Recombinant fibrinogen-based proteins

Abstract

The present invention relates to an isolated recombinant polypeptide comprising the gamma chain of fibrinogen and a His tag. The 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 biohybrid material comprising a conjugate comprising said polypeptide, uses of the above, and a method of making said polypeptide.

Description

[0001] This application is a divisional application of the Chinese patent application with application number 202180030887.7.

[0002] 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 the polypeptide, a vector comprising the nucleic acid, a host cell (preferably a bacterial host cell) comprising the nucleic acid or the vector, a biohybrid material comprising a conjugate containing the polypeptide, uses thereof, and a method for producing the polypeptide.

[0003] Proteins 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. The blood protein fibrinogen is a precursor to fibrin and plays an important role in wound healing, hemostasis, and angiogenesis. After blood clot formation, fibrin serves as a new, temporary ECM for tissue regeneration. Fibrinogen is a hexamer composed of a collection of two α, β, and γ chains, interconnected by intra- and interchain disulfide bridges. The natural polymerization of fibrinogen and fibrin is a multistep enzymatic process catalyzed by thrombin following vascular injury. Thrombin cleaves two short amino acid sequences at the amino termini of the Aα and Bβ chains of the fibrin precursor fibrinogen. This exposes polymerization sites within the fibrin molecule, leading to the polymerization of individual fibrinogen molecules into a 3D fibrin network. This network is further stabilized by covalent cross-linking of the γ chains catalyzed by factor XIIIa.

[0004] The most common medical use of fibrin is as a hemostatic agent and tissue sealant. The first FDA-approved fibrin glue on the market was Baxter's However, many other products have since been introduced to the market. These preparations are used as hemostatic agents, sealants, and tissue adhesives in various clinical settings. They are all produced from homologous fibrinogen and thrombin from pooled blood donations. Because fibrin for biomedical and cell culture applications is derived from human or animal blood, there is always a residual risk of pathogenic transmission. In addition, due to the source of the material, the material is subject to natural batch-to-batch variations, which makes it difficult to standardize fibrin materials in biomedical research. These variations may hinder the GMP (Good Manufacturing Practice) requirements for specific newly developed fibrin-based products.

[0005] A safer approach to obtaining pure protein of consistent quality without the risk of pathogenic transmission is recombinant production. Due to the complex structure and post-translational modifications involved, most heterologous expression systems for producing intact fibrinogen molecules are based on mammalian cells. Accordingly, recombinant fibrinogen has been expressed in baby hamster cells, primarily for the purpose of studying its subunit interactions and assembly. In addition, Fully assembled fibrinogen is produced in cell lines. CHO cells have also been used to produce recombinant fibrinogen, and yields have increased over the years. Another approach to obtaining fully assembled fibrinogen is to produce it in the milk of transgenic animals. This has been demonstrated, for example, in cows and mice.

[0006] The applications of fibrinogen are all based on the material's attractive properties, particularly its propensity to bind growth factors and its interaction with platelets, leukocytes, fibroblasts, and endothelial cells. Consequently, fibrinogen is a widely used biopolymer in tissue engineering for the repair of damaged tissues / organs, as a coating or 3D matrix in cell culture, but also as a cell and drug delivery agent, particularly for growth factor delivery. Fibrinogen binds with well-characterized affinities to a variety of growth factors, particularly fibroblast growth factor 2 (FGF-2) and vascular endothelial growth factor (VEGF), as well as heparin. The latter has been used as a linker for growth factors that do not naturally bind to fibrin (nerve growth factor (NGF), NGF-3). Such growth factor-immobilized constructs can be used to deliver growth factors for therapeutic or cell culture applications. For example, in the field of cell culture coatings, fibrin has been very successfully used for solid growth factor delivery, improving the survival and proliferation of MG-63 cells in contact with FGF-2 patterns, which are immobilized by fibrin's natural affinity for the growth factor. There are numerous reports that combining FGF-2 with fibrinogen or fibrin protects the growth factor from degradation, thereby increasing its long-term effects in cell culture. Similarly, the fibrinogen-FGF-2 pair appears to enhance the proliferative capacity of the growth factor compared to fibrinogen or FGF-2 alone.

[0007] Fibrinogen has multiple RGD, AGDV (SEQ ID NO: 7) and integrin binding sites, which enable it to interact with various types of cells. This biological activity makes it an attractive matrix for stem cell differentiation, cell delivery / cell enrichment and tissue engineering. In cell culture applications, fibrinogen coatings have been shown to be a viable alternative to laminin (Geltrex) in induced pluripotent stem cell (iPSC) culture. iPSCs cultured on fibrinogen coatings maintain 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 separation / 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 need for trypsinization. This technology can also be used to capture cancer cells from blood suspensions.

[0008] Despite their interesting bioactivity, fibrinogen-based materials often suffer from poor mechanical properties and rapid degradation. This sometimes necessitates modification of the materials with synthetic polymers to create biohybrid materials with superior properties. These semisynthetic materials combine the inherent bioactivity of fibrinogen with customizable, tunable properties derived from the synthetic moiety.

[0009] PEG is an attractive polymer for modification because it is bioinert and can be used to precisely tune mechanical properties by controlling chain length and degree of cross-linking. Amine-reactive PEG has been conjugated to fibrinogen-producing hydrogels for 3D cell culture that can be loaded with growth factors (PDGF, TGF-β) and support MSC viability and neovascularization. A different approach has been taken to produce PEGylated precursors by cross-linking denatured fibrinogen to PEG-DA. Photopolymerization of these PEGylated precursors results in scaffolds that enable migration, cell spreading, and controlled proteolytic degradation. Another promising approach is to develop scaffolds based on fibrin and F127( F127) copolymer combination thermosensitive hydrogel. F127 copolymers undergo reverse thermal gelation in response to temperature changes. Conjugation of F127 enables control of the physical properties while maintaining the intact cytocompatibility of the hybrid hydrogel.

[0010] As described above, recombinant fibrinogen-based materials could represent a cost-effective and regulatory-compliant source of fibrinogen, opening the door to a variety of cell culture and biomedical applications.

[0011] The technical problem underlying the present invention is therefore to provide a fibrinogen-like material and a corresponding method for producing such a material, which can be recombinantly produced in a rapid, simple and cost-effective manner while avoiding the use of human or animal blood and avoiding the use of mammalian cell cultures.

[0012] The solution to the above technical problem is achieved by the embodiments characterized in the claims.

[0013] In particular, in a first aspect, the present invention relates to an isolated recombinant polypeptide comprising:

[0014] (i) the gamma chain of fibrinogen, and

[0015] (ii) His tag.

[0016] As used herein, the term "recombinant polypeptide" refers to an artificially produced polypeptide, ie, a polypeptide produced by expressing recombinant, ie, artificially produced, DNA in a living cell.

[0017] 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 fibrinogen comprises binding sites for integrins, leukocytes, platelets, fibroblasts, endothelial cells, and / or fibroblast growth factor 2 (FGF-2). Specifically, the γ chain of fibrinogen may comprise one or more or all of the following specific binding sites:

[0018] -Platelet binding site: FGG 427-438 on the surface of the α-integrin IIb γC-dodecapeptide of β3 (HHLGGAKQAGDV; SEQ ID NO: 8)

[0019] -FGG 404-422 (YSMKKTTMKIIPFNRLTIG; SEQ ID NO: 9) at the leukocyte, neutrophil, monocyte integrin Mac-1 or α M β2 binding site

[0020] -FGG 217-229 (WTVFQKRLDGSV; SEQ ID NO:10) and FGG 373-385 Integrin alpha at (GVYYQGGTYSKAS; SEQ ID NO: 11) leading to binding of endothelial cells, fibroblasts and tumor cells V β3 binding site

[0021] -C-terminal region FGG: growth factor FGF-2 and interleukin-β binding site

[0022] - Factor XIII cross-linking sites, FGG lysine at 433 and FGG glutamine at 425 / 426

[0023] More preferably, the fibrinogen gamma chain comprises or consists of (i) an amino acid sequence according to SEQ ID NO: 1, or (ii) an amino acid sequence that has 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%, or at least 97. %, 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 gamma chain activity" refers to the characteristic of having a functional binding site for integrin, leukocytes, platelets, fibroblasts, endothelial cells and / or FGF-2.

[0024] In addition, the recombinant polypeptide of the present invention comprises a His tag, i.e. a polyhistidine tag, comprising an amino acid motif comprising at least 6 histidine (His) residues. Preferably, the His tag comprises or consists of: (i) an amino acid sequence according to SEQ ID NO: 2, or (ii) an amino acid sequence having the following sequence identity to 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 97. %, 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 the feature of having an amino acid motif comprising at least 6 (preferably consecutive) histidine (His) residues.

[0025] In preferred embodiments, the isolated recombinant polypeptide of the present invention comprises: (i) an amino acid sequence according to SEQ ID NO:3, or (ii) an amino acid sequence having the following sequence identity to SEQ ID NO:3: 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 97. %, at least 99.9%, 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 consisting 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. In addition, the amino acid sequence according to SEQ ID NO: 3 contains a Factor Xa protease cleavage site to allow removal of the His tag from FGG when desired.

[0026] In a specific embodiment, the isolated recombinant polypeptide of the present invention is expressed in a bacterial host cell, preferably an Escherichia coli (E. coli) host cell.

[0027] In a second aspect, the present invention relates to an isolated nucleic acid (preferably DNA) encoding a polypeptide according to the first aspect of the invention.

[0028] In preferred embodiments, the isolated nucleic acid of the present invention comprises: (i) a nucleotide sequence according to SEQ ID NO:4, or (ii) a nucleotide sequence having 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 97. or (iii) a nucleotide sequence that is complementary to the nucleotide sequence of (i) or (ii).

[0029] In other preferred embodiments, the isolated nucleic acid of the present invention comprises: (i) a nucleotide sequence according to SEQ ID NO:6, or (ii) a nucleotide sequence having 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 97. , 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 that is complementary to the nucleotide sequence of (i) or (ii).

[0030] The nucleic acids of the invention may also contain appropriate upstream and downstream regulatory sequences, including promoter sequences, enhancer sequences, stop codons, and other sequences known in the art.

[0031] In a third aspect, the present invention relates to a vector comprising the nucleic acid of the present invention. In this context, the term "vector" includes plasmid vectors, cosmid 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.

[0032] In a fourth aspect, the present invention relates to a host cell comprising the nucleic acid of the present invention 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.

[0033] In a fifth aspect, the present invention relates to a biohybrid material comprising a conjugate comprising the polypeptide of the present invention and one or more moieties selected from the group consisting of 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(vinyl pyrrolidone) (PVP), poly(ethyleneimine) (PEI), poly(acryloylmorpholine) (PAcM), poly(2-ethyl 2-oxazoline) (PEOZ), divinyl ether maleic anhydride / acid copolymer (DIVEMA), poly(styrene) -co-maleic acid / anhydride) (SMA), poly(vinyl alcohol) (PVA) and temperature sensitive polymers including poloxamers (Pluronic), 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 "poloxamer" 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 specific embodiment, poloxamers are manufactured under the trade name F-127 is a known compound having the molecular formula EO 100 PO 65 EO 100 , polyoxyethylene-polyoxypropylene block copolymer with CAS number 9003-11-6.

[0034] As used herein, the term "conjugate" refers to the fact that the polypeptide of the present invention and one or more moieties described above are covalently bound to each other.

[0035] In a sixth aspect, the present invention relates to the in vitro use of the polypeptide or biohybrid material of the present invention, i.e., use not performed on the human or animal body. Specifically, the present invention relates to the use of the isolated recombinant polypeptide of the present invention or the biohybrid material of the present invention as a cell culture plate coating, as a 3D cell culture matrix, as an in vitro tissue / organ model, an in vitro drug testing platform, as an in vitro cell and / or drug delivery platform, as a coating for cell enrichment and / or separation, as a coating for growth factor (FGF-2) enrichment and / or separation, as a bio-ink for bioprinting, or in enzyme-free cell separation.

[0036] In a related seventh aspect, the present invention relates to the use of the recombinant polypeptide of the invention or the biohybrid material of the invention for use 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.

[0037] In an eighth aspect, the present invention relates to a method for producing a polypeptide of the present invention, comprising the following steps:

[0038] (a) providing a host cell of the present invention,

[0039] (b) cultivating the host cell under conditions that allow expression of the polypeptide, and

[0040] (c) isolating the polypeptide from the host cell and / or culture medium.

[0041] In this aspect, the host cell is preferably a bacterial host cell, more preferably an E. coli host cell.

[0042] Methods for culturing the host cells under conditions allowing 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.

[0043] In this study, a partial sequence of human fibrinogen, consisting of the fibrinogen gamma chain (FGG) and a His tag for purification, was produced in Escherichia coli. Furthermore, studies have shown that FGG performs indistinguishably with human fibrinogen as a coating for cell culture plates. Studies have also shown that modification with synthetic polymers can produce a biohybrid material, FGG-PEG, which is photoactive and can be photocrosslinked. Both forms of the material, the basic biopolymer FGG and its biohybrid form, can be used in a variety of cell culture and laboratory solution applications.

[0044] The biological properties of FGG are derived from its parent molecule, fibrinogen. Fibrinogen is a blood protein and precursor of fibrin that physiologically serves as (a) a temporary cell matrix, (b) possesses hemostatic properties (for wound healing applications), (c) enhances stem cell differentiation / delivery, and (d) induces angiogenesis. FGG consists of the entire mature protein sequence of the fibrinogen gamma chain plus a His tag and contains binding sites for integrins, leukocytes, platelets, fibroblasts, endothelial cells, and fibroblast growth factor 2 (FGF-2).

[0045] Fibrinogen is widely used in biomedical applications (especially as tissue sealant) and is generally derived from blood. Although blood represents a widely available resource, there are several disadvantages in its use. Specifically, there is always a residual risk of pathogenicity transmission, and there is considerable batch-to-batch variability in the fibrin raw material produced. In order to address these shortcomings, the present invention provides a recombinant partial fibrinogen sequence produced in Escherichia coli. In contrast to mammalian cell culture, the advantage of using a simple host microorganism is that there is no virus source and more cost-effective production. In fact, the vast majority of recombinant human fibrinogens are produced in mammalian cell lines. The advantages of recombinant human FGG from Escherichia coli include (i) virus-free production, (ii) cost-effective production by a large number of microbial fermentations, (iii) uniform properties (e.g., with respect to molecular weight (MW) and / or pI), and (iv) if necessary, a clear structure for further chemical modification.

[0046] It is noteworthy that the isolated recombinant polypeptides of the present invention surprisingly and advantageously adopt a functionally active conformation (e.g., with respect to active binding sites for integrins, leukocytes, platelets, fibroblasts, endothelial cells, and FGF-2) in a spontaneous manner, for example, after buffer exchange from a buffer containing a large amount of chaotropic agents (such as, for example, 5M urea), or even directly from a corresponding solution containing a large amount of chaotropic agents. In addition, the inclusion bodies (IBs) of the polypeptides can even be used directly, for example, for coating plates, without the need for dissolution at all. This is even more surprising because the fibrinogen gamma chain itself, i.e., in the absence of other fibrinogen chains (α chain and β chain), has no native conformation. However, the polypeptides of the present invention spontaneously adopt a conformation that is indistinguishable from native fibrinogen, for example, when used as a coating for cell culture plates. Therefore, there is no need to perform any complex and time-consuming refolding procedures using the polypeptides of the present invention. In addition, the advantage of not having to perform chromatographic separation / isolation makes the product even more cost-effective.

[0047] Studies have shown that when used as a cell culture coating, FGG's performance is indistinguishable from native fibrinogen. The primary application of FGG is as a coating for cell plates used in mammalian cell culture. This is further distinguished from other coatings by its propensity to bind and stabilize FGF-2 (for solid growth factor delivery) and many types of specific cells. In the form of insoluble inclusion bodies, FGG can be used as a biodegradable matrix for harvesting anchorage-dependent cells from tissue or blood. Similarly, it can be used in this form to modify disposable bags or chromatography membranes, all with the goal of providing a specific cell- and growth factor-affinity surface.

[0048] The advantages of the basic material FGG according to the invention over existing solutions all relate to not having to use homologous fibrinogen from human or animal blood, i.e., a certifiable material with reproducible material properties and no risk of pathogenic transmission. Other advantages derive from the fact that production is not carried out in mammalian cell cultures but in Escherichia coli, thus offering an animal component-free process, virus-free production, and considerable cost-effectiveness.

[0049] To increase the range of applications for the FGGs according to the present invention, it is also possible to modify the base material to create biohybrid materials that provide additional functionality. These biohybrid FGG biopolymers can be used for 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.

[0050] The advantages of such modified biohybrid materials according to the present invention over existing solutions again relate to obtaining the biological component FGG from recombinant technology rather than from natural sources. Consequently, the modified biohybrid materials will possess more reproducible mechanical and biological properties. Furthermore, the creation of biohybrids from recombinant fibrinogen produced in mammalian cell culture is inherently limited by high costs.

[0051] The accompanying figure shows:

[0052] Figure 1 ：

[0053] The sequence of FGG, including the target binding site, disulfide bonds, and connections to other chains, is shown, along with the 3D FGG structural model.

[0054] Figure 2 ：

[0055] SDS PAGE (A) and Western blot (B) of soluble and insoluble fractions of cell lysates after 0 and 3 h of induction with IPTG, M: molecular weight marker (expressed in kDa), +: fibrinogen from human plasma as a positive control, the red box highlights the position of the γ chain.

[0056] Figure 3 ：

[0057] Purification procedure overview. A two-step purification can be used for the purification of FGG. Step 1: Chromatogram of the dissolved inclusion bodies run on an IMAC column (HisTrap, 5 mL, GE Healthcare). The gray area represents the sharp elution peak containing the labeled FGG protein. Step 2: The peak eluted from the affinity column is subjected to size exclusion chromatography (SEC, Superdex 75 pg, GE Healthcare), which results in the isolation of a peak highlighted in gray. Further analysis of the peak via SDS-PAGE shows that FGG is separated at approximately 55 kDa.

[0058] Figure 4A and Figure 4B :

[0059] ( Figure 4A ) Polymerization test of recombinant FGG and FXIII.0 (control). The time above the lane shows the duration of the reaction and the increase in the intensity of FGG dimer formation at 100 kDa. ( Figure 4B ) FGG in cell culture medium resulted in a dose-dependent increase in viability compared to the control (cell culture medium only).

[0060] Figure 5 ：

[0061] (A) CTB assay showing the proliferation of AD-MSCs on different cell culture coatings during 1, 3, and 7 days of culture; (B) Growth of AD-MSCs on FGG, fibrinogen, BSA, and uncoated cell culture wells, shown as confluence determined using Fiji. Cell concentration: 15,000 cells / well; using Cytation 5 TM System imaging (4× magnification).

[0062] Figure 6 ：

[0063] AD-MSCs were grown on FGG, fibrinogen, BSA, and uncoated cell culture wells after 1, 3, and 7 days of culture. 15,000 cells / well; Cytation 5 TM Fluorescence imaging (4× magnification) was performed using the system, cells were stained with Calcein-AM, and cells formed aggregates grown on BSA and uncoated substrates.

[0064] Figure 7 ：

[0065] Cell morphology of AD-MSCs after 24 h of growth on FGG, fibrinogen, BSA, and uncoated cell culture wells. Cell concentration: 15,000 cells / well in a 48-well plate. Cytation 5TM The system performs fluorescence imaging (left: 4×, right 20× magnification), live cell imaging (Calcein-AM, green), nuclear staining (DAPI, blue), and dead cell imaging (propidium iodide, red). FGG is a coating of purified soluble protein, and FGG2 coating is directly derived from unpurified soluble IB.

[0066] Figure 8 ：

[0067] Time sweep oscillation test of FGG-PEGDA (FGG biohybrid biopolymer modified with photoactive PEG-DA) showing the formation of a strong hydrogel during in situ UV light irradiation. Rheological parameters are: 1% amplitude, 1 Hz, 37°C, FGG-PEGDA 19 mg / ml.

[0068] The present invention relates to the following amino acid and nucleotide sequences:

[0069] SEQ ID NO: 1

[0070] Fibrinogen gamma chain (FGG)

[0071] MYVATRDNCCILDERFGSYCPTTCGIADFLSTYQTKVDKDLQSLEDILHQVENKTSEVKQLIKAIQLTYNPDESSKPNMIDAATLKSRKMLEEIMKYEASILT HDSSIRYLQEIYNSNNQKIVNLKEKVAQLEAQCQEPCKDTVQIHDITGKDCQDIANKGAKQSGLYFIKPLKANQQFLVYCEIDGSGNGWTVFQKRLDGSVDFK KNWIQYKEGFGHLSPTGTTEFWLGNEKIHLISTQSAIPYALRVELEDWNGRTSTADYAMFKVGPEADKYRLTYAYFAGGDAGDAFDGFDFGDDPSDKFFTSHN GMQFSTWDNDNDKFEGNCAEQDGSGWWMNKCHAGHLNGVYYQGGTYSKASTPNGYDNGIIWATWKTRWYSMKKTTMKIIPFNRLTIGEGQQHHLGGAKQAGDV

[0072] SEQ ID NO:2

[0073] His tag

[0074] MGHHHHHHHHHHSSGHIEGRHMLEDI

[0075] SEQ ID NO 3

[0076] His-tag-FGG

[0077] MGHHHHHHHHHHSSGHIEGRHMLEDIMYVATRDNCCILDERFGSYCPTTCGIADFLSTYQTKVDKDLQSLEDILHQVENKTSEVKQLIKAIQLTYNPDESSKPNMIDAATLKSRKMLEEIMKYEASILTHDSSIRYLQEIYNSNNQKIVNLKEKVAQLEAQCQEPCKDTVQIHDITGKDCQDIANKGAKQSGLYFIKPLKANQQFLVYCEIDGSGNGWTVFQKRLDGSVDFKKNWIQYKEGFGHLSPTGTTEFWLGNEKIHLISTQSAIPYALRVELEDWNGRTSTADYAMFKVGPEADKYRLTYAYFAGGDAGDAFDGFDFGDDPSDKFFTSHNGMQFSTWDNDNDKFEGNCAEQDGSGWWMNKCHAGHLNGVYYQGGTYSKASTPNGYDNGIIWATWKTRWYSMKKTTMKIIPFNRLTIGEGQQHHLGGAKQAGDV

[0078] SEQ ID NO:4

[0079] FGG coding sequence

[0080]

[0081] SEQ ID NO:5

[0082] Nucleic acid for FGG expression in E. coli

[0083]

[0084] SEQ ID NO:6

[0085] His tag-FGG coding sequence

[0086]

[0087] SEQ ID NO:7

[0088] AGDV binding site

[0089] AGDV

[0090] SEQ ID NO:8

[0091] FGG 427-438 Platelet binding site on

[0092] HHLGGAKQAGDV

[0093] SEQ ID NO:9

[0094] FGG 404-422 Mac-1 or αMβ2 binding site

[0095] YSMKKTTMKIIPFNRLTIG

[0096] SEQ ID NO: 10

[0097] FGG 217-229 The binding site of integrin αVβ3

[0098] WTVFQKRLDGSV

[0099] SEQ ID NO:11

[0100] FGG 373-385 The binding site of integrin αVβ3

[0101] GVYYQGGTYSKAS

[0102] The present invention will be further illustrated by the following examples, but is not limited thereto. Example

[0103] Example 1

[0104] Design of constructs and production strain Tuner TM (DE3)-Creation of FGG

[0105] The gene sequence used for recombinant expression of the human γA chain in E. coli corresponds to the mature protein (amino acids 27–437, gene accession number C9JC84; Figure 1 ) and was produced by gene synthesis service (Life Technologies). The expression vector pET-16b (Merck Millipore, USA) was selected to produce FGG using the E. coli expression system. The gene sequence of FGG was cloned in frame with the sequence of the N-terminal polyhistidine affinity tag (His tag). The integrity of the coding sequence was confirmed by sequencing the entire fragment in both directions. The confirmed plasmid was used to transform competent Tuner cells via heat shock transformation. TM(DE3) host cells, 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 (IBs) in the intracellular fraction at 35°C ( Figure 2 ).

[0106] Example 2

[0107] Production and purification of FGG

[0108] FGG was produced in a 10 L fed-batch culture, which can be scaled up or down as needed. TM The (DE3)-FGG preculture was inoculated, which had been cultured for 10-12 h (OD600 = 0.1). The main culture was carried out 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 a pH of 6.8, a temperature of 35°C, an air supply of 4 L*min-1, and a dissolved oxygen level of 30% air saturation. After the glucose in the main culture medium was depleted, glucose injection (0.8 g / min) was started. A typical fermentation lasted 18 h, after which the culture medium was removed from the biomass by centrifugation.

[0109] The cell pellet was washed, resuspended in lysis buffer (50 mM MOPS, 150 mM NaCl, 1 mM EDTA), and disrupted by multiple passages (8×) in a French press at 9 psi. The resulting mixture was homogenized and centrifuged at 10,000×g for 60 min at 4°C. The pellet containing the IBs was washed and centrifuged multiple times (3×) in a buffer of 50 mM Tris HCl, 100 mM NaCl, 1 M urea, 1 mM EDTA, and 1% Triton to remove residual impurities. The typical total yield of this process is 350 g wet weight of IBs containing FGG (70% of the total FGG protein in 1 g of IB).

[0110] Further processing of FGG depends on its final application. Since this protein is not present in its natural environment within the intact fibrinogen molecule, it is not expected to have a "native" conformation per se. Its biological activity is primarily related to its sequence. It has been shown that simple solubilization 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 to coat cell culture plates with excellent results (see Figure 7 ). Alternatively, IB can be used to directly coat plates without requiring solubilization at all. If the application requires highly pure (>90%) soluble FGG protein, a two-step chromatographic purification process has been developed consisting of ion metal affinity chromatography (IMAC) and size exclusion chromatography (SEC).

[0111] Details of the purification procedure

[0112] use Purification system (GE Healthcare, USA), soluble FGG is applied to a HisTrap HP column (GE Healthcare, USA, column volume, CV = 5 ml) previously equilibrated with 5CV equilibration buffer (5M urea, 50mM Tris-HCl, 150mM NaCl, 20mM imidazole, pH 8). Subsequently, the column is washed with 5CV equilibration buffer to remove impurities. Elution is performed with a buffer containing 5M urea, 50mM Tris-HCl, 150mM NaCl, 200mM imidazole, pH 8. The flow rate is maintained at 5 ml / min. The eluted IMAC fraction (5 ml) is further purified using size exclusion chromatography using HiLoad Superdex 75pg (GE Healthcare, USA, CV = 120 ml). The SEC column is equilibrated with 2CV equilibration buffer (10mM Tris-HCl). The sample was then applied and eluted from the column using 1.5 CV of running buffer (50 mM Tris HCl, 150 mM NaCl, 2 M urea, pH 8). The flow rate in 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. 20 (Sartorius, MWCO = 30 kDa) was used for buffer exchange and simultaneous urea removal against PBS pH 7.4. The final FGG product was filtered with a 0.22 μm PES sterile filter (Sartorius, Germany) and stored frozen before further use ( Figure 3 ).

[0113] Example 3

[0114] Characterization and application of FGG

[0115] One approach to characterizing recombinant FGG produced in E. coli was to investigate whether its cross-linking sites for factor XIII (Gln398 and Lys406) could be accessed in a manner similar to native fibrinogen. Purified soluble recombinant FGG (170 μg / mL) was initially polymerized with factor XIII (0.5 mg / mL, 1.1 U / mL) in the presence of human α-thrombin (0.1 U / mL). The reaction was run at 37°C in 50 mmol / L TBS buffer, pH 7.4, and terminated at selected intervals by the addition of 1% SDS and 2% 2-mercaptoethanol. A control sample (labeled 0) was prepared by adding SDS and 2-mercaptoethanol to the fibrinogen prior to thrombin and factor XIII. Using SDS-PAGE analysis, it was demonstrated that recombinant FGG could be cross-linked by factor XIII, generating dimers (approximately 100 kDa) and higher molecular weight FGG structures ( Figure 4A and Figure 4B ).

[0116] The bioactivity of the produced FGG was tested in mammalian cell culture. Mammalian cells, such as the adipose-derived mesenchymal stem cells (AD-MSCs) used in this experiment, need to be able to adhere to the extracellular matrix for growth, communication, and other cell-to-cell interactions. Cell culture plates were coated with recombinant FGG as well as human fibrinogen and BSA. Uncoated plates were used as controls. All proteins were coated at the same concentration (500 μg / ml).

[0117] MSCs were cultured on various coatings and examined for cell adhesion, confluence, morphology, and viability. It was demonstrated that FGG and fibrinogen coatings showed the highest activity (indirectly determined as proliferation using the CTB assay). Furthermore, FGG promoted cell proliferation in a dose-dependent manner ( Figure 5 Cells grown on FGG and fibrinogen showed higher confluence, whereas cells grown on integrin-free BSA or uncoated BSA developed aggregates ( Figure 6 In addition, cell morphology examination showed that AD-MSCs grown on FGG and fibrinogen had a spindle-like shape and morphology ( Figure 7 The performance of recombinant FGG in mammalian cell culture was indistinguishable from that of native fibrinogen. The results were confirmed in other cell types (data not shown).

[0118] Cell culture details

[0119] 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-glutamine, and 50 μg mL-1 gentamicin) and harvested by accutase treatment. Experiments were performed using hAD-MSCs from passages 2 to 8.

[0120] Example 4

[0121] Modification of FGG biopolymers

[0122] Although it has been shown that FGG can be cross-linked by factor XIII as a natural fibrinogen ( Figure 4A and Figure 4B ), but this does not provide sufficient stability for the generation of 3D networks. For example, in order to crosslink FGG to create hydrogels for 3D cell encapsulation, additional modifications are required. Crosslinking can be achieved by chemical modification with synthetic polymers (e.g. PEG). Another strategy is to use synthetic moieties to create biohybrids with tunable properties. For example, modification with PEG diacrylate (PEG-DA) provides FGG photoactive materials that can be used, for example, in 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, to create responsive sheets for enzyme-free cell separation. In all cases, FGG provides the bioactivity of the biohybrid, while the synthetic polymer contributes to the desired physicochemical properties. The purpose of modification with synthetic moieties (modified biohybrids) is to (i) introduce crosslinking or design / control mechanical properties (PEG) or (ii) add "tunable" properties such as temperature switches (e.g. Pluronic F127) or photopolymerization (e.g. PEG-DA) ( Figure 8 ).

Claims

1. An isolated recombinant polypeptide comprising: (i) the amino acid sequence according to SEQ ID NO: 3, or (ii) an amino acid sequence that has at least 99% sequence identity to SEQ ID NO: 3 and has fibrinogen γ chain activity and His-tag functionality.

2. The isolated recombinant polypeptide of claim 1, comprising binding sites for integrins, leukocytes, platelets, fibroblasts, endothelial cells and / or fibroblast growth factor 2 (FGF-2).

3. The isolated recombinant polypeptide of claim 1, wherein the polypeptide comprises the γ chain of fibrinogen, and the γ chain of fibrinogen consists of the amino acid sequence according to SEQ ID NO:

1.

4. The isolated recombinant polypeptide of claim 1, wherein the polypeptide comprises a His tag consisting of the amino acid sequence according to SEQ ID NO:

2.

5. An isolated nucleic acid encoding the polypeptide of any one of claims 1 to 4. A vector comprising the nucleic acid according to claim 5 .

7. A host cell comprising the nucleic acid of 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 an E. coli host cell.

10. A biohybrid material comprising a conjugate comprising a polypeptide according to any one of claims 1 to 4 and one or more moieties selected from the group consisting of polyethylene glycol (PEG), PEG derivatives, PEG diacrylate (PEG-DA), poly(oligo(ethylene glycol) methyl ether methacrylate (POEGMA), poly(N-(2-hydroxypropyl)-methacrylamide) (PHPMA) and HPMA copolymers, poly(vinyl pyrrolidone) (PVP), poly(ethyleneimine) (PEI), poly(acryloylmorpholine) (P AcM), poly(2-ethyl 2-oxazoline) (PEOZ), divinyl ether maleic anhydride / acid copolymer (DIVEMA), poly(styrene-co-maleic acid / anhydride) (SMA), poly(vinyl alcohol) (PVA) and temperature-sensitive polymers including poloxamers, poly(N-isopropylacrylamide) (PNIPAM), poly(N,N-diethylacrylamide) (PDEAM), poly(methyl vinyl ether) (PMVE), poly(N-vinylcaprolactam) (PNVCl), and mixtures thereof.

11. Use of the isolated recombinant polypeptide of any one of claims 1 to 4 or the biohybrid material of claim 10 as a cell culture plate coating, as a 3D cell culture matrix, as an in vitro tissue / organ model, as an in vitro drug testing platform, as an in vitro cell and / or drug delivery platform, as a coating for cell enrichment and / or separation, as a coating for growth factor (FGF-2) enrichment and / or separation, as a bio-ink for bioprinting, or in enzyme-free cell separation.

12. Use of the isolated recombinant polypeptide of any one of claims 1 to 4 or the biohybrid material of claim 10 in the preparation of a medicament for wound healing, in vivo tissue engineering, in vivo tissue / organ repair, or in vivo cell and / or drug delivery.

13. A method for producing a polypeptide according to any one of claims 1 to 4, comprising the following steps: (a) providing a host cell according to any one of claims 7 to 9, (b) cultivating the host cell under conditions allowing expression of the polypeptide, and (c) isolating the polypeptide from the host cell and / or the culture medium.

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