Photoresponse protein hydrogel with wide-range mechanical regulation characteristic and preparation method thereof

Abstract

The invention discloses photoresponsive protein hydrogel with wide-area mechanical regulation characteristics. The photoresponsive protein hydrogel is formed by polymerizing a specific mutant of photoactivated xanthoprotein (PYP) and a multi-arm polyethylene glycol derivative with the tail end modified with a maleimide group. Through rational design of molecular dynamics and material mechanics, cysteine mutation is carried out on the 48th site and the 85th site of a wild type PYP sequence, and the PYP (48 / 85) double mutant with an anisotropic unfolding energy scene is constructed. According to the mutant, exposed sulfydryl and a multi-arm polyethylene glycol derivative are subjected to Michael addition reaction to form a cross-linked network. Compared with the prior art (such as PYP 36 / 128 hydrogel), the method has the advantages that higher mechanical sensitivity and unfolding probability of PYP 48 / 85 under network tension are utilized, the limitation that traditional design only depends on chain length change is overcome, and remarkably larger macroscopic rigidity adjusting amplitude (the mechanical switch ratio can reach 70% or above) is achieved. The material has the characteristics of good biocompatibility, high response speed (millisecond level), complete reversibility and wide rigidity dynamic range, and can meet the biological application requirements of stem cell differentiation, tissue fibrosis model construction and the like with high requirements on a mechanical signal threshold.

Description

Technical Field

[0001] This invention relates to the field of hydrogel materials technology, specifically to a photoresponsive protein hydrogel with wide-range mechanical modulation properties, its preparation method, and its applications. Background Technology

[0002] The extracellular matrix (ECM) is not a static scaffold, but a highly dynamic microenvironment. During physiological and pathological processes such as embryonic development, wound healing, and fibrotic lesions, the mechanical properties of the ECM undergo significant spatiotemporal changes. These dynamic mechanical signals are crucial for regulating cell diffusion, migration, proliferation, and differentiation. To simulate this process in vitro, researchers have developed various dynamic hydrogel systems. Among them, photoresponsive hydrogels have attracted considerable attention due to their advantages such as non-contact control and high spatiotemporal resolution.

[0003] Early designs primarily relied on photolytic crosslinking agents, which achieved softening but the process was irreversible. Subsequently, reversibly crosslinked hydrogels based on photosensitive proteins such as LOV2 or Dronpa were proposed, but these typically suffer from slow response times (minutes) or small modulus changes (<20%). In recent years, hydrogels based on photoactivated flavin (PYP) have shown great potential due to their millisecond-level rapid photocycling properties. Existing research (including the applicant's previous work) has utilized mutants at positions 36 and 128 of PYP (PYP 36 / 128) to construct photoresponsive hydrogels.

[0004] However, in-depth mechanobiological studies have revealed that although PYP 36 / 128 hydrogels achieve reversible regulation, the magnitude of stiffness change before and after light exposure (i.e., the mechanical on / off ratio) is relatively limited (typically less than 50%). Traditional rubber elasticity theory tends to suggest that the longer the amino acid sequence spanned between mutation sites (e.g., 92 residues in 36 / 128), the longer the unfolded chain length, theoretically resulting in a greater modulus change. However, practical applications show that this limited dynamic range is often insufficient to induce significant cellular phenotypic changes in many biological processes sensitive to mechanical signal thresholds (such as the transdifferentiation of fibroblasts to myofibroblasts and the induction of specific stem cell lineages), thus limiting its application in precision medicine and disease models.

[0005] This raises a critical technical challenge in this field: how to break through the intuition of traditional theories and construct photoresponsive hydrogels with a wider range of stiffness regulation through molecular design? This cannot be achieved through simple random mutation attempts, as most mutants will disrupt protein folding stability and prevent gelation. More importantly, a new molecular mechanism is needed to overcome the limitations of relying solely on "chain length release." Through extensive molecular biology screening and single-molecule force spectroscopy studies, the inventors discovered that changes in macroscopic modulus depend not only on changes in chain length but also on the protein's unfolding probability under network tension. Based on this, this invention proposes a PYP 48 / 85 hydrogel system based on mechanical sensitivity and rational design, achieving wide-range mechanical regulation properties through specific topological design. Summary of the Invention

[0006] The main objective of this invention is to solve the technical problem of limited stiffness adjustment range of existing photoresponsive hydrogels and to provide a photoresponsive protein hydrogel with wide-range mechanical adjustment characteristics.

[0007] To solve the above problems, the present invention adopts the following solution: a photoresponsive protein hydrogel with wide-range mechanical regulation properties, characterized in that the photoresponsive protein hydrogel with wide-range mechanical regulation properties is polymerized from a photoactivated flavin PYP (48 / 85) double mutant and a multi-arm polyethylene glycol derivative with maleimide groups at the ends, wherein the multi-arm polyethylene glycol derivative with maleimide groups at the ends is preferably 8-arm-maleimide-polyethylene glycol (8-armedPEG-Mal, 20kDa).

[0008] The PYP 48 / 85 double mutant was obtained by genetic engineering, which involved mutating amino acid residues at positions 48 and 85 of the wild-type photoactivated flavin (PYP) amino acid sequence to cysteine. These two sites are located at specific positions on the protein surface, forming a unique mechanical connection geometry.

[0009] The scientific principle of this invention lies in: This invention overcomes the limitations of traditional rubber elasticity theory in the design of photoresponsive hydrogels. Existing technologies and traditional theories generally hold that the magnitude of modulus change in polymer networks primarily depends on the change in polymer chain length (ΔL) between crosslinking points. Therefore, previous design approaches tended to select mutation sites that release longer chains after unfolding (such as PYP 36 / 128). However, the applicant, through single-molecule force spectroscopy combined with polymer physics models, discovered that in hydrogel networks at swelling equilibrium, the dominant factor determining the magnitude of macroscopic modulus change is not simply the change in chain length, but rather the change in the "unfolding probability" of protein crosslinking points under network background tension.

[0010] Based on this novel mechanical mechanism, the applicant rationally designed the PYP 48 / 85 double mutant. Compared with existing technologies, although the profile length released during unfolding of PYP 48 / 85 is shorter (approximately 10-14 nm, less than the approximately 28-33 nm of existing technologies), it possesses a unique anisotropic mechanical energy profile: lower mechanical stability and a larger transition distance parameter. This special molecular mechanical property makes the folding / unfolding equilibrium of PYP 48 / 85 extremely sensitive to conformational changes induced by light. Under light, the unfolding probability of PYP 48 / 85 undergoes a dramatic transition. This high-probability conformational switching significantly amplifies the change in the effective chain length of the network, thus manifesting as a significant softening on a macroscopic scale; while in the dark, it can efficiently fold back to a rigid state. This molecular lever mechanism of "compensating for short chain length with high sensitivity" enables the material of this invention to achieve a stiffness adjustment range far exceeding that of existing technologies (such as PYP 36 / 128) on a macroscopic scale, thus solving the technical bottleneck of insufficient dynamic range of existing photoresponsive hydrogels.

[0011] A method for preparing a photoresponsive protein hydrogel with wide-range mechanical modulatory properties, characterized by comprising the following steps: Step 1: Obtaining the recombinant protein variant: The original wild-type photoactivated flavin sequence was subjected to site mutation and sequence expansion to construct an expression vector containing the PYP(48 / 85) coding sequence. The PYP 48 / 85 gene fragment was cloned into the pQE80L vector after restriction endonuclease treatment and induced expression in an E. coli system. The PYP(48 / 85) protein solution with photoresponsive conformational change ability was obtained by affinity chromatography separation and purification. The PYP(48 / 85) coding sequence was obtained by site-directed mutation modification of the coding regions at positions 48 and 85 of the wild-type PYP gene. Step 2: In-situ crosslinking to form a gel: The PYP (48 / 85) protein solution and the multi-arm polyethylene glycol derivative solution with maleimide terminal modification are mixed in a preset ratio, and a crosslinking reaction is initiated in a physiological buffer system to obtain the hydrogel.

[0012] Furthermore, the method for preparing the photoresponsive protein hydrogel with wide-range mechanical regulation properties is characterized in that, in step one, a commercial site-directed mutagenesis kit is used to perform site-directed mutagenesis and sequence expansion on the wild-type photoactivated flavonoid protein PYP gene fragment, mutating the aspartic acid at positions 48 and 85 of the wild-type photoactivated flavonoid protein PYP to cysteine. The resulting mutant protein PYP 48 / 85 gene fragment is treated with restriction endonucleases BamHI and KpnI, and the pQE80L vector is treated with restriction endonucleases BglII and KpnI. The protein gene sequence carries an N-terminal 6×His tag from the pQE80L vector for subsequent purification of the protein using metal affinity chromatography.

[0013] Furthermore, the method for preparing the photoresponsive protein hydrogel with wide-range mechanical modulation properties is characterized in that, in step two, the protein expression temperature is 20-37℃, the expression is induced for 4-8 hours, and Co is used... 2+ -NTA protein resin purification, dialyzed to 1× phosphate buffer, and stored at 4°C or below before use.

[0014] Furthermore, the method for preparing the photoresponsive protein hydrogel with wide-range mechanical modulation properties is characterized in that, in step three, the photoresponse amplitude of the hydrogel can be controlled by adjusting the molar ratio of the PYP(48 / 85) protein to the multi-arm polyethylene glycol derivative with maleimide-terminal modification; when the molar ratio of the PYP(48 / 85) protein to the multi-arm polyethylene glycol derivative with maleimide-terminal modification is 1:1, the photoresponse amplitude of the hydrogel is the largest.

[0015] Furthermore, the method for preparing the photoresponsive protein hydrogel with wide-range mechanical modulation properties is characterized in that the density of crosslinking points in the hydrogel can be controlled by adjusting the reaction concentration of the PYP(48 / 85) protein with a multi-arm polyethylene glycol derivative modified with maleimide groups at the ends, thereby controlling the initial stiffness of the hydrogel. The initial stiffness of the hydrogel is positively correlated with the reaction concentration; that is, as the concentration of protein PYP 48 / 85 and the multi-arm polyethylene glycol derivative modified with maleimide groups at the ends increases, the initial stiffness of the resulting hydrogel also increases.

[0016] Furthermore, the method for preparing the photoresponsive protein hydrogel with wide-range mechanical regulation properties is characterized in that the 1× phosphate buffer has a molar concentration of 10 mM and a pH of 6.2-8.2.

[0017] An application of a photoresponsive protein hydrogel with wide-range mechanical modulatory properties, characterized in that the photoresponsive protein hydrogel with wide-range mechanical modulatory properties is applied to cell culture, drug release, and tissue engineering.

[0018] Inspired by the rapid and reversible conformational and mechanical changes of photoactivated flavonoid protein (PYP), this invention proposes a photoresponsive protein hydrogel with wide-range mechanical modulatory properties. The main idea is to design a hydrogel material whose mechanical properties undergo rapid and reversible changes in response to blue light irradiation. First, the original wild-type PYP sequence is subjected to site mutations and sequence expansion. The thiol groups in the cysteine ​​residues exposed at the mutation sites and in the expanded sequences can react with the maleimide groups at the ends of 8-armed PEG-Mal (20kDa). In terms of macroscopic regulation, controlling the concentration of 8-armed PEG-Mal and the mutant protein can affect the crosslinking rate and initial mechanical properties of the protein hydrogel. When the protein hydrogel is exposed to blue light, the conformation of the mutant protein changes rapidly, resulting in a rapid and reversible change in the mechanical properties of the gelled hydrogel.

[0019] When blue light irradiation is present, the stiffness of the protein hydrogel decreases; when the blue light irradiation disappears, the stiffness of the protein hydrogel recovers. Furthermore, increasing the concentration of 8-arm-maleimide-polyethylene glycol and protein PYP 48 / 85 increases the stiffness of the protein hydrogel and accelerates the gelation rate; conversely, decreasing the concentration of 8-arm-maleimide-polyethylene glycol and protein PYP 48 / 85 decreases the stiffness of the protein hydrogel and slows down the gelation rate.

[0020] The technical effects of this invention are as follows: The photoresponsive protein hydrogel with wide-range mechanical regulation characteristics demonstrated by this invention can regulate the mechanical properties of the hydrogel in time and space as needed; it is non-cytotoxic; and it can rapidly and reversibly regulate the mechanical properties to simulate the dynamic changes of the extracellular matrix in the absence or presence of blue light irradiation.

[0021] (1) In terms of regulation speed, since the conformational change of protein PYP 48 / 85 is rapid when exposed to blue light and disappears, the present invention can achieve rapid and reversible regulation of mechanical properties through the presence and disappearance of blue light.

[0022] (2) Regarding the speed of regulation, since the conformational changes of protein PYP 48 / 85 caused by blue light irradiation and disappearance occur throughout the entire protein domain, the present invention can achieve a significant regulation of mechanical properties by the presence and disappearance of blue light irradiation.

[0023] (3) In terms of stability, the mechanical properties of the present invention can be quickly adjusted by blue light irradiation and almost will not lose reversibility. Attached Figure Description

[0024] Figure 1 This is a schematic diagram illustrating the principle of how the stiffness of the photoresponsive protein hydrogel with wide-range mechanical regulation characteristics changes when exposed to blue light, according to the present invention.

[0025] Figure 2 This is a single-molecule force spectrum of the PYP 48 / 85 mutant protein and the control group PYP36 / 128 mutant protein of the present invention.

[0026] Figure 3 The graph shows the Young's modulus statistics of the photoresponsive protein (PYP 48 / 85) hydrogel with wide-range mechanical regulation properties and the control group (PYP36 / 128) hydrogel under cyclic blue light irradiation.

[0027] Figure 4 This is a statistical chart showing the detection of luciferase activity inducing cell transdifferentiation when the hydrogel of this invention is applied to cell culture. Detailed Implementation

[0028] The present invention will now be described in further detail with reference to the accompanying drawings. Example

[0029] like Figure 1 As shown, photoactivated flavin (PYP) in a photoresponsive protein hydrogel with wide-range mechanical regulation properties can undergo conformational changes when blue light irradiation is present and absent, causing changes in the density of cross-linking points in the hydrogel network, thereby altering the mechanical properties of the hydrogel.

[0030] The present invention provides a photoresponsive protein hydrogel with wide-range mechanical regulation properties. The preparation method includes the following steps: (1) Plasmid construction: The protein PYP 48 / 85 is based on the amino acid sequence of wild-type photoactivated flavonoid PYP, with the aspartic acid at positions 48 and 85 mutated to cysteine. The gene fragment of PYP 48 / 85 is treated with restriction endonucleases BamHI and KpnI, and the pQE80L vector is treated with restriction endonucleases BglII and KpnI. The protein gene sequence carries the N-terminal 6×His label of the pQE80L vector for subsequent protein purification.

[0031] (2) Protein expression and purification: The plasmid obtained in step (1) was transformed into E. coli BL21(DE3) competent cells to express the protein and the resulting protein was purified; the protein expression temperature was 37℃ for 4 hours, and Co was used to express the protein. 2+ -NTA protein was purified with resin, dialyzed to 1× phosphate buffer, and stored at 4°C before use. The 1× phosphate buffer had a molar concentration of 10 mM and a pH of 8.0.

[0032] (3) Preparation of photoresponsive protein hydrogel with wide-range mechanical regulation properties: The protein PYP48 / 85 obtained in step (2) was mixed with 8-arm-maleimide-polyethylene glycol at a molar ratio of 1:1 to prepare a photoresponsive protein hydrogel with wide-range mechanical regulation properties.

[0033] The photoresponsive protein hydrogel prepared by the method described in this invention has a wide range of mechanical modulating properties. When blue light irradiation is present, the stiffness of the protein hydrogel decreases; when the blue light irradiation disappears, the stiffness of the protein hydrogel recovers.

[0034] Using this protein hydrogel design, the materials employed are non-cytotoxic, and the mechanical properties of the hydrogel can be controlled as needed in time and space. Under the absence or presence of blue light irradiation, the mechanical properties can be rapidly and reversibly adjusted to simulate the dynamic changes of the extracellular matrix, thus enabling applications in multiple fields such as cell culture, drug delivery, and tissue engineering.

[0035] Materials preparation: The experimental group was prepared according to Example 1: PYP48 / 85 mutant protein and PYP 48 / 85 hydrogel. The control group was prepared according to existing technology: PYP 36 / 128 mutant protein and PYP 36 / 128 hydrogel.

[0036] Experimental Example 1 The following are examples of performance tests for various aspects of the present invention: This experimental example characterizes the mechanical properties of the photoresponsive protein hydrogel described in this invention at both the molecular and macroscopic levels.

[0037] The specific experimental methods are as follows: First, single-molecule force spectroscopy was performed: purified PYP 48 / 85 mutant protein and PYP 36 / 128 mutant protein were converted into multi-polymer chains (Poly-PYP) through cysteine-mediated oxidation to simulate the cross-linking state in the hydrogel network; the multi-polymer protein solution was deposited on a cleaned gold-coated substrate, and single-molecule stretching experiments were performed using atomic force microscopy (JPK Nanowizard II) under dark conditions and blue light irradiation (405 nm laser, intensity of about 15.6 mW / cm²); force-stretch curves showing characteristic serrated patterns and at least three consecutive unfolding events were recorded, and the unfolding force and unfolding length of the protein domains were analyzed. Next, macroscopic mechanical and cyclic photoresponse tests of the hydrogels were conducted: The prepared PYP 48 / 85 hydrogels and PYP 36 / 128 hydrogels were placed in a PBS buffer environment at 37°C. The Young's modulus of the hydrogels was measured using the nanoindentation mode of an atomic force microscope. For each condition, force-deformation curves were collected at different locations on the hydrogel surface, and the Young's modulus value was calculated by fitting the close part of the curves using the Hertz model. At the same time, a light cycle program was set to perform multiple rounds of alternating "dark-blue light irradiation" treatment on the hydrogels (with an interval of 10 minutes), and the modulus changes were monitored and recorded in real time.

[0038] The test results are analyzed as follows: Figure 2The single-molecule force spectroscopy results show that, under dark conditions, the characteristic unfolding force of the PYP48 / 85 mutant protein is significantly lower than that of the PYP36 / 128 mutant protein, indicating its lower overall mechanical stability. Furthermore, the experiment observed that the unfolded profile length of the PYP48 / 85 mutant protein is shorter (approximately 10-14 nm, less than the approximately 28-33 nm of the PYP36 / 128 mutant protein). Under blue light irradiation, the unfolding forces of both mutants further decreased, and this change was reversible. Further comparison of the changes before and after light irradiation revealed that the change in unfolding force of the PYP48 / 85 mutant protein was significantly greater than that of the PYP36 / 128 mutant protein, indicating a higher mechanical sensitivity to conformational changes induced by light stimulation. This high sensitivity at the single-molecule level is the molecular basis for the large-scale stiffness adjustment of macroscopic hydrogels.

[0039] like Figure 3 The macroscopic mechanical test results of the hydrogels shown indicate that the Young's modulus of the two hydrogels was quantified using the AFM nanoindentation method. Statistical analysis confirmed that both PYP 48 / 85 and PYP 36 / 128 hydrogels exhibited significant changes in photoinduced modulus. Specifically, the Young's modulus of the experimental group, PYP 48 / 85 hydrogel, softened from approximately 2.15 kPa in the dark to approximately 1.26 kPa under blue light irradiation, demonstrating a large range of stiffness adjustment. In contrast, the Young's modulus of the control group, PYP 36 / 128 hydrogel, decreased from approximately 2.19 kPa in the dark to approximately 1.62 kPa, with a relatively smaller change. In the cyclic light irradiation test (10-minute intervals), both hydrogels exhibited completely reversible changes in photoinduced modulus across multiple cycles, consistently hardening in the dark and softening under blue light, with no obvious fatigue decay observed. Of particular note is the significant technological breakthrough achieved by comparing the relative mechanical response magnitudes of the two: the existing PYP 36 / 128 hydrogel hardens by only about 35% from its photosoftened state, while the PYP 48 / 85 hydrogel of this invention exhibits a significantly larger change, with a hardening magnitude of approximately 71%, roughly twice that observed in the PYP 36 / 128 variant. This performance multiplication effect strongly demonstrates the unique and unexpected technological advantages of the specific connectivity geometry of the PYP 48 / 85 double mutant in constructing wide-range mechanically modulated materials.

[0040] Experimental Example 2 This experiment investigates the effects of dynamic mechanical stimulation of different frequencies and amplitudes on cell transdifferentiation under cyclic light irradiation.

[0041] The specific experimental methods are as follows: First, cell transfection and seeding were performed. Using Lipofectamine 3000, luciferase reporter plasmids containing myofibroblast marker promoters (α-SMA-luciferase and Postn-luciferase, respectively) were transfected into NIH / 3T3 fibroblasts. The transfected cells were then seeded at a rate of 1×10⁻⁶ cells / cells. 6 Cells were seeded at a density of [number] cells / mL on PYP 48 / 85 and PYP 36 / 128 hydrogels and cultured in the dark for 24 hours to allow them to adhere. Subsequently, grouped light stimulation was performed using a custom-designed 405 nm LED array for differential light stimulation. The experiment consisted of five groups: a static rigid group (constant darkness), a static soft group (constant blue light), and three dynamic cycling groups. The duty cycle of the dynamic cycling groups was 50% (i.e., light time equals dark time), with cycle periods of 30 min (30 min on / 30 min off), 5 min (5 min on / 5 min off), and 1 min (1 min on / 1 min off). Finally, luciferase activity was measured. After light stimulation, cells were lysed, and luciferase activity in each group was measured using a Firefly Luciferase Glow Assay Kit and a microplate reader to quantitatively reflect the expression levels of α-SMA and Postn, and to assess the degree of cell transdifferentiation.

[0042] The test results are analyzed as follows: Figure 4 The luciferase reporter gene assay results revealed the cellular response to dynamic mechanical signals. First, a significant frequency-dependent response was observed: under constant conditions (static rigidity or static softness), phenotypic changes were limited; even the statically rigid group with the highest cumulative stiffness did not induce significant transdifferentiation. However, under dynamic light stimulation, cells exhibited a clear frequency-dependent response, particularly under high-frequency light switching conditions (1-minute cycles), where the expression levels of intracellular myofibroblast-related markers (α-SMA and Postn) were significantly upregulated, indicating strong transdifferentiation towards myofibroblasts. This transdifferentiation effect gradually weakened with increasing cycle length. Second, a significant amplitude-dependent response was observed: further comparison of different hydrogel systems revealed that, at the same stimulation frequency (e.g., 1-minute cycles), cells cultured on PYP 48 / 85 hydrogels with a larger stiffness variation exhibited significantly higher transdifferentiation levels (luciferase activity) than cells cultured on the control group (PYP 36 / 128 hydrogels) with a smaller stiffness variation. In summary, the PYP 48 / 85 hydrogel of the present invention not only provides a large degree of mechanical regulation, but also effectively induces cell transdifferentiation through high-frequency dynamic stimulation.

[0043] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above-described experimental examples. The experimental examples and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A photoresponsive protein hydrogel with wide-range mechanical modulation properties, characterized in that, The hydrogel comprises a three-dimensional cross-linked network constructed from a multi-arm polyethylene glycol derivative and a recombinant photoactivated flavin (PYP variant); wherein the PYP variant is a PYP (48 / 85) double mutant formed by genetically engineering the PYP sequence by mutating amino acids at positions 48 and 85 to cysteine; the multi-arm polyethylene glycol derivative is terminally modified with maleimide groups, and the two are covalently cross-linked through a thiol-maleimide Michael addition reaction.

2. The method for preparing a photoresponsive protein hydrogel with wide-range mechanical modulation properties according to claim 1, characterized in that, Includes the following steps: (1) Obtaining recombinant protein variants: An expression vector containing the PYP(48 / 85) coding sequence was constructed and induced to express in an E. coli system. The PYP(48 / 85) protein solution with photoresponsive conformational change capability was obtained by affinity chromatography separation and purification. The PYP(48 / 85) coding sequence was obtained by site-directed mutation modification of the coding regions at positions 48 and 85 of the wild-type PYP gene. (2) In situ crosslinking to form a gel: The PYP(48 / 85) protein solution was mixed with a multi-arm polyethylene glycol derivative solution with maleimide terminal modification in a preset ratio, and a crosslinking reaction was initiated in a physiological buffer system to obtain the hydrogel.

3. The method for preparing a photoresponsive protein hydrogel with wide-range mechanical modulation properties according to claim 2, characterized in that, The PYP(48 / 85) variant was obtained by replacing the 48th aspartic acid and the 85th serine in the wild-type PYP sequence (UniProt ID: P16113) with cysteine, respectively. This two-site mutation endowed the protein with the ability to change the terminal distance significantly under light. The gene fragment of the resulting mutant protein PYP 48 / 85 was spliced ​​by treating with restriction endonucleases BamHI and KpnI. The pQE80L vector was treated with restriction endonucleases BglII and KpnI. The protein gene sequence was marked with the N-terminus 6×His tag of the pQE80L vector for subsequent protein purification.

4. The method for preparing a photoresponsive protein hydrogel with wide-range mechanical modulation properties according to claim 2, characterized in that, In step (1), the protein expression temperature is 20-37℃, the expression is induced for 4-8 hours, the protein is purified by metal affinity chromatography with Co2+-NTA or Ni2+-NTA resin, dialyzed into phosphate buffer, and stored at 4℃ or below before use.

5. The method for preparing a photoresponsive protein hydrogel with wide-range mechanical modulation properties according to claim 2, characterized in that, The light response amplitude of the hydrogel can be controlled by adjusting the molar ratio of the PYP(48 / 85) protein to the multi-arm polyethylene glycol derivative with maleimide-terminal modification. The light response amplitude of the hydrogel is maximized when the molar ratio of the PYP(48 / 85) protein to the multi-arm polyethylene glycol derivative with maleimide-terminal modification is 1:

1.

6. The method for preparing a photoresponsive protein hydrogel with wide-range mechanical modulation properties according to claim 2, characterized in that, The initial stiffness of the hydrogel can be controlled by adjusting the reaction concentration of the PYP(48 / 85) protein with a multi-arm polyethylene glycol derivative modified with maleimide groups at the ends, thereby controlling the density of crosslinking points.

7. The method for preparing a photoresponsive protein hydrogel with wide-range mechanical modulation properties according to claim 2, characterized in that, The physiological buffer system is phosphate-buffered saline (PBS) with a pH between 6.2 and 8.2, and the reaction process is carried out under light-protected or low-light conditions to keep the PYP protein in the ground state.

8. An application of a photoresponsive protein hydrogel with wide-range mechanical modulation properties as described in claim 1, characterized in that, Photoresponsive protein hydrogels with wide-range mechanical modulation properties can be applied to cell culture, drug release, and tissue engineering.

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