Preparation method and application of whole-protein woven network hydrogel

Abstract

The invention discloses a preparation method and application of whole-protein woven network hydrogel, and belongs to the technical field of biological materials. According to the preparation method disclosed by the invention, by designing a pseudo-cable hydrocarbon dimer (BXA ') and utilizing stimulation factors such as concentration, calmodulin (CaM), photoresponse CaM (hv), temperature and / or light to induce topological conversion of the pseudo-cable hydrocarbon dimer into a star-shaped molecule, intermolecular crosslinking is realized, and then spontaneous polymerization is performed to form a whole-protein woven network, so that the whole-protein woven network has dynamically adjustable mechanical properties; the ultrahigh-modulus full-protein woven network hydrogel with multiple stimulation responsiveness is realized. Further, the hydrogel can be used for immobilization and controllable release of enzymes (such as mannase). The whole-protein woven network hydrogel provided by the invention has the advantages of high biocompatibility, controllable structure, diversified response mechanisms and the like, and has wide application prospects in the fields of biomedicine, intelligent drug loading, tissue engineering and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a method for preparing a fully protein-woven network hydrogel and its application as a controlled-release gel for target proteins such as mannanase. Background Technology

[0002] As the core substance of life, proteins are considered a highly promising next-generation biomaterial due to their precisely programmable structures, outstanding multifunctional bioactivity, and inherent biodegradability. Among them, all-protein hydrogels have emerged as a unique class of gene-encoded biomaterials. Currently, there are two main methods for cross-linking pure protein components: chemical reactions and physical aggregation. Chemical cross-linking utilizes the inherent reactivity of natural or non-natural amino acid residues; these reactions can proceed spontaneously or be mediated by enzymes and photosensitive modules. Gene-encoded click chemistry is particularly prominent due to its ability to achieve site-specific cross-linking within the biological reaction space, and can construct all-protein chemically cross-linked hydrogels with pre-defined multifunctionality (Zhang, W.-B., J. Am. Chem. Soc. 2013, 135, 13988-13997). The Tirrell team pioneered the development of all-protein hydrogels using protein interactions such as coils and helices (Shen, W., Nat. Mater. (2006, 5, 153-158). Other physical cross-linking strategies, such as pairing involving adhesion proteins, aggregation mediated by vitamin B12-dependent photosensitive transcription regulators, and assembly at the Tax-interacting protein-1 interface, have also been used in the design of dynamic protein materials. However, these physical cross-links are susceptible to solution erosion. Despite progress, existing protein hydrogels still generally suffer from low cross-linking density and weak mechanical properties due to uncontrollable structural defects.

[0003] Recent advancements in the field of synthetic polymers have focused on mechanical reinforcement strategies, such as dual-network, interpenetrating networks, microphase separation, and topology engineering (Wei, J., Sci. China Chem. (2022, 65, 486-496), providing new ideas for protein materials. Especially based on rotaxane (Zhang, Z., Acc. Chem. Res. Mechanically interlocked polymers (2024, 57, 992-1006) exhibit topological entanglement that can be significantly enhanced through slip-ring effects. Researchers constructed highly entangled all-protein hydrogels using the tumor suppressor p53dim motif, achieving genetically programmable weaving networks and extended relaxation times (Yang, Z., ACS Macro Lett. (2018, 7, 1468-1474) The triple helix structure of collagen provides another type of natural entanglement template for weaving protein networks. However, this type of biomimetic woven hydrogel is still limited by insufficient cross-linking density and bottlenecks in practical applications.

[0004] Therefore, it is crucial to develop synergistic strategies that can both enhance mechanical properties and preserve the natural functions of proteins (such as stimulus responsiveness and biocatalytic activity). Summary of the Invention

[0005] The purpose of this invention is to controllably form a fully protein-woven network hydrogel with multiple stimulus responses within a topologically restricted network, thereby achieving a multi-level enhanced ultra-high modulus hydrogel for enzyme immobilization and release.

[0006] To achieve the above technical objectives, this invention provides a method for preparing a full-protein hydrogel based on topological weaving, see [link to relevant documentation]. Figure 1 First, pseudo-needle dimers are designed and prepared: topological proteins are designed by fusing gene-encoded polypeptide-protein reaction pairs, entanglement domains, and protein-short peptide binding systems to express fusion proteins that form pseudo-needle dimers; then, gelation is performed: under the stimulation of short peptide binding proteins (such as calmodulin), temperature, and / or light, the fusion protein induces the pseudo-needle dimer to topologically transform into star-shaped molecules, which then spontaneously polymerize to form a fully protein-woven network.

[0007] The genetically encoded peptide-protein reaction pair can be split into two fragments, commonly referred to as the tag peptide (Tag) and the catcher protein (Catcher). Commonly used peptide-protein reaction pairs include various covalently binding and corresponding physically binding domains of SpyTag-SpyCatcher, as well as domains such as SdyTag-SdyCatcher, SnoopTag-SnoopCatcher, and Split-GFP that exhibit similar coupling reactions.

[0008] The entangled structural domain includes homogeneous entangled primitives such as X(p53dim), which are composed of two identical X's. + (p53dimT4R / Q6R) and X - Heteroentangled primitives composed of two different Xs, such as (p53dimT4E / Q6E), and mutants from the above versions, such as X'(p53dimK20P).

[0009] To form a network, the intramolecular binding of peptide-protein reaction pairs, such as SpyCatcher (B) and SpyTag (A), needs to be transformed into intermolecular binding. If the constructed fusion protein can form intramolecular covalent bonds, such as the BXA system fusion protein constructed using the covalently bound Spy domain (BA), it will form a nepheloid dimer after expression and purification, rather than a pseudonepheloid dimer, and will not be able to induce topological transformation into a star-shaped molecule to form a network. However, if A is mutated to A' to obtain the physically bound Spy domain (BA'), the dimer formed by the corresponding BXA' system fusion protein is a pseudonepheloid, which can then topologically transform into a star-shaped molecule under stimulation and assemble into a network. Another strategy is to construct fusion proteins of BXB and AXA systems, express these two fusion proteins separately, and then assemble them. This avoids the need to consider topological transformation into a star-shaped molecule during induction (because BXB and AXA are naturally star-shaped molecules) and can utilize the covalent binding between B and A to weave a network.

[0010] The protein-short peptide binding system includes physical domains such as calmodulin (CaM)-M13 peptide that can trigger topological protein conformational transitions.

[0011] The genetically encoded polypeptide-protein reaction pair and entanglement domain are essential building blocks for constructing the topologically transformable fusion protein of this invention. These building blocks are linked by connecting peptides. The two segments of the polypeptide-protein reaction pair are located at the N-terminus and C-terminus of the fusion protein, respectively, and these two segments can form a dynamically tunable physical binding domain. After expressing and purifying the fusion protein, a pseudo-nepote dimer is obtained under the guidance of the entanglement domain.

[0012] In this invention, the genetically encoded polypeptide-protein reaction pair is preferably a mutated tag polypeptide-catcher protein reaction pair (such as SpyTag-SpyCatcher), that is, one or more residues in the amino acid sequence of the tag polypeptide or catcher protein are mutated so that the catcher protein and the tag polypeptide do not covalently couple to form a physical binding domain.

[0013] Furthermore, by adding protein-binding short peptides as building blocks in the protein-short peptide binding system to the fusion protein, network formation can be promoted during subsequent gelation using short peptide-binding proteins (such as calmodulin). A typical topologically transformable fusion protein of this invention has the following structural formula: reaction pair fragment one - tangled domain - protein-binding short peptide - reaction pair fragment two, wherein reaction pair fragment one and reaction pair fragment two refer to two fragments of a polypeptide-protein reaction pair capable of forming dynamically tunable physically binding domains. The building blocks are linked by connecting peptides; preferably, the connecting peptides are flexible chains, such as GGS, GGSGG, GSGG, etc. More preferably, a histidine purification tag (6×His) and / or a solubilization tag is added to the N-terminus or C-terminus of the fusion protein.

[0014] In some embodiments of the present invention, a Figure 1 The fusion protein shown comprises the following building block BX-M13-A', where B represents SpyCatcher, X represents p53dim, M13 represents the M13 peptide, and A' represents the SpyTag mutant SpyTag(DA). The amino acid sequences of SpyCatcher, SpyTag, p53dim, and M13 are shown in SEQ ID NO:1, 2, 4, and 6 of the sequence listing, respectively. SpyTag(DA) is a D7A mutant of SpyTag (SEQ ID NO:3), which aims to disrupt the covalently binding domain between SpyCatcher and SpyTag, replacing it with a dynamically tunable physically binding domain. In other embodiments of the invention, a mutant of p53dim, p53dim(KP), is used as a control. This K19P variant of p53dim aims to disrupt the entanglement between p53dim molecules, preventing them from forming dimers, and its sequence is shown in SEQ ID NO:5. The amino acid sequence of the short peptide-binding protein CaM is shown in SEQ ID NO:7 in the sequence listing.

[0015] Methods to achieve gelation and network enhancement include utilizing CaM and photo-regulated CaM(hv), high temperature and photo-regulated heating, among which CaM(hv) is regulated by ultraviolet light irradiation such as UV365. Since the CaM sequence lacks Cys, a thiol group is introduced through an N60C mutation to enable a click reaction with compounds containing azobenzene side groups, such as 4-phenylazomaleinanil (4-benzomycin, a maleimide containing an azobenzene side group). The reaction product is the photoresponsive protein CaM(hv). If the azobenzene on CaM is in the trans conformation, it will extend into the binding pocket of CaM, reducing its binding ability to M13. If UV365 light treatment is used to convert the azobenzene to the cis conformation, more space is available for the binding pocket, increasing the binding ability of CaM(hv) to M13. The light-controlled heating is achieved by using near-infrared dyes with high molar extinction coefficients to convert light of a specific wavelength into heat. Near-infrared dye molecules, such as IR-780, are typical near-infrared heptamethyl anthocyanins with high molar extinction coefficients.

[0016] The all-protein woven network hydrogel obtained by the method for preparing all-protein hydrogels based on topological weaving according to the present invention is also within the scope of protection of the present invention.

[0017] Using the aforementioned all-protein woven network hydrogel as a carrier, target proteins can be loaded onto it through physical or chemical immobilization, enabling the immobilization and controlled release of the target proteins. A fragment of the genetically encoded peptide-protein reaction pair (such as SpyTag or its D7A mutant) is fused to the target protein, and then blended with the pseudocarbohydrate dimer to prepare the hydrogel.

[0018] In some specific embodiments of the present invention, network enhancement is achieved by controlling the unfolding and aggregation of the SpyTag (DA) (abbreviated as A')-SpyCatcher (abbreviated as B) complex, ultimately developing a full-protein hydrogel with multi-stimulus responsiveness, suitable for applications such as immobilization and controlled release of mannanase. The gel can be loaded with enzymes such as mannanase through different loading methods (e.g., physical and chemical immobilization), and the enzyme release rate can be regulated, thereby enabling programmed breaking of guar gum in oil and gas extraction.

[0019] The research of this invention includes the following aspects: 1) Design of interlocked dimer proteins by fusing protein motifs. The protein motifs involved in this invention include the SpyTag-SpyCatcher domain, the tumor suppressor p53dim (abbreviated as X), and the CaM-M13 complex.

[0020] 2) Topological proteins were characterized at the small molecule level using sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE), size exclusion chromatography (SEC), and circular dichroism spectroscopy (CD).

[0021] 3) Hydrogels were prepared using lyophilized topoproteins, with Tris-HCl buffer promoting the dissolution of the dry powder and molecular exchange, which could then be used to investigate the subsequent stimulus response.

[0022] 4) By utilizing the non-covalent binding of calmodulin CaM to M13 peptide, the internal tension of the topological protein ring is increased, promoting the transformation of pseudo-chain hydrocarbon molecules into star-shaped molecules, thereby increasing the cross-linking sites in the network.

[0023] 5) Explore the thermal responsiveness of whole protein woven networks, use a rheometer to detect changes in mechanical properties during thermal shock, realize network enhancement induced by protein unfolding, and reveal the mechanism of its micro-phase separation.

[0024] 6) Unlocking the photoactivated version of the full protein weaving network: Azophenyl is used to achieve photomodulation of calmodulin. As the UV irradiation time increases, the modulus of the protein network increases accordingly. Near-infrared dyes are introduced and near-infrared light irradiation is used for heating to enhance the protein network.

[0025] 7) Expand the applications of this protein network, such as using it as an immobilization and controlled release carrier for mannanase. Hydrogels were prepared by blending the topological protein with target proteins containing SpyTag, and the sustained-release effect of the gel was evaluated using methods such as protein concentration detection in the sustained-release solution and mechanical property measurement.

[0026] In summary, this invention utilizes stimulation-response factors such as concentration, calmodulin (CaM), photoresponsive CaM (hv), temperature, and / or light to achieve intermolecular cross-linking of the physical binding domains of peptide-protein reactions, thereby endowing it with dynamically tunable mechanical properties and realizing an ultra-high modulus all-protein woven hydrogel. Furthermore, this hydrogel can be used for the immobilization and controlled release of enzymes (such as mannanase). The all-protein hydrogel provided by this invention has advantages such as high biocompatibility, controllable structure, and diverse response mechanisms, and has broad application prospects in biomedicine, intelligent drug delivery, and tissue engineering. Attached Figure Description

[0027] Figure 1 This paper presents a design and fabrication method for a whole protein woven network, including a three-dimensional structural model and a schematic diagram of the secondary structure arrangement.

[0028] Figure 2The following is a series of characterizations of the constructed topological protein in Example 1, wherein (a) and (b) are volume exclusion chromatography (SEC) curves of BXA' and BX'A', respectively; (c) is a sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (SDS-PAGE) image; and (d) is a circular dichroism (CD) spectrum.

[0029] Figure 3 The following are the characterizations of the concentration-regulated BXA' network in Example 2, where (a) is the frequency scan curve of the protein hydrogel containing only 20% w / v BXA'; and (b) is the frequency scan curve of the protein hydrogel containing only 30% w / v BXA'.

[0030] Figure 4 The following are characterizations of the CaM-regulated BXA' network in Example 2, where (a) is the frequency scan curve of a protein hydrogel containing only 30% w / v BXA'; and (b) is the frequency scan curve of a protein hydrogel containing 30% w / v BXA' and an equivalent amount of CaM.

[0031] Figure 5 The following are characterizations of the CaM(hv)-regulated BXA' network in Example 2: (a) is the frequency scan curve of a protein hydrogel containing 30% w / v BXA' and an equivalent amount of CaM(hv); (b) is the frequency scan curve of a protein hydrogel containing 30% w / v BXA' and an equivalent amount of CaM(hv) after UV365 irradiation for 50 min.

[0032] Figure 6 The following are characterizations of the thermotropically enhanced all-protein braided network in Example 3, where (a) is the dynamic mechanical property scan curve of the protein hydrogel containing only 20% w / v BXA'; (b) is the dynamic mechanical property scan curve of the protein hydrogel containing only 30% w / v BXA'; and (c) is the storage modulus (G') of the 30% w / v BXA' protein hydrogel after treatment at different temperatures (16 ℃, 48 ℃, 70 ℃) under conditions of no / with CaM excitation.

[0033] Figure 7 The thermotropically enhanced protein hydrogel used in Example 4 is employed for the immobilization and release of mannanase (TpMan), wherein (a) is the release curve of the red fluorescent protein mCherry-A'; and (b) is the loss factor Tan of guar gum. δ (c) is the curve of (G' / G'') changing with time; (d) is the curve of the storage modulus (G') of guar gum changing with time. Detailed Implementation

[0034] To better understand the above technical solutions, the following will provide a detailed explanation of the technical solutions in conjunction with the accompanying drawings and specific implementation methods.

[0035] Example 1: This example provides a method for designing, synthesizing, and characterizing topological proteins for forming whole protein braiding networks.

[0036] In this invention, the constituent motifs of the pseudo-nephrite dimer, from N-terminus to C-terminus, include SpyCatcher-p53dim-M13-SpyTag(DA), abbreviated as BXA', and a peptide segment MKGSSHHHHHHVEASA containing a histidine purification tag and a solubilization tag is introduced at its N-terminus. Its amino acid sequence is shown in SEQ ID NO:8 of the sequence listing. The constituent motifs of the control protein BX'A' in this invention are SpyCatcher-p53dim(KP)-M13-SpyTag(DA), as shown in SEQ ID NO:9 of the sequence listing; the constituent motif of BA' is SpyCatcher-SpyTag(DA), as shown in SEQ ID NO:10 of the sequence listing. Wherein, p53dim(KP) is the K19P variant of p53dim, and SpyTag(DA) is the D7A variant of SpyTag. BX'A' is a monomer without entanglement structure and cannot form a dimer; while BXA' can form a pseudo-chain dimer.

[0037] The experimental methods and specific operations in this embodiment are as follows: a. Gene Construction and Transformation: The target gene was amplified by PCR, and NdeI (GCTAGC) and XhoI (GGTACC) restriction sites were introduced (forward primer: 5'-GCTAGCGCCATGGTTGATACCTTATC-3'; reverse primer: 5'-GGTACCTTACTTCGTCGGCTTGTATGCA-3'). The amplified fragment was cloned into the pQE-80L vector, and verified by double enzyme digestion and sequencing (sequencing company: Suzhou Genewiz Biotechnology Co., Ltd.). Recombinant plasmid transformation. E. coli BL21(DE3) competent cells were spread on 2×YT solid plates containing 100 μg / mL ampicillin.

[0038] b. Protein expression and purification: Single colonies were picked and inoculated into 2×YT liquid medium, and cultured at 37 ℃ with shaking until OD... 600 =0.6-0.8 Add 1 mM isopropyl- βInduced with β-D-thiogalactoside (IPTG), cultured at 16 °C for 16 h. Cells were collected by centrifugation at 6000 × g for 10 min at 4 °C, and resuspended at a 1:5 (w / v) ratio in lysis buffer (40 mM Tris-HCl, 50 mM NaCl, 10 mM imidazole, pH 8.0). The cells were then sonicated on ice (300 W, 5 s on, 8 s off, total 20 min). The supernatant was collected by centrifugation at 12000 × g for 30 min at 4 °C. The equilibration buffer (40 mM Tris-HCl, 50 mM NaCl, 20 mM imidazole, pH 8.0) and elution buffer (40 mM Tris-HCl, 50 mM NaCl, 250 mM Mimidazole, pH 8.0) were used for nickel column affinity chromatography purification.

[0039] c. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis: Prepare a 12% separating gel (containing 4.0 mL of 30% acrylamide / methylenebisacrylamide (29:1), 2.5 mL of 1.5 M Tris-HCl (pH 8.8), 0.1 mL of 10% SDS, 0.1 mL of 10% ammonium persulfate, and 0.004 mL of TEMED, and bring the volume to 10 mL) and a 5% stacking gel (containing 0.83 mL of 30% acrylamide / methylenebisacrylamide (29:1), 0.63 mL of 1.0 M Tris-HCl (pH 6.8), 0.05 mL of 10% SDS, 0.05 mL of 10% ammonium persulfate, and 0.005 mL of TEMED, and bring the volume to 5 mL). Samples were loaded with 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 30% glycerol, 5%). β A mixture of mercaptoethanol and 0.02% bromophenol blue (4:1) was boiled for 10 minutes before loading the sample. Electrophoresis was then performed in electrophoresis buffer (6.06 g Tris-HCl, 36.03 g glycine, 2 g SDS, adjusted to 2 L with ddH2O) at 80 V until the sample entered the separating gel. The electrophoresis was then continued at 120 V. Protein bands were observed after Coomassie Brilliant Blue R-250 staining.

[0040] d. Volume exclusion chromatography: An ÄKTA pure 25 protein purification system (GE Healthcare) equipped with a Superdex 200 Increase 10 / 300 GL column (24 mL column volume) was used. The mobile phase was Tris-HCl buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.4, filtered through a 0.22 μm filter and degassed by sonication). The flow rate was set to 0.5 mL / min, and the sample loading volume was 1 mL. UV absorption was monitored at 280 nm. The column was pre-equilibrated with 2 column volumes (35 mL) of mobile phase. After baseline stabilization, the sample was injected, and the main peak fraction was collected (retention time approximately 24-32 minutes).

[0041] e. Circular dichroism spectroscopy: A J-1500 circular dichroism spectrometer (JASCO) was used at a constant temperature of 25 ℃. Detection was performed using quartz cuvettes with a 1 mm optical path (ultrasonically cleaned with ethanol and ultrapure water for 15 minutes). Protein samples were dissolved in 20 mM potassium phosphate buffer (pH 7.4) and the concentration was adjusted to 0.2 mg / mL. The sample loading volume was 200 μL. The scan range was set to 200-260 nm, the speed to 1 nm / s, the bandwidth to 1 nm, the response time to 1 s, and the data interval to 0.1 nm. A total of 3 scans were performed. Before each detection, baseline correction with buffer was performed and background subtraction was performed.

[0042] Figure 2 The SEC curves in (a) and (b) show the outflow volumes of BXA' and BX'A', respectively, proving that the former is a dimer in its natural state. Figure 2 The SDS-PAGE gel image in (c) shows that BXA' forms a pseudo-chain dimer through physical bonding, and under denaturing conditions, it behaves as a monomer, similar to BX'A' and BA'. Furthermore, Figure 2 The CD absorption curve in (d) shows that the BXA' pseudohelical dimer exhibits secondary structures such as α-helix (negative peaks at 208 nm and 220 nm) and β-turn (positive peak at 230 nm) in its natural state. The above characterization results are consistent with expectations.

[0043] Example 2: This example provides a calmodulin (CaM) regulation strategy for a whole protein weaving network, and develops a light-responsive version of CaM, CaM(hv), for regulating the network.

[0044] The experimental methods and specific operations in this embodiment are as follows: a. Preparation of protein hydrogel: The elution product obtained in Example 1b was desalted using a PD10 desalting column (Sephadex G-25 packing). Specifically, 2.5 mL of eluent was loaded onto a chromatography column with a packing height of 5 cm, followed by elution with 3 mL of ultrapure water. The eluent was collected and lyophilized (pre-frozen in liquid nitrogen for 1 hour, cold trap temperature -60°C, vacuum degree 10 Pa) to obtain protein powder, which was stored at -20°C for later use. The protein powder was transferred to a 1.5 mL EP tube, centrifuged at 12000 g for 2 minutes to allow the powder to adhere to the tube wall, Tris-HCl buffer was added, and the tube was centrifuged again (12000 g, 2 minutes, 4°C). The solution was then sonicated (50 W, 4°C) for 30 minutes until clear and transparent, and placed in a humidity-controlled 4°C freezer for later use.

[0045] b. Measurement of frequency sweep curves: An ARES-G2 rheometer (TA Instruments) with an 8 mm parallel plate fixture was used for testing. Paraffin oil was applied to the sample edges before the experiment to prevent evaporation of the hydrogel. First, the linear viscoelastic region was determined by strain scanning (10 rad / s, strain range 0.1-100%). Then, frequency sweep tests were performed at 16 ℃: with a fixed strain of 1%, the angular frequency range was 100-0.01 rad / s, and the curves of storage modulus (G') and loss modulus (G') as a function of frequency were recorded. Data acquisition time for each frequency point was no less than 10 oscillation cycles, and the experiment was repeated three times, with the average value taken.

[0046] c. Synthesis of the light-responsive protein CaM(hv): The CaM mutant (10 mg / mL) was dissolved in Tris-HCl buffer and mixed with 4-phenylazomaleinanil (4-benzodiazepine, 10 mg / mL) dissolved in DMF at a volume ratio of 1:1000. The mixture was stirred magnetically for 3 hours under light-protected conditions, achieving specific monosubstituted modification of cysteine ​​residues via free radical reaction to obtain CaM(hv). This optimized condition avoided the problems of multi-substituted byproducts caused by the pure DMF system and incomplete reaction in the pure aqueous system.

[0047] d. UV365 irradiation excitation of photoresponsive protein hydrogels: CaM(hv) and BXA' protein were mixed at a molar ratio of 1:1 and dissolved in 20 mM Tris-HCl buffer, adjusted to the target concentration (30% w / v as an example). The hydrogel was prepared according to the method described in specific operation a of this embodiment. Under light-protected conditions, the frequency sweep curve of the unexcited hydrogel was measured using an ARES-G2 rheometer (8 mm parallel plate clamp) (angular frequency range 0.01-100 rad / s, strain 1%). The paraffin oil at the edge of the sample was removed, and the surface of the hydrogel was vertically irradiated with a UV365 laser (power 5 mW, spot diameter 8 mm) for 50 minutes. The frequency sweep curve of the excited hydrogel was measured under the same rheological conditions.

[0048] like Figure 3 As shown, the BXA' network exhibits a significant concentration response, forming a gel only when the w / v fraction is greater than 20%. The BXA' network before and after CaM excitation displays drastically different mechanical properties. Figure 4 The frequency scanning curves show that CaM effectively promotes the ring-opening of pseudo-chain hydrocarbon molecules to form star-shaped molecules, thereby enhancing the network. CaM(hv) is based on CaM with the introduction of azobenzene, thereby achieving UV365 irradiation regulation. The three variants of CaM, N60C, D64C, and M124C, have sequences as shown in SEQ ID NO:11-13 of the sequence listing. Preferably, the reaction product of the N60C variant of CaM with 4-phenylazomaleinanil is used as CaM(hv). Azobenzene is inserted into a cysteine ​​residue, and when it is in the trans (… trans In its - conformation, it hinders the binding of CaM(hv) to the M13 peptide on BXA'; upon exposure to UV365 irradiation, it transforms into the cis-( cis -) The conformation opens the binding pocket, increasing the affinity for the M13 peptide. Figure 5 The frequency scan curves show that the mechanical properties of the protein hydrogels are enhanced with the extension of UV irradiation time.

[0049] Example 3: In this example, a temperature-responsive protein gel system was established by synergistically utilizing the unfolding of the Spy physical binding domain and the entanglement effect of p53dim.

[0050] The experimental methods and specific operations in this embodiment are as follows: a. NanoDSF (Micro-Diffusion Differential Scanning Fluorescence) for the determination of topological proteins T mThe purified topoprotein sample (0.5 mg / mL, dissolved in 20 mM Tris-HCl buffer, pH 7.4) was loaded into a standard capillary tube, and the temperature was increased from 25 °C to 95 °C at a rate of 1 °C / min, while simultaneously monitoring the fluorescence intensity ratio at 350 nm and 330 nm. The first derivative curve was calculated using the instrument's built-in software to determine the protein unfolding temperature. Each sample was tested three times to ensure data reliability.

[0051] b. DMA (Dynamic Thermomechanical Analysis) Test: An ARES-G2 rheometer (TA Instruments) with an 8 mm parallel plate fixture was used for testing. First, the linear viscoelastic region was determined by strain scanning (fixed frequency 1 Hz, strain range 0.1%-100%), and 1% strain was selected as the subsequent test condition. Temperature scanning was performed from 4 °C to 70 °C (heating rate 1 °C / min, frequency 1 Hz), and changes in storage modulus (G') and loss modulus (G'') were recorded in real time. Before testing, 20% or 30% w / v BXA' protein hydrogel was uniformly spread on the test platform, and silicone oil was applied to the edges to prevent moisture evaporation. Each experiment was repeated three times.

[0052] c. Temperature Annealing Experiment: A multi-stage temperature cycling procedure was implemented on an ARES-G2 rheometer. First, Sample 1 containing only 30% w / v BXA' and Sample 2 containing 30% w / v BXA' and an equimolar amount of CaM were equilibrated at 16 °C for 30 minutes to establish a baseline for initial measurements. Subsequently, the temperature was increased to 48 °C at a rate of 1 °C / min and held for 60 minutes, then cooled to 16 °C and equilibrated for 30 minutes to observe structural recovery for a second measurement. Finally, the temperature was increased to 70 °C and held for 60 minutes to induce Spy domain aggregation, and finally cooled to 16 °C for the final test. Throughout the process, a 1% oscillating strain (angular frequency 10 rad / s) was continuously applied to monitor the evolution of mechanical properties. Sufficient equilibration time was set for each temperature transition stage, and all experimental conditions were independently repeated three times.

[0053] The topological protein BXA' exhibited two characteristic unfolding transitions during the heating process as measured by NanoDSF: p53dim unfolded at 40 °C, while the Spy domain underwent dominant unfolding at 65 °C. Figure 6 Dynamic mechanical property analysis in (a) shows that the 20% w / v BXA' protein hydrogel exhibits a unique solid-liquid-solid phase transition behavior during heating: the first phase transition point (40 ℃) corresponds to network relaxation caused by p53dim unfolding, while the second phase transition point (65 ℃) originates from enhanced crosslinking induced by Spy domain unfolding. It is noteworthy that... Figure 6The 30% w / v BXA' system in (b) exhibited a similar mechanical behavior transformation, where the entanglement effect of p53dim provided stable crosslinking sites for the network, effectively preventing network collapse caused by the unfolding of the Spy domains, thereby achieving microphase separation-induced mechanical enhancement. Compared to 16 °C, at Figure 6 Under annealing at 48 °C, the increase in storage modulus G' for both Sample 1 and Sample 2 was less than 100%, indicating that annealing at 48 °C only caused a slight reorganization of the network structure. Compared to 48 °C, under annealing at 70 °C, the increase in storage modulus (G') for Sample 1 was greater than 200%, indicating that annealing at 70 °C can promote the aggregation between Spy domains, forming new crosslinking points and increasing the mechanical properties of the network. However, the increase in storage modulus (G') for Sample 2 was an order of magnitude higher, indicating that the proportion of star-shaped molecules in the CaM-excited hydrogel network was significantly higher than that in the hydrogel network containing only BXA', thus fully utilizing the synergistic effect of the Spy domain aggregation effect and the p53dim entanglement effect to effectively enhance the mechanical properties. This phenomenon reveals the dynamic evolution mechanism of protein network structure under temperature regulation.

[0054] Example 4: This example provides a method for thermally enhanced protein networks using IR780 dye-NIR808 nm laser, and uses a buffer containing mannanase to prepare a gel for controlled gel breaking of guar gum.

[0055] The experimental methods and specific operations in this embodiment are as follows: a. Preparation of thermotropically enhanced protein hydrogels: 5% w / v near-infrared dye IR780 (a highly efficient photothermal conversion material with characteristic excitation wavelengths of 780 / 808 nm) was incorporated into the gelation buffer and mixed thoroughly with 20% w / v BXA' solution before being transferred to an EP tube. The sample was vertically irradiated for 5 seconds using an 808 nm near-infrared laser (power density 2 W / cm²), inducing localized heating through the photothermal conversion effect of IR780, achieving rapid gelation. The gelation effect was verified by pipetting experiments: the untreated BXA' solution could be easily aspirated (significant drop in liquid level), while the sample after near-infrared irradiation formed a stable gel network that could not be aspirated by pipette. The target protein (POI) can be pre-dissolved in 20 mM Tris-HCl buffer for gel preparation, ensuring the final POI concentration does not exceed 5% of the total gel mass, thus obtaining a POI-loaded thermotropically enhanced BXA' gel.

[0056] b. Red Fluorescent Protein Release Curve: A thermo-enhanced BXA' gel (20% w / v) loaded with mCherry-A' was placed in a 1.5 mL EP tube, and 1 mL of Tris-HCl buffer was added. The release experiment was conducted by incubation at 37 ℃ with shaking (100 rpm). The protein concentration in the release medium was detected using a fluorescence spectrophotometer (excitation wavelength 587 nm, emission wavelength 607 nm). At each sampling time, all release medium was removed (centrifuged at 12000g for 2 minutes to remove residual gel particles), and an equal volume of fresh buffer was added before further incubation. The experimental data were validated through three independent replicates, and a curve was finally plotted based on the cumulative release mass.

[0057] c. Guar gum cross-linking-breaking: Guar gum loaded with mannanase TpMan (a enzyme derived from...) Thermotoga petrophila Thermostable mannanase in bacteria T m >90 ℃, Santos, C.-R. J. Struct. Biol. The thermotropically enhanced BXA' gel (2012, 177, 469-476) was chopped into small pieces, dispersed in a 2% guar gum solution, and crosslinking was initiated by adding borax crosslinking agent (final concentration 0.1%). Monitoring indicators for gel breaking efficiency included a decrease in storage modulus (G') to 50-100% of its pre-crosslinking state and G' ≤ 50 Pa, or a loss factor (tan... δ The concentration of tan ions increased to 100-120% of that before crosslinking and tan δ ≥ 0.5. The storage modulus (G') and loss factor (tan φ) were monitored in real time using an ARES-G2 rheometer. δ Changes: The control group (without enzyme) and the chemically immobilized group completed cross-linking within 300 seconds, while the physical adsorption group experienced incomplete cross-linking due to premature enzyme release. During the gel breaking phase, the physical adsorption group was completely degraded within 700 seconds (tan...). δ (Restore the initial plateau value), the chemical fixation group takes 4000 seconds to degrade, and thus the programmed breaking of guar gum gel can be achieved through molecular design.

[0058] Adding 5% w / v IR780 to the gelation buffer, combined with 808 nm near-infrared laser irradiation, can induce rapid gelation of a 20% w / v BXA' solution within 5 seconds. Figure 7 As shown in (a), the thermo-enhanced protein network effectively immobilizes SpyTag-tagged red fluorescent protein and achieves its controlled release. TpMan was immobilized in the BXA' network using both physical (the added TpMan-A' sequence is shown in SEQ ID NO:14) and chemical (the added TpMan-A sequence is shown in SEQ ID NO:15) methods, as shown in (a). Figure 7As shown in (b) and (c), rheological analysis reveals that physically adsorbed TpMan exhibits faster enzyme release kinetics, providing a new approach for developing stimulus-responsive biomaterials.

[0059] Table 1. Amino acid sequences involved in this invention

[0060]

Claims

1. A method for preparing a fully protein-woven network hydrogel, comprising the following steps: 1) Design and preparation of pseudo-nephrite dimers: First, a fusion protein constituting the pseudo-nephrite dimer is constructed. Its constituent units include gene-encoded polypeptide-protein reaction pairs and entanglement domains. The constituent units are linked by linker peptides. The two fragments of the gene-encoded polypeptide-protein reaction pairs are located at the N-terminus and C-terminus of the fusion protein, respectively, and the two fragments can form a dynamically tunable physical binding domain. The fusion protein is expressed and purified to obtain the pseudo-nephrite dimer. 2) Gel formation: In solution, pseudo-needle dimers are topologically transformed into star-shaped molecules by short peptide binding proteins, temperature and / or light stimulation, and then spontaneously polymerize to form a fully protein-woven network hydrogel.

2. The preparation method according to claim 1, characterized in that, The genetically encoded polypeptide-protein reaction pair is a mutated tag polypeptide-catcher protein reaction pair, that is, one or more residues in the amino acid sequence of the tag polypeptide or catcher protein are mutated so that the catcher protein and the tag polypeptide do not covalently couple and form a physical binding domain.

3. The preparation method according to claim 1, characterized in that, The constituent building blocks of the fusion protein also include short peptides that can be bound to proteins.

4. The preparation method according to claim 1, characterized in that, The fusion protein comprises the following building blocks: reaction pair fragment 1 - entanglement domain - short peptide that can be bound to proteins - reaction pair fragment 2, wherein reaction pair fragment 1 and reaction pair fragment 2 refer to two fragments of a polypeptide-protein reaction pair, and the building blocks are connected by flexible linker peptides; a histidine purification tag and / or a solubilization tag are added to the N-terminus or C-terminus of the fusion protein.

5. The preparation method according to claim 1, characterized in that, The entanglement domain is p53dim or a mutant thereof.

6. The preparation method according to claim 2, characterized in that, The tag peptide is SpyTag or its D7A mutant, and the catcher protein is SpyCatcher.

7. The preparation method according to claim 3, characterized in that, The short peptide that can be bound to proteins is the M13 peptide.

8. The preparation method according to claim 7, characterized in that, In step 2), CaM or the photoresponsive protein CaM(hv) is added to the solution of the pseudo-nephrite dimer to promote the topological transformation of the pseudo-nephrite dimer into star-shaped molecules and polymerize to form a fully protein-woven network hydrogel; wherein, the photoresponsive protein CaM(hv) is a product of the N60C mutant of CaM modified with azobenzene, and the formation of the fully protein-woven network hydrogel is promoted by irradiating the photoresponsive protein CaM(hv) with ultraviolet light.

9. The preparation method according to claim 8, characterized in that, The amino acid sequence of the fusion protein is shown in SEQ ID NO:8 in the sequence listing.

10. The preparation method according to claim 1, characterized in that, In step 2), the formation of the all-protein woven network hydrogel is induced by heating or light-controlled heating, wherein the light-controlled heating is achieved by adding a near-infrared dye with a high molar extinction coefficient to a solution of pseudo-toluene dimer, which converts light energy into heat energy under specific wavelength light irradiation.

11. The all-protein woven network hydrogel obtained by the preparation method according to any one of claims 1 to 10.

12. The application of the all-protein woven network hydrogel of claim 11 as a carrier for the immobilization and controlled release of target proteins, characterized in that, The target protein is loaded into the whole protein woven network hydrogel by physical or chemical immobilization.

13. The application as described in claim 12, characterized in that, A fragment of the genetically encoded polypeptide-protein reaction pair is fused to the target protein and then blended with the pseudocarbohydrate dimer to prepare a hydrogel.

14. The application as described in claim 12, characterized in that, The target protein is mannanase, and the whole protein woven network hydrogel loaded with mannanase is used for programmed breaking of guar gum in oil and gas extraction.

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