Photocrosslinkable recombinant bacterial collagen-like proteins

Abstract

A recombinant bacterial collagen-like protein having an amino acid sequence having at least > = 60% identity to the amino acid sequence of SEQ ID NO: 1, characterized in that the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1, and the recombinant collagen-like protein is functionalized with an ethylenically unsaturated group.

Description

[0001] The present invention relates to photo-crosslinkable functionalized recombinant collagen-like proteins (CLPs) of bacterial origin, which can be used as bio-inks for cell encapsulation, and for the preparation of 3D scaffolds, in vitro models and tissue grafts.

[0002] Bioinks commonly used for bioprinting are largely synthetic thermoplastic polymers or animal-derived proteins such as collagen, gelatin, (meth)acryloyl-functionalized animal-derived collagen, or -gelatin methacrylate.

[0003] US 2016 / 0051727 discloses collagen-based polymer materials comprising collagen molecules and / or collagen-derived molecules which are functionalized by adding one or more ethylenically unsaturated groups and are cross-linked via said groups.

[0004] US 2020 / 0179562 discloses curable recombinant human collagen functionalized with methacryloyl groups, which can be formulated into inks for additive manufacturing and printed into 3D objects.

[0005] US 2016 / 0193384 discloses the production of hydrogels by photocrosslinking methacrylated or acrylated collagen and synthetic polymers. Such hydrogels can be used as 3D scaffolds and implants, suitable as inks for 3D printing to prepare complex 3D structures, or for incorporating cells into structures.

[0006] The collagen in the prior art mentioned above is derived from human, mammalian or avian sources. Such human or animal-derived collagen is not well defined, viscous and has weak water solubility under physiological conditions. Due to viscosity, a large force is required to extrude or spray the viscous collagen mixture during the printing process. If cells are co-printed in the same mixture, this leads to greater shear stress (and therefore lower cell viability). The low pH required to dissolve animal-derived collagen also makes it challenging to increase the concentration.

[0007] Therefore, the object of the present invention is to provide a protein that can be formulated into a bio-ink and cross-linked with a hydrogel and that can at least reduce the disadvantages of prior art materials, in particular animal-derived collagen.

[0008] This object is achieved by a recombinant bacterial collagen-like protein having an amino acid sequence that is at least ≥60% identical to the amino acid sequence of SEQ ID NO:1, characterized in that the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO:1, and the recombinant collagen-like protein is functionalized with an ethylenically unsaturated group.

[0009] In the bio-ink composition, the ethylenically unsaturated groups can form a polymer cross-linked network with the functionalized recombinant bacterial collagen-like protein molecules or other components in the bio-ink composition in the presence of free radicals, anions, nucleophiles, or a combination thereof.

[0010] There is an unmet need for GMP-compliant bioinks that facilitate the translation of bioprinted clinical products from bench to bedside, particularly water-soluble biomaterials that can form hydrogels and thus support cell encapsulation and bioprinting.

[0011] Collagen-like proteins (CLPs) of bacterial origin have attractive mechanical properties similar to collagens of higher eukaryotes without the complex maturation steps required by their eukaryotic counterparts. CLPs present a common structure: two α-helices that stabilize each other constitute the "V domain", which is followed by a rod-like structure collagen domain (CL). After the collagen domain, there is usually a membrane anchor (GPI-like) at the C-terminal end of the protein.

[0012] The most industrially relevant CLP is the product of Streptococcus pyogenes and is called Scl2.

[0013] The current understanding of this process, as described in various publications (Lukomski et al. 2002, Brodsky et al. 2009), is that the V domain is required for the folding of three Scl2 protein monomers into a triple helical structure in vitro.

[0014] Since the V domain is considered critical for the generation of triple-helical Scl2, removal of this sequence has never been considered, leading to the following challenges:

[0015] The V domain constitutes approximately one third of the entire sequence and impedes protein export from the Pichia pastoris host, requiring a complex downstream process involving cell lysis to remove the target protein from the cell.

[0016] The V domain itself has pathogenic properties and needs to be removed during the purification process. This is done by protease digestion. The use of proteases is quite expensive and it also needs to be removed in downstream processes.

[0017] Surprisingly, it was found that truncated variants of collagen-like proteins, including variants with truncated V domains or without any V domains, resulted in increased production of collagen-like proteins and secretion into the fermentation medium. Further surprisingly, the truncated variants can fold correctly in the absence of the V domain.

[0018] This is preferred when the amino acid sequence comprises a deletion of 38 to 74 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1. This includes a complete deletion of the N-terminal V domain (comprising 74 amino acids) and different truncations of the V domain of at least 38 amino acids.

[0019] The amino acid sequence preferably has at least 60% identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4. SEQ ID NO: 2 and SEQ ID NO: 3 are truncated forms of Scl2, and in SEQ ID NO: 4, the N-terminal V domain is completely deleted.

[0020] The sequence IDs indicated herein therefore relate to the amino acid sequence before functionalization with ethylenically unsaturated groups and not to the functionalized recombinant bacterial collagen-like protein.

[0021] In a preferred configuration, the amino acid sequence has at least ≥90%, ≥92%, ≥94%, ≥96%, ≥97%, ≥98%, ≥99% or 100% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, preferably ≥97%, particularly preferably ≥98%, very particularly preferably ≥99% and extremely preferably 100%.

[0022] In a preferred embodiment, truncated variants of the collagen-like protein from Streptococcus pyogenes are used for the recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups according to the invention.

[0023] The functionalized recombinant bacterial collagen-like protein of the present invention accordingly also encompasses polypeptide variants of SEQ ID NO: 1 to 4, which contain one or more insertions or deletions. Preferably, the polypeptide contains at most 5, at most 4, at most 3 or at most 2 amino acid insertions or deletions.

[0024] Therefore, the following functionalized recombinant bacterial collagen-like proteins also form part of the present invention:

[0025] The recombinant bacterial collagen-like protein has an amino acid sequence that is at least 94% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the recombinant collagen-like protein is functionalized with methacrylate, acrylate, methacrylamide, acrylamide groups or mixtures thereof.

[0026] The recombinant bacterial collagen-like protein has an amino acid sequence that is at least 96% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and the recombinant collagen-like protein is functionalized with methacrylate, acrylate, methacrylamide, acrylamide groups or a mixture thereof.

[0027] The recombinant bacterial collagen-like protein has an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the recombinant collagen-like protein is functionalized with methacrylate, acrylate, methacrylamide, acrylamide groups or mixtures thereof.

[0028] The recombinant bacterial collagen-like protein has an amino acid sequence that is at least 99% identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, and the recombinant collagen-like protein is functionalized with methacrylate, acrylate, methacrylamide, acrylamide groups or mixtures thereof.

[0029] A recombinant bacterial collagen-like protein according to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4, wherein the amino acid sequence contains one to five, one to four, or one to three insertions or deletions, and the recombinant collagen-like protein is functionalized with methacrylate, acrylate, methacrylamide, acrylamide groups or a mixture thereof.

[0030] Recombinant bacterial collagen-like protein (CLP) can be produced in a method comprising the following steps:

[0031] a) fermenting in a culture medium a bacterial, yeast or plant host cell expressing a CLP having an amino acid sequence that is at least ≥ 60% identical to the amino acid sequence of SEQ ID NO: 1, wherein the amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1,

[0032] b) accumulating bacterial CLP in a culture medium, wherein a fermentation broth is obtained,

[0033] c) separating the bacterial, yeast or plant host cells from the fermentation broth,

[0034] d) incubating the fermentation broth at no more than 25° C. for at least 1 hour for folding of CLP,

[0035] e) optionally purifying the bacterial CLP by at least one of the following: solvent precipitation, tangential flow filtration (TFF), ion exchange chromatography, reverse phase chromatography.

[0036] The present invention accordingly also relates to polypeptide variants of SEQ ID NO: 1 to 4, which contain one or more insertions or deletions. Preferably, the polypeptide contains at most 5, at most 4, at most 3 or at most 2 amino acid insertions or deletions.

[0037] In a preferred embodiment, the CLP folding in step d) is performed at a temperature between -80°C and 25°C, preferably between 0°C and 20°C. In a preferred configuration, the folding is performed in the presence of glycerol or a salt.

[0038] In another preferred embodiment, the CLP folding in step d) is performed for a time of 1 hour to 48 hours, preferably 1 hour to 24 hours.

[0039] In another preferred embodiment, the CLP folding in step d) is performed with a CLP concentration of at least 1 mg / ml, preferably at least 4 mg / ml.

[0040] In a preferred embodiment, the host cell is a microorganism of the species Pichia pastoris, E. coli, P. putida or C. glutamicum comprising any of the polypeptides according to the present invention.

[0041] In a preferred embodiment, the microorganism is a yeast or bacterial cell of the genus Pichia pastoris, preferably Escherichia coli, Corynebacterium or Brevibactetium.

[0042] The microorganism may be one in which the nucleotide sequence encoding CLP is present in an overexpressed form.

[0043] Functionalization

[0044] The recombinant bacterial collagen-like protein is functionalized with ethylenically unsaturated groups. The term "ethylenically unsaturated groups" refers to formate, vinyl, allyl, itaconate, fumarate or (meth)acryloyl groups. Preferably, the ethylenically unsaturated groups are selected from (meth)acryloyl, fumarate or vinyl.

[0045] More preferably, the recombinant bacterial collagen-like protein is functionalized with a (meth)acryloyl group. The term "(meth)acryloyl group" means a methacrylate group, an acrylate group, a methacrylamide or an acrylamide or a mixture thereof.

[0046] If a (meth)acryloyl group is selected for functionalization, the functionalized recombinant bacterial collagen-like protein according to the present invention can be prepared by reacting a recombinant bacterial collagen-like protein (which has at least ≥60% identity with the amino acid sequence of SEQ ID NO: 1 and contains a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1) with (meth)acrylic anhydride in an alkaline carbonate buffer. Amino acids containing primary amine and hydroxyl groups can react with (meth)acrylic anhydride to form (meth)acrylamide and methacrylate groups, respectively. The (meth)acryloyl functional group is coupled to the primary amine group at the N-terminus, the primary amine group of a lysine residue, and the hydroxyl group of a tyrosine residue.

[0047] The degree of functionalization can be controlled via the molar ratio of the recombinant bacterial collagen-like protein and (meth) acrylic anhydride. After functionalization, the mixture is preferably subjected to diafiltration or dialysis to remove by-products. The degree of functionalization can be determined by trinitrobenzenesulfonic acid assay, NMR or HPLC-MS.

[0048] Due to the high solubility of the recombinant bacterial collagen-like protein in neutral and alkaline buffers, an unexpectedly high degree of functionalization of over 95% could be achieved with minimal or no damage to the triple helical protein structure.

[0049] In a preferred embodiment of the present invention, the recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups has a degree of functionalization of the primary amine groups at the N-terminus of the recombinant collagen-like protein, the primary amine groups of lysine residues and the hydroxyl groups of tyrosine residues in the range of 5% to 99%, preferably 50% to 99% and more preferably 60% to 99%.

[0050] In addition, the degree of functionalization can be adjusted by adjusting the reaction conditions. By adjusting the molar ratio of (meth)acrylic anhydride to recombinant collagen, (meth)acryloyl recombinant collagen with different degrees of functionalization can be obtained. For example, when the ratio of (meth)acrylic anhydride to the amine group of recombinant collagen is maintained at 0.6:1, (meth)acryloyl recombinant collagen with a functionalization degree of about 60% is synthesized.

[0051] In contrast, animal-derived collagen is typically only 10% to 20% substituted because the extreme reaction conditions denature and hydrolyze the triple-helical protein structure, producing a less mechanically rigid gelatin.

[0052] The present invention accordingly also relates to a method for producing functionalized recombinant bacterial collagen-like protein using a recombinant bacterial collagen-like protein whose amino acid sequence has at least 98% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 and (meth)acrylic anhydride, characterized in that the functionalization is carried out at a molar ratio of (meth)acrylic anhydride to the amine groups of the recombinant bacterial collagen-like protein of 0.5:1 to 5:1.

[0053] The functionalization is performed under mild conditions at room temperature and a pH of 7-10, preferably 8 to 10. Preferably, the functionalization is performed in an alkaline buffer at a buffer concentration of 0.1 M to 1 M, more preferably 0.1 M to 0.6 M, most preferably 0.1 M to 0.4 M. Preferably, the alkaline buffer is a carbonate / bicarbonate buffer.

[0054] The present invention also relates to a method for producing functionalized recombinant bacterial collagen-like protein using a recombinant bacterial collagen-like protein whose amino acid sequence has at least 99% identity with the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 and (meth)acrylic anhydride, characterized in that the functionalization is carried out at a molar ratio of (meth)acrylic anhydride to the amine groups of the recombinant bacterial collagen-like protein of 0.5:1 to 5:1.

[0055] Functionalization is performed as described above under mild conditions at room temperature and a pH of 7-10, preferably 8 to 10. Preferably, the functionalization is performed in an alkaline buffer at a buffer concentration of 0.1 M to 1 M, more preferably 0.1 M to 0.6 M, most preferably 0.1 M to 0.4 M. Preferably, the alkaline buffer is a carbonate / bicarbonate buffer.

[0056] The present invention also relates to a method for producing functionalized recombinant bacterial collagen-like protein using a recombinant bacterial collagen-like protein according to the amino acid sequence SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 (the amino acid sequence contains one to five, one to four, or one to three insertions or deletions) and (meth)acrylic anhydride, characterized in that the functionalization is carried out at a molar ratio of (meth)acrylic anhydride to the amine groups of the recombinant bacterial collagen-like protein of 0.5:1 to 5:1.

[0057] Functionalization is performed as described above under mild conditions at room temperature and a pH of 7-10, preferably 8 to 10. Preferably, the functionalization is performed in an alkaline buffer at a buffer concentration of 0.1 M to 1 M, more preferably 0.1 M to 0.6 M, most preferably 0.1 M to 0.4 M. Preferably, the alkaline buffer is a carbonate / bicarbonate buffer.

[0058] During the functionalization process, what often happens with the animal or human collagen or collagen-like proteins disclosed in the prior art is that gelatin is formed due to the hydrolysis of the collagen or collagen-like proteins. This hydrolysis can take place under acidic or alkaline functionalization conditions. Gelatin has a lower molecular weight than collagen or collagen-like proteins.

[0059] Surprisingly, using the method for producing functionalized recombinant bacterial collagen-like proteins according to the invention, the recombinant bacterial collagen-like proteins according to the invention are not hydrolyzed into smaller sequences. The combination of the recombinant bacterial collagen-like proteins according to the invention and mild functionalization conditions has an unexpected effect: the molecular weight of the recombinant bacterial collagen-like proteins according to the invention is not reduced. No hydrolysis of the molecule occurs.

[0060] The functionalized recombinant bacterial collagen-like proteins according to the present invention thus produced are particularly suitable for use in bio-ink compositions. Such compositions have lower viscosity than compositions using functionalized animal-derived collagen or collagen-like proteins of the prior art, and therefore exert less shear stress on the cells and reduce nozzle clogging during bioprinting. Without wishing to be bound by theory, it is assumed that the unique combination of the recombinant bacterial collagen-like protein according to the present invention and the method for functionalizing it partially unfolds the protein, thereby reducing viscosity, while avoiding hydrolysis and therefore not forming gelatin. The skilled person could not foresee this beneficial effect of functionalization.

[0061] The functionalized recombinant bacterial collagen-like protein according to the present invention has the following advantages over animal-derived collagen with the same functionalization, as exemplified by comparing the recombinant bacterial collagen-like protein functionalized with (meth)acryloyl groups and the animal-derived collagen functionalized with (meth)acryloyl groups.

[0062]

[0063] The recombinant bacterial collagen-like protein (meth)acryloyl according to the invention is soluble in water, in aqueous buffers at neutral or alkaline pH and in particular at a physiologically relevant pH of 7 to 7.4.

[0064] Bioactivity broadly describes cell attachment and enzymatic degradation. Bioactivity can be tailored via the addition of cell-interactive ligands during the photocrosslinking process.

[0065] The present invention encompasses bio-ink compositions preferably comprising 1% to 10% by weight of recombinant bacterial collagen-like proteins functionalized with ethylenically unsaturated groups. Within this concentration range, formulations containing 1.5% to 6% bio-ink are preferred. In the absence of additives, the viscosity of the bio-ink composition according to the present invention is less than 10 centipoise when dissolved in water or a physiological buffer.

[0066] In addition, the bio-ink composition contains a photoinitiator, preferably a free radical photoinitiator. The amount of photoinitiator added to the bio-ink composition formulation ranges from 0.01 wt % to 2 wt % of the total liquid formulation. The photoinitiator is capable of generating free radicals when irradiated with actinic radiation.

[0067] The bio-ink composition according to the present invention can be applied to prepare photo-crosslinked hydrogels and sponges. Therefore, the present invention also relates to a method for producing a hydrogel by photo-crosslinking a functionalized recombinant bacterial collagen-like protein according to the present invention. The bio-ink composition can undergo photo-crosslinking in the presence of actinic radiation, blue light or UV light to form a transparent and colorless hydrogel. The resulting pH of the bio-ink composition and therefore the hydrogel is preferably between 6.5 and 8. A hydrogel comprising a photo-crosslinked functionalized recombinant bacterial collagen-like protein according to the present invention is therefore also part of the present invention.

[0068] The bio-ink composition according to the present invention comprises

[0069] a) 1 to 10% by weight of a functionalized recombinant bacterial collagen-like protein according to the invention;

[0070] b) 80 to 99% by weight of an aqueous solvent;

[0071] c) 0.01 to 2 wt % of a photoinitiator;

[0072] d) 0 to 5 wt % of a photocrosslinkable polymer or other photocrosslinkable peptide;

[0073] e) 0 to 10 wt. % additives, rheology modifiers, biopolymers, gelation promoters, bioactive moieties, peptides, nanocellulose and / or cells;

[0074] The condition is that the sum of all components of the bio-ink is 100%.

[0075] Component d) is optional, since the functionalized recombinant bacterial collagen-like protein according to the present invention can be cross-linked to itself.

[0076] Preferably, the photocrosslinkable polymer of component d) is selected from natural (e.g. hyaluronic acid methacrylate) and synthetic (e.g. acrylate and methacrylate derivatives of poly(ethylene glycol)) polymers and is mixed into the composition to impart additional biological properties (e.g. antifouling properties) and to adjust physical properties (e.g. degradability and swelling behavior in water).

[0077] Synthetic peptides with photocrosslinkable groups can be incorporated to impart additional biological activities such as cell adhesion, stem cell differentiation, and enzymatic degradability.

[0078] The bio-ink composition can be prepared by dissolving the functionalized recombinant bacterial collagen-like protein according to the present invention in an aqueous solution, and adding a water-soluble photoinitiator such as lithium phenyl-2,4,6-trimethylbenzoylphosphinate or 1-[4-(2-hydroxyethoxy)-phenyl]-2-hydroxy-2-methyl-1-propan-1-one.

[0079] Depending on the end use application, components d) and e) may optionally be included in the composition.

[0080] The bio-ink composition can be dispensed using a bioprinter, or poured into a mold for photocuring to form a hydrogel scaffold.

[0081] Depending on the selected photoinitiator, photopolymerization or photocrosslinking is preferably triggered by exposure to UV or visible light. A method for producing a hydrogel by photocrosslinking a functionalized recombinant bacterial collagen-like protein according to the invention also forms part of the present invention. Similarly, a method for producing a hydrogel by photocrosslinking a bio-ink formulation according to the invention also forms part of the present invention. Preferably, the bio-ink formulation is photocrosslinked using UV light.

[0082] Compared to preparations using functionalized animal-derived collagen, the bio-ink composition according to the present invention has a lower viscosity and therefore applies less shear stress to cells and has a lower chance of nozzle clogging during the bioprinting process.

[0083] Another advantage of the bio-ink composition according to the present invention is that the mechanical properties of the resulting hydrogel are significantly more rigid compared to functionalized animal-derived collagen hydrogels, and the stiffness can be adjusted by varying the concentration of the functionalized recombinant bacterial collagen-like protein according to the present invention from 1 wt % to 10 wt % of the bio-ink composition (as determined by storage modulus measured using rheology).

[0084] This is in part due to the higher solubility of recombinant collagen, enabling the formation of high concentration hydrogels. In contrast, due to solubility considerations, bioinks derived from collagen of animal origin are typically less than 0.6%. The stiffness of recombinant collagen hydrogels (as determined by the storage modulus measured using rheology) can be as high as 8.2 kPa, as exemplified by a 9% hydrogel measured under 0.1% strain conditions. Hydrogels comprising functionalized recombinant bacterial collagen-like proteins photocrosslinked according to the present invention therefore also form part of the present invention.

[0085] Additives that can be incorporated into the bio-ink formulation as component e) include rheology modifiers, gelation promoters and / or bioactive moieties. These additives can enhance the mechanical, viscoelastic and biological properties of the bio-ink composition and / or the resulting photo-crosslinked hydrogel.

[0086] Self-assembling peptides and biopolymers such as nanocellulose can also be incorporated into the bio-ink composition as component e). Such molecules enhance gelation and impart additional mechanical properties. Therefore, the present invention also relates to a hydrogel comprising a functionalized recombinant bacterial collagen-like protein cross-linked by light according to the present invention, the hydrogel further comprising nanocellulose, peptides or a mixture thereof.

[0087] Cells can be encapsulated by adding them to the bio-ink composition during the photo-crosslinking process. The cells can then also be incorporated into the photo-crosslinked hydrogel scaffold. Whether printed or cast, a scaffold for tissue engineering comprising the above hydrogel is also the subject of the present invention.

[0088] The resulting photocrosslinked hydrogels supported the in vitro proliferation of different cell types. Cells can also be advantageously incorporated into the bioprinting process. Cell viability, proliferation, and spreading in bioprinted hydrogels were significantly better than those in batch-cast hydrogels. Therefore, photocrosslinked hydrogels are particularly suitable as scaffolds for tissue engineering.

[0089] Hydrogels formed from the bio-ink compositions according to the present invention, whether bioprinted or cast, demonstrated good in vivo stability for more than three months, compared to animal-derived collagen, which was absorbed by native tissue within one month.

[0090] The bio-ink composition according to the present invention can be formulated for different 3D printing or bio-printing techniques, in particular drop-on-demand inkjet and digital light printing / stereolithography. Due to its low viscosity, the bio-ink is particularly suitable for drop-on-demand inkjet printing.

[0091] The bio-ink is also suitable for digital light printing, where a bio-ink composition containing 1.5 wt % to 6 wt % of the functionalized recombinant bacterial collagen-like protein according to the present invention has been successfully printed into a 3D hydrogel structure.

[0092] Preferably, the bio-ink composition containing 1.5 wt % to 6 wt %, more preferably 3 wt % to 6 wt % of the functionalized recombinant bacterial collagen-like protein according to the present invention can be printed into a 3D hydrogel structure. Example:

[0093] Example 1

[0094] The functionalized recombinant bacterial collagen-like protein according to the present invention is prepared by reacting a recombinant bacterial collagen-like protein having SEQ ID NO.4 with methacrylic anhydride in a 0.25M alkaline carbonate / bicarbonate buffer at pH 9.5. Amino acids containing primary amines and hydroxyl groups can react with methacrylic anhydride to form methacryloyl derivatives of methacrylamide and methacrylate, respectively. The degree of functionalization is controlled by the molar ratio of the recombinant bacterial collagen-like protein and methacrylic anhydride. After functionalization, the mixture undergoes diafiltration or dialysis to remove by-products. The degree of functionalization can be determined by trinitrobenzenesulfonic acid assay, NMR or HPLC-MS. In the present embodiment, trinitrobenzenesulfonic acid assay and NMR were used, and the degree of functionalization was found to be 64% to 97.3% of primary amine groups on the recombinant bacterial collagen-like protein.

[0095]

[0096]

[0097] Table 1.1: Degree of functionalization at various molar ratios of methacrylic anhydride to primary amine groups of the recombinant bacterial collagen-like protein according to the invention.

[0098] The degree of substitution is based on the TNBS assay, which measures the change in primary amine groups.

[0099] Example 2

[0100] The bio-ink according to the present invention was formulated by dissolving the functionalized recombinant bacterial collagen-like protein from Example 1 in phosphate buffered saline and then adding the water-soluble photoinitiator lithium phenyl-2,4,6-trimethylbenzoylphosphinate at a concentration of 0.2 wt % of the bio-ink formulation.

[0101] Photocrosslinkable polymers such as PEG-AR were incorporated as shown in Example 8. The cell-free mixture was vortexed for optimal mixing to obtain a homogenous solution. Due to the low viscosity and lack of pH or temperature sensitivity, obtaining a homogenous solution and subsequent hydrogel is much easier than with animal-derived collagen.

[0102] As described in Examples 6, 9 and 10, other ingredients such as peptides, polymers and cells are also incorporated into the formulations depending on the end application.

[0103] Example 3

[0104] The bio-ink composition was dispensed using a bio-printer or poured into a mold for photocuring to form a hydrogel scaffold. Photopolymerization was triggered by exposure to UV light. There was a phase transition from solution to hydrogel. The resulting hydrogel was transparent with good optical clarity and shape fidelity. Two hydrogels were produced using bio-ink compositions comprising 3% and 6% of the functionalized recombinant bacterial collagen-like protein prepared according to Example 1.

[0105] Shape fidelity refers to the shape retention of a bioink after extrusion or molding and crosslinking (via light or chemical methods). It compares the final structure of the hydrogel to the intended design or shape of the mold. The shape fidelity of cast hydrogels is reflected by their well-defined edges and smooth surfaces, especially at higher concentrations (≥3 mg / mL).

[0106] In contrast, shape fidelity and uniformity of animal-derived collagenmethacrylol are quite difficult to achieve because photocrosslinking, pH, and temperature must be well controlled to form the hydrogel. Inhomogeneous mixing often results in inhomogeneous hydrogels with poorly defined edges and uneven surfaces.

[0107] Figure 3.1 shows that transparent disc-shaped hydrogels with good shape fidelity were obtained using 3% (left) and 6% (right) functionalized recombinant bacterial collagen-like protein according to the present invention (photocross-linked in a mold using a full-spectrum UV light source).

[0108] As shown in Figure 3.2, the shape fidelity and printability of the material were confirmed by 3D printing a hollow cylinder with a height of 10.8 mm and a wall thickness of 1.6 mm. The bio-ink containing 6% functionalized recombinant bacterial collagen-like protein according to the present invention was dispensed onto a glass coverslip in a layer-by-layer manner by an on-demand inkjet bioprinter. After the deposition of each layer, the sample was photocrosslinked with UV light to induce hydrogel formation. No delamination was observed, and the hollow cylinder was 3D printed without relying on a support.

[0109] FIG. 3.2 shows a 3D printed hollow cylinder using the bio-ink according to the present invention prepared as described in Examples 1 and 2.

[0110] Example 4

[0111] A porous scaffold / sponge can be prepared by freeze drying a hydrogel. The hydrogel prepared using the same method as described in Example 3 with water as the solvent is flash frozen in an ultra-low temperature (-80°C) freezer. The frozen sample is then placed in a freeze dryer. During the freeze drying process, the sample is subjected to low temperatures and vacuum, which causes the water in the sample to sublime.

[0112] Sample 4.1 was prepared using the bio-ink composition as described in Example 2, using 0.45 wt % of animal collagen functionalized with methacryloyl groups (comparative sample).

[0113] Sample 4.2 was prepared using the bio-ink composition described in Example 2, which contained 6 wt % of the recombinant bacterial collagen-like protein functionalized with methacryloyl groups as described in Example 1.

[0114] Sample 4.3 was prepared using the bio-ink composition described in Example 2, which contained 6 wt % of a recombinant bacterial collagen-like protein functionalized with methacryloyl groups as described in Example 1 and 5 wt % of a branched (8-arm) polyethylene glycol acrylate having a molecular weight of 10 kDa.

[0115] All samples were photocrosslinked by UV exposure.

[0116] The sponge is then rehydrated by immersion in water. Good rehydration requires overnight soaking and can be accelerated by sonication.

[0117]

[0118]

[0119] Table 4.1: Hydrogels, freeze-dried sponges, and rehydrated hydrogels prepared using methacryloylated bovine collagen and recombinant bacterial collagen-like proteins functionalized with methacryloyl groups.

[0120] The sponge is a structure resulting from freeze drying. It is a porous, opaque sponge-like scaffold. Compared with the starting hydrogel, the sponge has a similar shape and size (diameter and area). If a polymer such as PEG is incorporated into the hydrogel formulation, more shrinkage is observed, but since the shrinkage is uniform, the overall shape is well maintained.

[0121] Example 5

[0122] To determine viscoelastic properties, 15 mm disks of hydrogels produced as described in Example 3 were subjected to dynamic time, strain and frequency sweep experiments using an Anton Paar MCR 302 rheometer with a 15.0 mm diameter contoured parallel plate geometry.

[0123] The strain sweep experiment characterized the hydrogel by using an oscillating strain increasing from 0.01% to 100% at a constant frequency of 1 Hz. The results of the strain sweep test are expressed via the storage (G') and loss (G") moduli of the hydrogel over an increasing strain range and show a linear viscoelastic region of the material where elastic behavior (G') dominates. The intersection of G' and G" characterizes the condition where the sample has decomposed and is no longer considered a hydrogel. The storage modulus (G') represents the rigidity of the hydrogel and its ability to store deformation energy in an elastic manner. This is directly related to the degree of cross-linking, where the higher the degree of cross-linking, the higher the storage modulus. The loss modulus (G") represents the deformation energy lost due to internal friction during shearing of the hydrogel sample.

[0124] Frequency sweep testing is another rheological method that reflects the relationship between the test frequency and the storage (G’) and loss (G”) moduli of the material. The frequency is varied from 0.1 Hz to 10 Hz while keeping the strain constant at 0.1%. By comparing the two G’ and G” values ​​over the frequency range, the results reflect the viscoelastic properties and state of the hydrogel.

[0125] Samples were prepared using a bio-ink composition as described in Example 2, comprising 9 wt %, 6 wt %, 4.5 wt % and 3 wt % of a recombinant bacterial collagen-like protein functionalized with methacryloyl groups as described in Example 1.

[0126] All samples were photocrosslinked by UV exposure.

[0127] Figure 5.1 shows the average strain sweep at 1 Hz of hydrogels comprising a functionalized recombinant bacterial collagen-like protein according to the present invention at concentrations of 9%, 6%, 4.5% and 3%.

[0128] Figure 5.1 shows that the rigidity of the hydrogel can be adjusted by changing the concentration of the functionalized recombinant bacterial collagen-like protein according to the present invention. At a concentration of 3 wt% of the recombinant bacterial collagen-like protein functionalized with methacryloyl groups, the rigidity (G') was found to be 340 Pa, and at a concentration of 9%, its G' was 8200 Pa.

[0129] The rigidity (G') of the hydrogel increases with increasing concentration of recombinant bacterial collagen-like protein functionalized with methacryloyl groups as described in Example 1. However, this increase is not linear. Interestingly, as the concentration of recombinant bacterial collagen-like protein functionalized with methacryloyl groups increases, the strain at which the crossover point occurs decreases. This confirms that the higher the concentration, the more brittle the hydrogel. Compared with the 4.5%, 6% and 9% hydrogels, the 3% hydrogel can withstand higher strains before the structure breaks.

[0130] Hydrogels produced from methacryloylated animal-derived collagen are typically less than 0.6% w / v due to solubility limitations and pH-induced gelation. As a result, mechanical properties are significantly weaker, with a G' value of 118 Pa.

[0131] Example 6

[0132] Example 6 shows that hydrogels comprising functionalized recombinant bacterial collagen-like proteins according to the present invention are cell compatible. The experiments were performed with human dermal fibroblasts under standard cell culture conditions. The hydrogels were produced as described in Examples 1 to 3.

[0133] The cytocompatibility of recombinant bacterial collagen-like proteins functionalized with methacryloyl groups as described in Example 1 was evaluated and compared against controls which were standard culture (no protein) and non-functionalized recombinant bacterial collagen-like proteins. The test samples were first dissolved in cell culture medium and subsequently diluted to different concentrations. Human dermal fibroblasts cultured in 96-well plates were exposed to different dilutions of the test samples for 24 hours under standard culture conditions. Cell metabolic activity (an indicator of cell viability), proliferation and cytotoxicity of the test samples were quantified. A colorimetric XTT assay was performed because metabolically active cells reduce yellow tetrazolium salts to orange formazan. Dye. Orange nail The concentration of the dye was measured by a microplate reader via absorbance measurement at 450 nm.

[0134] Figure 6.1 shows the metabolic activity of human dermal fibroblasts (HDF) incubated with increasing concentrations of non-functionalized recombinant bacterial collagen-like protein of SEQ ID NO 4 (rCol) and recombinant bacterial collagen-like protein of SEQ ID NO 4 functionalized with methacryloyl groups (rCol-MA), normalized to cells cultured in culture medium.

[0135] Solutions of non-functionalized recombinant bacterial collagen-like protein of SEQ ID NO: 4 (rCol) and recombinant bacterial collagen-like protein of SEQ ID NO: 4 functionalized with methacryloyl groups (rCol-MA) are cell compatible as assessed by incubating human dermal fibroblasts (HDF) with increasing concentrations of samples dissolved in culture medium for 24 hours under standard cell culture conditions. Metabolic activity was quantified using the XTT assay and then normalized to cells cultured in culture medium.

[0136] The metabolic activity of cells exposed to the test solutions at all tested concentrations was comparable to that of cells cultured under normal conditions. This indicates that the recombinant bacterial collagen-like protein and the recombinant bacterial collagen-like protein functionalized with methacryloyl groups are cell-compatible and do not induce cytotoxicity.

[0137] The cytocompatibility of hydrogels prepared from photocrosslinked recombinant bacterial collagen-like proteins functionalized with methacryloyl groups, prepared as described in Example 2, was also evaluated. High-density polyethylene film (HDPE) and commercial bovine collagen methacrylate (bCol-MA) were used as negative controls, while polyurethane film (ZDEC) was used as a positive control to evaluate cytotoxicity. Extracts of the samples were prepared by incubating the samples in cell culture medium at 37°C for 72 hours. The protocol is based on the international standard DIN EN ISO 10993-12. The extracts were then applied to human dermal fibroblasts for 24 hours, after which metabolic activity was measured using the XTT assay as described above.

[0138] Figure 6.2 shows the metabolic activity of human dermal fibroblasts cultured in sample extracts of high-density polyethylene film (HDPE), polyurethane film (ZDEC), commercial methacryloyl-ated bovine collagen, and photo-crosslinked hydrogel (rCol-MA), wherein the photo-crosslinked hydrogel contains a recombinant bacterial collagen-like protein having SEQ ID NO 4 functionalized with methacryloyl groups.

[0139] Hydrogels prepared from photocrosslinked recombinant bacterial collagen-like proteins functionalized with methacryloyl groups were cytocompatible regardless of concentration. The metabolic activity of cells exposed to extracts from these hydrogels was comparable to that of cells cultured under normal conditions and in extracts of HDPE. Extracts prepared from hydrogels of bovine collagen functionalized with methacryloyl groups induced higher metabolic activity and cell proliferation. In contrast, the positive control ZDEC induced significant cytotoxicity as cell metabolic activity dropped to baseline. The results of XTT were confirmed by fluorescence microscopy observations of living cells.

[0140] Cells were also cultured on and encapsulated in hydrogels prepared from photo-crosslinked recombinant bacterial collagen-like proteins functionalized with methacryloyl groups according to the present invention. The hydrogels were prepared as described in Example 3. When human dermal fibroblasts were cultured on the hydrogels, increasing the concentration of recombinant bacterial collagen-like proteins functionalized with methacryloyl groups from 3% to 6% promoted faster proliferation and greater spreading of the cells. This was observed via fluorescent imaging of cells seeded on the hydrogels. In contrast, when human dermal fibroblasts were encapsulated in hydrogels, faster cell proliferation and increased cell spreading were observed for cells encapsulated in a lower concentration of 1.5% hydrogel compared to cells cultured in 3% and 6% hydrogels. This is attributed to the lower rigidity of the 1.5% hydrogel, which promotes cell migration and spreading.

[0141] Example 7

[0142] Example 7 was conducted to investigate the biocompatibility of hydrogels and sponges comprising functionalized recombinant bacterial collagen-like proteins according to the present invention in vivo. The hydrogels and sponges were produced as described in Examples 1-3.

[0143] The hydrogels and sponges prepared from the functionalized recombinant bacterial collagen-like proteins according to the present invention were implanted into the subcutaneous space of immunocompetent C57BL / 6 mice. Controls included biocompatible high-density polyethylene films (HDPE) and sham surgeries without implants.

[0144] The implants and surrounding tissues were explanted after 1 and 3 months to determine the biocompatibility, stability, and stimulation potential of the implanted materials. Histological examination of the explants was performed according to ISO 10993-6:2009.

[0145] At the 1 month time point, all samples could still be observed in the explanted tissue. Cellular infiltration was observed to varying degrees by polymorphonuclear cells, lymphocytes, plasma cells, macrophages and some giant cells. Newly formed blood vessels were also observed.

[0146] At the 3-month time point, all photo-cross-linked scaffolds and sponges were still observed in the explants, suggesting in vivo stability.

[0147] The immune responses of hydrogel and sponge samples were compared with those of HDPE and sham controls, and the photo-cross-linked recombinant bacterial collagen-like protein functionalized with methacryloyl groups was considered non-irritating.

[0148] Example 8

[0149] Recombinant bacterial collagen-like protein of SEQ ID NO 4 functionalized with methacryloyl groups (rCol-MA) was mixed with polyethylene glycol acrylate (PEG-AR) and subsequently photocrosslinked to form a hydrogel. Such a formulation allows to reduce the amount of functionalized recombinant bacterial collagen-like protein according to the present invention required for hydrogel formation, and enables to adjust mechanical and physical properties.

[0150] Different hydrogels were produced using various ratios of recombinant bacterial collagen-like protein functionalized with methacryloyl groups as shown in Example 1 and branched (8-arm) PEG-AR of molecular weight 10 kDa.

[0151] Sample 8.1 was prepared using the bio-ink composition as described in Example 2, comprising 6% of the recombinant bacterial collagen-like protein of SEQ ID NO 4 functionalized with methacryloyl groups.

[0152] Sample 8.2 was prepared using a bio-ink composition comprising 6% of a 10 kDa molecular weight branched polyethylene glycol acrylate.

[0153] Sample 8.3 was prepared using the bio-ink composition as described in Example 2, which contained 3% of the recombinant bacterial collagen-like protein of SEQ ID NO 4 functionalized with methacryloyl groups and 3% of branched polyethylene glycol acrylate with a molecular weight of 10 kDa.

[0154] Sample 8.4 was prepared using the bio-ink composition as described in Example 2, which contained 6% of the recombinant bacterial collagen-like protein of SEQ ID NO 4 functionalized with methacryloyl groups and 3% of branched polyethylene glycol acrylate with a molecular weight of 10 kDa.

[0155] Sample 8.5 was prepared using the bio-ink composition as described in Example 2, which contained 6% of the recombinant bacterial collagen-like protein of SEQ ID NO 4 functionalized with methacryloyl groups and 6% of branched polyethylene glycol acrylate with a molecular weight of 10 kDa.

[0156]

[0157] Table 8.1 Average storage modulus, loss modulus and corresponding standard deviation of hydrogels formulated with different ratios of functionalized recombinant bacterial collagen-like protein according to the present invention and 10 kDa molecular weight branched polyethylene glycol acrylate at 0.1% strain. The values ​​provided are the average of three measurements.

[0158] At the same concentration of 6%, the branched polyethylene glycol acrylate hydrogel and the functionalized recombinant bacterial collagen-like protein according to the present invention have comparable rigidity. When 3% of the functionalized recombinant bacterial collagen-like protein according to the present invention are formulated with 3% branched polyethylene glycol acrylate, the resulting hydrogel has a similar G'. This shows that the addition of branched polyethylene glycol acrylate compensates for the reduction in the concentration of the functionalized recombinant bacterial collagen-like protein according to the present invention while maintaining the mechanical properties.

[0159] Keeping the concentration of the functionalized recombinant bacterial collagen-like protein according to the present invention constant at 6%, increasing the concentration of branched polyethylene glycol acrylate increased the rigidity of the resulting hydrogel.

[0160] Example 9

[0161] The recombinant bacterial collagen-like protein of SEQ ID NO 4 functionalized with methacryloyl groups was mixed with a suspension of biosynthesized nanocellulose fibers (NF) and subsequently photo-crosslinked to form a composite hydrogel.

[0162] NF is produced by bacterial fermentation. A stock solution of NF is mixed with a stock solution of a recombinant bacterial collagen-like protein (rCol-MA) functionalized with a methacryloyl group as shown in Example 1 and a photoinitiator. Up to 0.6 wt % of NF is incorporated into the composite mixture together with 1.5% of a recombinant bacterial collagen-like protein of SEQ ID NO 4 functionalized with a methacryloyl group and 0.2% of a phenyl-2,4,6-trimethylbenzoyl lithium phosphinate (LAP) photoinitiator. The addition of NF increases the viscosity of the mixture, as determined by rheological measurements using an Anton Paar MCR 302 rheometer with a 15 mm profile measuring plate. Shear thinning properties are also observed for NF / rCol-MA mixtures. All of these viscosity properties make the formulations highly suitable for potential applications such as injectable hydrogels and bio-inks for extrusion bioprinting.

[0163] Figure 9.1 illustrates how the bio-ink viscosity increases with the incorporation of biosynthesized nanocellulose fibers (NF) into a solution of recombinant bacterial collagen-like protein (rCol-MA) functionalized with methacryloyl groups. The viscosity of the NF / rCol-MA solution is significantly higher than that of the rCol-MA solution. For all solution mixtures, the viscosity increases with increasing NF content. A shear-thinning response, i.e., a decrease in viscosity with increasing shear rate, is observed in all NF / rCol-MA solutions.

[0164] After photocrosslinking, the nanocellulose fibers mechanically reinforced the soft hydrogels, especially at a low concentration of 1.5% of recombinant bacterial collagen-like protein (rCol-MA) functionalized with methacryloyl groups as shown in Example 1. In a strain sweep study, the stiffness of the different formulations was compared at 0.1% strain (in the linear viscoelastic region) and 1 Hz frequency.

[0165] Figure 9.2 depicts how the incorporation of NFs increases the stiffness (G') of the composite hydrogels in a concentration-dependent manner.

[0166] The storage modulus of the hydrogel increases with the concentration of the functionalized recombinant bacterial collagen-like protein according to the present invention. For 1.5% rCol-MA hydrogel, the average storage modulus is around 70Pa, while for 3%, the value increases to around 760Pa. The incorporation of NF also increases the storage modulus of the hydrogel containing rCol-MA. The storage modulus of the composite hydrogel containing 0.3% NF and 1.5% rCol-MA is similar to that of the 2% rCol-MA hydrogel.

[0167] The reinforced composite hydrogel provided a favorable microenvironment for the proliferation, spreading, and migration of encapsulated human dermal fibroblasts.

[0168] Example 10

[0169] The functionalized recombinant bacterial collagen-like protein prepared as shown in Example 1 was blended with the short self-assembling peptide Ac-KGAVLI-NH2 to form a chemically well-defined composite hydrogel. Such a composite system combines the advantages of both the photo-crosslinkable functionalized recombinant bacterial collagen-like protein according to the present invention and the self-assembling peptide. The tendency of the peptide to self-assemble under defined conditions (addition of saline buffer or elevated temperature) allows hydrogel formation, even when the concentration of the functionalized recombinant bacterial collagen-like protein according to the present invention is below the gelation limit or in the absence of photo-crosslinking.

[0170] Surprisingly, the functionalized recombinant bacterial collagen-like protein according to the present invention (despite its significantly larger molecular weight) does not interfere with the self-assembly of the peptide, because the minimum gelation concentration of 5 mg / mL (0.5 wt %) peptide is comparable in the presence and absence of the functionalized recombinant bacterial collagen-like protein according to the present invention. This is an unexpected result because the peptide self-assembly is driven by intermolecular interactions that are disrupted by the addition of macromolecular proteins such as serum albumin. In addition, significantly higher concentrations of the functionalized recombinant bacterial collagen-like protein according to the present invention can be encapsulated even at the lowest gelation concentration of the peptide compared to the concentration of the peptide. Post-gelation UV can be applied to further "cure" the hydrogel. The photo-crosslinked composite hydrogel has improved mechanical properties compared to hydrogels containing only peptides and hydrogels containing only photo-crosslinked functionalized recombinant bacterial collagen-like proteins prepared as shown in Example 1.

[0171] The hexameric N-terminally acetylated, C-terminally amidated peptide lysine-glycine-alanine-valine-leucine-isoleucine (Ac-KGAVLI-NH2) was selected as the optimal peptide component due to its tendency to consistently form hydrogels under physiological conditions at concentrations as low as 5 mg / mL (0.5%). This peptide can encapsulate significantly larger amounts of the functionalized recombinant bacterial collagen-like protein according to the present invention.

[0172] The following hydrogels were visually evaluated:

[0173] - Contains 0.5% peptide Ac-KGAVLI-NH2,

[0174] - containing 0.5% of the peptide Ac-KGAVLI-NH2 and 3% of a functionalized recombinant bacterial collagen-like protein according to the invention, and

[0175] - contains 0.5% of the peptide Ac-KGAVLI-NH2 and 3% of the functionalized recombinant bacterial collagen-like protein according to the invention, which was UV-cured.

[0176] Incorporation of functionalized recombinant bacterial collagen-like proteins according to the present invention produces less opaque hydrogels that gel more rapidly.

[0177] In addition, the composite hydrogels had higher rigidity compared to the hydrogels containing peptides alone and the hydrogels containing the photo-crosslinked functionalized recombinant bacterial collagen-like protein prepared as shown in Example 1.

[0178] Figure 10.1 shows the storage (G') modulus of different hydrogels containing 6% functionalized recombinant bacterial collagen-like protein (rCol-MA) prepared as shown in Example 1, 0.6% peptide Ac-KGAVLI-NH2, 0.6% peptide Ac-KGAVLI-NH2 and 1% functionalized recombinant bacterial collagen-like protein prepared as shown in Example 1, and photo-crosslinked hydrogels containing 0.6% peptide Ac-KGAVLI-NH2 and 1% functionalized recombinant bacterial collagen-like protein prepared as shown in Example 1.

[0179] The hydrogel containing 0.6% peptide Ac-KGAVLI-NH2 had a significantly higher storage modulus value of 3 kPa compared to the hydrogel containing 6 wt% of photo-crosslinked functionalized recombinant bacterial collagen-like protein prepared as shown in Example 1 (0.5 kPa). The composite hydrogel containing 0.6% peptide Ac-KGAVLI-NH2 and 1% functionalized recombinant bacterial collagen-like protein prepared as shown in Example 1 increased the rigidity by 3 times. The rigidity of the composite hydrogel containing functionalized recombinant bacterial collagen-like protein prepared as shown in Example 1 can be further enhanced via photo-crosslinking after exposure to UV in the presence of a photoinitiator.

[0180] The composite hydrogel containing the hexameric peptide Ac-KGAVLI-NH2 and the functionalized recombinant bacterial collagen-like protein according to the present invention is also biocompatible. Human dermal fibroblasts were uniformly encapsulated in the composite hydrogel of 0.6% peptide Ac-KGAVLI-NH2 + 1% functionalized recombinant bacterial collagen-like protein prepared as shown in Example 1. The cells proliferated and spread normally and were clearly shown by their spindle-like morphology. Therefore, the composite hydrogel is suitable for long-term culture.

[0181] Protein sequence

[0182] SEQ ID NO: 1 Streptococcus pyogenes collagen-like protein (CLP), full-length protein

[0183] SEQ ID NO:2 Streptococcus pyogenes CLP, truncated 3

[0184] SEQ ID NO:3 Streptococcus pyogenes CLP, truncated 5

[0185] SEQ ID NO:4 Streptococcus pyogenes CLP, without V domain

Claims

1. A recombinant bacterial collagen-like protein having an amino acid sequence that is at least ≥60% identical to the amino acid sequence of SEQ ID NO: 1, characterized in that The amino acid sequence comprises a deletion of at least 38 amino acids at the N-terminus of the amino acid sequence of SEQ ID NO: 1, and the recombinant collagen-like protein is functionalized with an ethylenically unsaturated group.

2. The recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups according to claim 1, characterized in that The amino acid sequence has at least 60% identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

3. The recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups according to claim 2, characterized in that The amino acid sequence has at least 90% identity to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:

4.

4. The recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups according to any one of claims 1 to 3, characterized in that The functionalization degree ranges from 5% to 99% of the primary amine groups on the N-terminus of the recombinant collagen-like protein, the primary amine groups of the lysine residues, and the hydroxyl groups of the tyrosine residues.

5. The recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups according to any one of claims 2 to 4, characterized in that The ethylenically unsaturated group is a (meth)acryloyl group, a fumarate group or a vinyl group.

6. The recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups according to claim 5, characterized in that The ethylenically unsaturated group is methacrylate, acrylate, methacrylamide, acrylamide or a mixture thereof.

7. The recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups according to claim 6, characterized in that The amino acid sequence has at least 98% identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the recombinant collagen-like protein is functionalized with methacrylate, acrylate, methacrylamide, acrylamide groups or a mixture thereof.

8. The recombinant bacterial collagen-like protein functionalized with ethylenically unsaturated groups according to claim 7, characterized in that The amino acid sequence has at least 99% identity with the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the recombinant collagen-like protein is functionalized with methacrylate, acrylate, methacrylamide, acrylamide groups or a mixture thereof.

9. A method for functionalizing a recombinant bacterial collagen-like protein with (meth)acrylic anhydride, wherein the recombinant bacterial collagen-like protein has an amino acid sequence that is at least 98% identical to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, characterized in that Functionalization was performed at a molar ratio of (meth)acrylic anhydride to amine groups of the recombinant bacterial collagen-like protein of 0.5:1 to 5:

1.

10. A bio-ink preparation comprising a) 1 to 10% by weight of a functionalized recombinant bacterial collagen-like protein according to any one of claims 1 to 8 b) 80 to 99% by weight of an aqueous solvent c) 0.01 to 2 wt% photoinitiator d) 0 to 5 wt. % of a photocrosslinkable polymer or other photocrosslinkable peptide e) 0 to 10 wt.% additives, rheology modifiers, biopolymers, gelation promoters, bioactive moieties, peptides, nanocellulose and / or cells Provided that the sum of all components of the bio-ink is 100%.

11. Method for producing a hydrogel by photocrosslinking the functionalized recombinant bacterial collagen-like protein according to any one of claims 1 to 8.

12. A method for producing a hydrogel by photocrosslinking the bio-ink formulation according to claim 10.

13. The method for producing a hydrogel according to claim 12, characterized in that The bio-ink formulation was photo-crosslinked using UV light.

14. A hydrogel comprising the photo-crosslinked functionalized recombinant bacterial collagen-like protein according to any one of claims 1 to 8.

15. The hydrogel according to claim 14, characterized in that The hydrogel further comprises nanocellulose, peptides or a mixture thereof.

16. A scaffold for tissue engineering, comprising the hydrogel according to claim 11 or 12.

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