at the same concentration (500 μg / ml).

[0116] MSCs were cultured on various coatings, and cell adhesion, confluence, morphology, and viability were examined. It was demonstrated that the FGG and fibrinogen coatings exhibited the highest activity (indirectly determined as proliferation using CTB assays). Furthermore, FGG promoted cell proliferation in a dose-dependent manner. Figure 5 Cells growing on FGG and fibrinogen showed higher confluence, while cells growing on integrin-free BSA or uncoated BSA developed aggregation. Figure 6 In addition, cell morphology examination showed that AD-MSCs growing on FGG and fibrinogen exhibited a spindle-shaped shape and morphology. Figure 7 The performance of recombinant FGG in mammalian cell cultures was indistinguishable from that of native fibrinogen. Results were also confirmed for other cell types (data not shown).

[0117] Cell culture details

[0118] The cell experiments shown were performed using cultures of human adipose-derived mesenchymal stem cells (hAD-MSCs). Cells were expanded in α-MEM medium (1 g L⁻¹ glucose, 10% human serum, 2 mM L⁻¹ glutamine, and 50 μg mL⁻¹ gentamicin) and harvested by accutase treatment. The experiments were performed using hAD-MSCs from passage 2 to passage 8.

[0119] Example 4

[0120] Modification of FGG biopolymers

[0121] While FGG has been shown to be crosslinked as Factor XIII of natural fibrinogen (Figure 4), this does not provide sufficient stability for generating 3D networks. For example, additional modifications are required to crosslink FGG to create hydrogels for 3D cell encapsulation. Crosslinking can be achieved through chemical modification using synthetic polymers such as PEG. Another strategy is to use the synthetic component to create biohybrid materials with tunable properties. For example, modification with PEG diacrylate (PEG-DA) provides photoactive FGG materials that can be used for applications such as bioprinting, stereolithography, and 3D cell culture. Modification with polymers such as Pluronic F127 provides FGG temperature switches that can be used for cell delivery or, in cell culture applications, for creating responsive sheets for enzyme-free cell separation. In all cases, FGG provides the bioactivity of the biohybrid material, while the synthetic polymer contributes to the desired physicochemical properties. The purpose of modifying the synthetic portion (modified biohybrid material) is to (i) introduce crosslinking or design / control mechanical properties (PEG) or (ii) add "tunable" properties, such as temperature switching (e.g., Pluronic F127) or photopolymerization (e.g., PEG-DA). Figure 8 ). sequence list <110> Sartorius Lab Instruments GmbH & Co. KG <120> Recombinant protein based on fibrinogen <130> S15165WO - kl <140> EP 20 176 426.3 <141> 26.05.2020 <160> 11 <170> PatentIn version 3.5 <210> 1 <211> 412 <212> PRT <213> Artificial sequence <220> <223> Fibrinogen gamma chain (FGG) <400> 1 Met Tyr Val Ala Thr Arg Asp Asn Cys Cys Ile Leu Asp Glu Arg Phe 1 5 10 15 Gly Ser Tyr Cys Pro Thr Thr Cys Gly Ile Ala Asp Phe Leu Ser Thr 20 25 30 Tyr Gln Thr Lys Val Asp Lys Asp Leu Gln Ser Leu Glu Asp Ile Leu 35 40 45 His Gln Val Glu Asn Lys Thr Ser Glu Val Lys Gln Leu Ile Lys Ala 50 55 60 Ile Gln Leu Thr Tyr Asn Pro Asp Glu Ser Ser Lys Pro Asn Met Ile 65 70 75 80 Asp Ala Ala Thr Leu Lys Ser Arg Lys Met Leu Glu Glu Ile Met Lys 85 90 95 Tyr Glu Ala Ser Ile Leu Thr His Asp Ser Ser Ile Arg Tyr Leu Gln 100 105 110 Glu Ile Tyr Asn Ser Asn Asn Gln Lys Ile Val Asn Leu Lys Glu Lys 115 120 125 Val Ala Gln Leu Glu Ala Gln Cys Gln Glu Pro Cys Lys Asp Thr Val 130 135 140 Gln Ile His Asp Ile Thr Gly Lys Asp Cys Gln Asp Ile Ala Asn Lys 145 150 155 160 Gly Ala Lys Gln Ser Gly Leu Tyr Phe Ile Lys Pro Leu Lys Ala Asn 165 170 175 Gln Gln Phe Leu Val Tyr Cys Glu Ile Asp Gly Ser Gly Asn Gly Trp 180 185 190 Thr Val Phe Gln Lys Arg Leu Asp Gly Ser Val Asp Phe Lys Lys Asn 195 200 205 Trp Ile Gln Tyr Lys Glu Gly Phe Gly His Leu Ser Pro Thr Gly Thr 210 215 220 Thr Glu Phe Trp Leu Gly Asn Glu Lys Ile His Leu Ile Ser Thr Gln 225 230 235 240 Ser Ala Ile Pro Tyr Ala Leu Arg Val Glu Leu Glu Asp Trp Asn Gly 245 250 255 Arg Thr Ser Thr Ala Asp Tyr Ala Met Phe Lys Val Gly Pro Glu Ala 260 265 270 Asp Lys Tyr Arg Leu Thr Tyr Ala Tyr Phe Ala Gly Gly Asp Ala Gly 275 280 285 Asp Ala Phe Asp Gly Phe Asp Phe Gly Asp Asp Pro Ser Asp Lys Phe 290 295 300 Phe Thr Ser His Asn Gly Met Gln Phe Ser Thr Trp Asp Asn Asp Asn 305 310 315 320 Asp Lys Phe Glu Gly Asn Cys Ala Glu Gln Asp Gly Ser Gly Trp Trp 325 330 335 Met Asn Lys Cys His Ala Gly His Leu Asn Gly Val Tyr Tyr Gln Gly 340 345 350 Gly Thr Tyr Ser Lys Ala Ser Thr Pro Asn Gly Tyr Asp Asn Gly Ile 355 360 365 Ile Trp Ala Thr Trp Lys Thr Arg Trp Tyr Ser Met Lys Lys Thr Thr 370 375 380 Met Lys Ile Ile Pro Phe Asn Arg Leu Thr Ile Gly Glu Gly Gln Gln 385 390 395 400 His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 405 410 <210> 2 <211> 26 <212> PRT <213> Artificial sequence <220> <223> His tag <400> 2 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Leu Glu Asp Ile 20 25 <210> 3 <211> 438 <212> PRT <213> Artificial sequence <220> <223> His-tag-FGG <400> 3 Met Gly His His His His His His His His His His Ser Ser Gly His 1 5 10 15 Ile Glu Gly Arg His Met Leu Glu Asp Ile Met Tyr Val Ala Thr Arg 20 25 30 Asp Asn Cys Cys Ile Leu Asp Glu Arg Phe Gly Ser Tyr Cys Pro Thr 35 40 45 Thr Cys Gly Ile Ala Asp Phe Leu Ser Thr Tyr Gln Thr Lys Val Asp 50 55 60 Lys Asp Leu Gln Ser Leu Glu Asp Ile Leu His Gln Val Glu Asn Lys 65 70 75 80 Thr Ser Glu Val Lys Gln Leu Ile Lys Ala Ile Gln Leu Thr Tyr Asn 85 90 95 Pro Asp Glu Ser Ser Lys Pro Asn Met Ile Asp Ala Ala Thr Leu Lys 100 105 110 Ser Arg Lys Met Leu Glu Glu Ile Met Lys Tyr Glu Ala Ser Ile Leu 115 120 125 Thr His Asp Ser Ser Ile Arg Tyr Leu Gln Glu Ile Tyr Asn Ser Asn 130 135 140 Asn Gln Lys Ile Val Asn Leu Lys Glu Lys Val Ala Gln Leu Glu Ala 145 150 155 160 Gln Cys Gln Glu Pro Cys Lys Asp Thr Val Gln Ile His Asp Ile Thr 165 170 175 Gly Lys Asp Cys Gln Asp Ile Ala Asn Lys Gly Ala Lys Gln Ser Gly 180 185 190 Leu Tyr Phe Ile Lys Pro Leu Lys Ala Asn Gln Gln Phe Leu Val Tyr 195 200 205 Cys Glu Ile Asp Gly Ser Gly Asn Gly Trp Thr Val Phe Gln Lys Arg 210 215 220 Leu Asp Gly Ser Val Asp Phe Lys Lys Asn Trp Ile Gln Tyr Lys Glu 225 230 235 240 Gly Phe Gly His Leu Ser Pro Thr Gly Thr Thr Glu Phe Trp Leu Gly 245 250 255 Asn Glu Lys Ile His Leu Ile Ser Thr Gln Ser Ala Ile Pro Tyr Ala 260 265 270 Leu Arg Val Glu Leu Glu Asp Trp Asn Gly Arg Thr Ser Thr Ala Asp 275 280 285 Tyr Ala Met Phe Lys Val Gly Pro Glu Ala Asp Lys Tyr Arg Leu Thr 290 295 300 Tyr Ala Tyr Phe Ala Gly Gly Asp Ala Gly Asp Ala Phe Asp Gly Phe 305 310 315 320 Asp Phe Gly Asp Asp Pro Ser Asp Lys Phe Phe Thr Ser His Asn Gly 325 330 335 Met Gln Phe Ser Thr Trp Asp Asn Asp Asn Asp Lys Phe Glu Gly Asn 340 345 350 Cys Ala Glu Gln Asp Gly Ser Gly Trp Trp Met Asn Lys Cys His Ala 355 360 365 Gly His Leu Asn Gly Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala 370 375 380 Ser Thr Pro Asn Gly Tyr Asp Asn Gly Ile Ile Trp Ala Thr Trp Lys 385 390 395 400 Thr Arg Trp Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro Phe 405 410 415 Asn Arg Leu Thr Ile Gly Glu Gly Gln Gln His His Leu Gly Gly Ala 420 425 430 Lys Gln Ala Gly Asp Val 435 <210> 4 <211> 1236 <212> DNA <213> Artificial sequence <220> <223> FGG coding sequence <400> 4 atgtatgttg caacccgtga taattgctgc attctggatg aacgttttgg tagctattgt 60 ccgaccacct gtggtattgc agattttctg agcacctatc agaccaaagt tgataaagat 120 ctgcagagcc tggaagatat tctgcatcag gttgaaaaca aaaccagcga agttaaacag 180 ctgattaaag caattcagct gacctataat ccggatgaaa gcagcaaacc gaatatgatt 240 gatgcagcaa ccctgaaaag ccgtaaaatg ctggaagaga tcatgaaata tgaagccagc 300 attctgaccc atgatagcag cattcgttat ctgcaagaaa tctacaatag caataatcag 360 aaaattgtga atctgaaaga aaaagtggca cagctggaag cacagtgtca agaaccgtgt 420 aaagataccg ttcagattca tgatatcacc ggtaaagatt gtcaggatat tgcaaacaaa 480 ggtgcaaaac agagcggtct gtattttatc aaaccgctga aagcaaatca gcagtttctg 540 gtgtattgcg aaattgatgg tagcggtaat ggttggaccg tttttcagaa acgtctggat 600 ggtagcgtgg acttcaaaaa aaactggatt cagtataaag aaggctttgg tcatctgagc 660 ccgaccggca ccaccgaatt ttggctgggt aatgaaaaaa ttcatctgat tagcacccag 720 agcgcaattc cgtatgcact gcgtgttgaa ctggaagatt ggaatggtcg taccagcacc 780 gcagattatg caatgtttaa agttggtccg gaagccgata aatatcgtct gacctatgca 840 tattttgccg gtggtgatgc cggtgatgca tttgatggtt ttgattttgg tgatgatccg 900 agcgataaat tctttaccag ccataatggt atgcagttta gcacctggga taacgataac 960 gataaatttg aaggcaattg tgccgaacag gatggtagtg gttggtggat gaataaatgt 1020 catgcaggtc atctgaacgg cgtttattat cagggtggca cctatagcaa agcaagcacc 1080 ccgaatggtt atgataatgg tattatttgg gcaacctgga aaacccgttg gtacagcatg 1140 aaaaaaacca ccatgaaaat catcccgttt aaccgtctga ccattggtga aggtcagcag 1200 catcatctgg gtggtgcaaa acaagccggt gatgtg <210> 5 <211> 1268 <212> DNA <213> Artificial sequence (Artificial sequence) <220> <223> Remove your FGG license plate <400> 5 ctcgaggata tcatgtatgt tgcaacccgt gataattgct gcattctgga tgaacgtttt ggtagctatt gtccgaccac ctgtggtatt gcagattttc tgagcaccta tcagaccaaa 180. gttgataaag atctgcagag cctggaagat attctgcatc aggttgaaaa caaaaccagc gagttaaac agctgattaa agcaattcag ctgacctata atccggatga aagcagcaaa ccgaatatga ttgatgcagc aaccctgaaa agccgtaaaa tgctggaaga gatcatgaaa sight gcattctgac ccatgatagc agcattcgtt atctgcaaga aatctacaat agcaataatc agaaaattgt gaatctgaaa gaaaaagtgg cacagctgga agcacagtgt caagaccgt gtaaagatac cgttcagatt catgatatca ccggtaaaga ttgtcaggat attgcaaaca aaggtgcaaa acagagcggt ctgtatttta tcaaaccgct gaaagcaaat cagcagtttc tggtgtattg cgaaattgat ggtagcggta atggttggac cgtttttcag 600 aaacgtctgg atggtagcgt ggacttcaaa aaaaactgga ttcagtata agaaggcttt ggtcatctga gcccgaccgg crack ttttggctgg gtaatgaaaa aattcatctg attagcaccc agagcgcaat tccgtatgca ctgcgtgttg aactggaaga ttggaatggt 780 cgtaccagca ccgcagatta tgcaatgttt aaagttggtc cggagccga taaatatcgt ctgacctatg catattttgc cggtggtgat gccggtgatg catttgatgg ttttgatttt 900 ggtgatgatc cgagcgataa attctttacc agccataatg gtatgcagtt tagcacctgg gataacgata acgataatt tgaaggcaat tgtgccgaac aggatggtag tggttggtgg atgaataaat gtcatgcagg tcatctgaac ggcgtttatt atcagggtgg cacctatagc 1140. aaagcaagca ccccgaatgg ttatgataat ggtattattt gggcaacctg gaaaaacccgt tggtacagca tgaaaaaaac caccatgaaa atcatcccgt ttaaccgtct caccattggt gaaggtcagc agcatcatct gggtggtgca aaacaagccg gtgatgtgta ataaaaggat ccgtcgac 1268 <210> 6 <211> 1334 <212> DNA <213> Artificial sequence (Artificial sequence) <220> <223> His groundbreaking-FGG cycle <400> 6 atgggccatc atcatcatca tcatcatcatcacacagca gcggccatat cgaaggtcgt catatgctcg aggatatcat gtatgttgca acccgtgata attgctgcat tctggatgaa cgttttggta gctattgtcc gaccacctgt ggtattgcag attttctgag cacctatcag 180 accaaagttg father gcagagcctg father tgcatcaggt tgaaaacaaa accagcgaag ttaaacagct gattaaagc attcagctga cctataatcc ggatgaaagc agcaaaccga atatgattga tgcagcaacc ctgaaaagcc gtaaaatgct ggaagagatc atgaaatatg aagccagcat tctgacccat gatagcagca ttcgttatct gcaagaaatc 480. 480. 480. 480. 480. 480. 480. 480. 480. 480. 480 cagtgtcaag aaccgtgtaa agataccgtt cagattcatg atatcaccgg taaagattgt caggatattg caaacaaagg tgcaaaacag agcggtctgt attttatcaa accgctgaaa gcaaatcagc agtttctggt gtattgcgaa attgatggta gcggtaatgg ttggaccgtt ttcagaaac gtctggatgg tagcgtggac ttcaaaaaaa actggattca gtataaagaa ggctttggtc atctgagccc gaccggcacc accgaatttt ggctgggtaa tgaaaaaatt 780 catctgatta gcacccagag cgcaattccg tatgcactgc gtgttgaact ggaagattgg aatggtcgta ccagcaccgc agattatgca atgtttaaag ttggtccgga agccgataaa tatcgtctga cctatgcata ttttgccggt ggtgatgccg gtgatgcatt tgatggtttt 960 gattttggtg atgatccgag cgataaattc tttaccagcc ataatggtat gcagtttagc acctgggata accgataacga taatttgaa ggcaattgtg ccgaacagga tggtagtggt tggtggatga ataaatgtca tgcaggtcat ctgaacggcg tttattatca gggtggcacc tatagcaaag caagcacccc gaatggttat ttatttgggc aacctggaaa acccgttggt acagcatgaa aaaaaccacc atgaaaatca tcccgtttaa ccgtctgacc attggtgaag gtcagcagca tcatctgggt ggtgcaaaac aagccggtga tgtgtaataa 1320 aaggatccgt cgac 1334 <210> 7 <211> 4 <212> PRT <213> Artificial sequence <220> <223> binding site <400> 7 Ala Gly Asp Val 1 <210> 8 <211> 12 <212> PRT <213> Artificial sequence <220> <223> binding sites on FGG <400> 8 His His Leu Gly Gly Ala Lys Gln Ala Gly Asp Val 1 5 10 <210> 9 <211> 19 <212> PRT <213> Artificial sequence <220> <223> binding sites on FGG <400> 9 Tyr Ser Met Lys Lys Thr Thr Met Lys Ile Ile Pro Phe Asn Arg Leu 1 5 10 15 Thr Ile Gly <210> 10 <211> 12 <212> PRT <213> Artificial sequence <220> <223> binding sites on FGG <400> 10 Trp Thr Val Phe Gln Lys Arg Leu Asp Gly Ser Val 1 5 10 <210> 11 <211> 13 <212> PRT <213> Artificial sequence <220> <223> binding sites on FGG <400> 11 Gly Val Tyr Tyr Gln Gly Gly Thr Tyr Ser Lys Ala Ser 1 5 10

Claims

1. The isolated recombinant polypeptide, which consists of the following: (i) The γ chain of fibrinogen, and (ii) His tag, The γ chain of the fibrinogen is composed of the amino acid sequence according to SEQ ID NO: 1, and the polypeptide is composed of the amino acid sequence according to SEQ ID NO:

3.

2. The isolated recombinant polypeptide of claim 1, wherein the γ chain of the fibrinogen contains binding sites targeting integrins, leukocytes, platelets, fibroblasts, endothelial cells, and / or fibroblast growth factor 2 (FGF-2).

3. The isolated recombinant polypeptide of claim 1, wherein the His tag is composed of... Based on the amino acid sequence composition of SEQ ID NO:

2.

4. An isolated nucleic acid encoding a polypeptide as described in any one of claims 1 to 3.

5. The isolated nucleic acid as described in claim 4, comprising the following: (i) Based on the nucleotide sequence of SEQ ID NO: 6, or (ii) is a nucleotide sequence complementary to the nucleotide sequence of (i).

6. A vector comprising the nucleic acid of claim 4 or claim 5.

7. A host cell comprising the nucleic acid of claim 4 or claim 5 or the vector of claim 6.

8. The host cell of claim 7, wherein the host cell is a bacterial host cell.

9. The host cell of claim 7 or claim 8, wherein the host cell is *Escherichia coli* (E. coli). E. coli Host cell.

10. A biohybrid material comprising a polypeptide comprising any one of claims 1 to 3 and a conjugate selected from one or more of the following: polyethylene glycol (PEG), PEG derivatives, PEG diacrylate, poly(oligo(ethylene glycol) methyl ether methacrylate), poly(N-(2-hydroxypropyl)-methacrylamide) and HPMA copolymer, poly(vinylpyrrolidone), poly(ethyleneimine), poly(acryloylmorpholine), poly(2-ethyl-2-oxazoline), divinyl ether maleic anhydride / acid copolymer, poly(styrene-copolymer-maleic acid / anhydride), poly(vinyl alcohol), and temperature-sensitive polymers including poloxamer, poly(N-isopropylacrylamide), poly(N,N-diethylacrylamide), poly(methyl vinyl ether), poly(N-vinylcaprolactam), and mixtures thereof.

11. Use of the isolated recombinant polypeptide of any one of claims 1 to 3 or the biohybrid material of claim 10 in the preparation of cell culture plate coatings, 3D cell culture substrates, in vitro tissue / organ models, in vitro drug testing platforms, in vitro cell and / or drug delivery platforms, coatings for cell enrichment and / or isolation, coatings for enrichment and / or isolation of growth factor FGF-2, or bio-inks for bioprinting, or in enzyme-free cell isolation.

12. Use of the isolated recombinant polypeptide of any one of claims 1 to 3 or the biohybrid material of claim 10 in the preparation of medicaments for wound healing, in vivo tissue engineering, in vivo tissue / organ repair, or in vivo cell delivery and / or drug delivery.

13. A method for producing the polypeptide according to any one of claims 1 to 3, comprising the following steps: (a) Providing a host cell according to any one of claims 7 to 9, (b) Culturing the host cells under conditions that allow expression of the polypeptide, and (c) Isolate the polypeptide from the host cell and / or the culture medium.

Images