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CN-122011723-A - Photoresponse protein hydrogel with wide-range mechanical regulation characteristic and preparation method thereof

CN122011723ACN 122011723 ACN122011723 ACN 122011723ACN-122011723-A

Abstract

The invention discloses a photoresponsive protein hydrogel with wide-domain mechanical regulation property, which is formed by polymerizing a specific mutant of photo-activated yellow protein (PYP) and a multi-arm polyethylene glycol derivative with a terminal modified with a maleimide group. According to the invention, through molecular dynamics and rational design of material mechanics, cysteine mutation is carried out on the 48 th and 85 th positions of a wild PYP sequence, and a PYP (48/85) double mutant with anisotropic unfolding prospect is constructed. The mutant utilizes the Michael addition reaction of the exposed sulfhydryl group and the multi-arm polyethylene glycol derivative to form a crosslinked network. Compared with the prior art (such as PYP 36/128 hydrogel), the invention overcomes the limitation that the traditional design only depends on chain length change by utilizing the higher mechanical sensitivity and unfolding probability of PYP 48/85 under the network tension, and realizes the remarkably larger macroscopic rigidity adjustment range (the mechanical switch ratio can reach more than 70%). 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 on high requirements on mechanical signal thresholds.

Inventors

  • YANG JIAPENG
  • GUO PENG
  • ZHANG YU
  • JIANG CHUNPING
  • CAO YI
  • XUE BIN

Assignees

  • 济南微生态生物医学省实验室

Dates

Publication Date
20260512
Application Date
20260126

Claims (8)

  1. 1. A photoresponsive protein hydrogel with wide-range mechanical regulation property is characterized by comprising a three-dimensional crosslinked network constructed by a multi-arm polyethylene glycol derivative and recombinant photo-activated yellow protein (PYP variant), wherein the PYP variant is a PYP (48/85) double mutant which is formed by mutating 48 th and 85 th amino acids into cysteine on the basis of the sequence of the photo-activated yellow protein (PYP) through a genetic engineering means, and the tail end of the multi-arm polyethylene glycol derivative is modified with a maleimide group, and covalent crosslinking is formed by thiol-maleimide Michael addition reaction.
  2. 2. The preparation method of the photoresponse protein hydrogel with the wide-range mechanical regulation characteristic is characterized by comprising the following steps of (1) obtaining a recombinant protein variant, constructing an expression vector containing a PYP (48/85) coding sequence, performing induced expression in an escherichia coli system, separating and purifying by affinity chromatography to obtain a PYP (48/85) protein solution with photoresponse conformational change capability, wherein the PYP (48/85) coding sequence is obtained by carrying out site-directed mutation modification on 48 th and 85 th coding regions of a wild PYP gene, and (2) carrying out in-situ crosslinking and gel formation, namely mixing the PYP (48/85) protein solution with a multi-arm polyethylene glycol derivative solution with a maleimide group modified at the tail end according to a preset proportion, and initiating a crosslinking reaction in a physiological buffer system to obtain the hydrogel.
  3. 3. The method for preparing a photoresponsive protein hydrogel with wide-range mechanical regulatory properties according to claim 2, wherein the PYP (48/85) variant is obtained by replacing the 48 th aspartic acid and the 85 th serine in the wild-type PYP sequence (UniProt ID: P16113) with cysteines respectively, the double-site mutation endows the protein with remarkable end-to-end distance variation capability under illumination, the gene fragment of the obtained mutant protein PYP 48/85 is subjected to treatment by restriction enzymes BamHI and KpnI to complete splicing, the pQE80L vector is subjected to treatment by restriction enzymes BglII and KpnI, and the protein gene sequence is provided with the N-terminal 6 XHis tag of the pQE80L vector for subsequent purification of the protein.
  4. 4. The method for preparing a photoresponsive protein hydrogel having broad-range mechanical properties according to claim 2, wherein in the step (1), the protein expression temperature is 20 to 37 ℃, the expression is induced for 4 to 8 hours, and the protein is purified by using a metal affinity chromatography co2+ -NTA or ni2+ -NTA resin, dialyzed into a phosphate buffer, and stored at 4 ℃ or below before use.
  5. 5. The method for preparing a photo-responsive protein hydrogel with wide-range mechanical adjustment properties according to claim 2, wherein the photo-responsive 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 the maleimide group at the end, and the photo-responsive amplitude of the hydrogel is maximum when the mixed molar ratio of the PYP (48/85) protein to the multi-arm polyethylene glycol derivative with the maleimide group at the end is 1:1.
  6. 6. The method for preparing a photoresponsive protein hydrogel with wide-range mechanical adjusting property according to claim 2, wherein the initial rigidity of the hydrogel can be adjusted by adjusting the reaction concentration of the PYP (48/85) protein and the multi-arm polyethylene glycol derivative with the terminal modified with the maleimide group.
  7. 7. The method for preparing the photoresponsive protein hydrogel with wide-range mechanical adjusting property according to claim 2, wherein the physiological buffer system is Phosphate Buffer Solution (PBS) with pH value of 6.2-8.2, and the reaction process is carried out under the condition of light shielding or weak light so as to keep the PYP protein in a ground state.
  8. 8. Use of the light response protein hydrogel with wide-range mechanical tuning properties according to claim 1, wherein the light response protein hydrogel with wide-range mechanical tuning properties is applied to cell culture, drug release, tissue engineering.

Description

Photoresponse protein hydrogel with wide-range mechanical regulation characteristic and preparation method thereof Technical Field The invention relates to the technical field of hydrogel materials, in particular to a photoresponsive protein hydrogel with wide-range mechanical regulation characteristics, and a preparation method and application thereof. Background 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, fibrosis and the like, significant temporal and spatial changes in the mechanical properties of ECM can occur. These dynamic mechanical signals are critical to regulate the diffusion, migration, proliferation and differentiation behavior of cells. To simulate this process in vitro, researchers have developed a variety of dynamic hydrogel systems. Among them, photo-responsive hydrogels are attracting attention because of their advantages of non-contact control, high space-time resolution, and the like. Early designs relied primarily on photolytic crosslinkers, which achieved softening but were irreversible in process. Subsequently, a reversibly crosslinked hydrogel based on a photosensitive protein such as LOV2 or Dronpa has been proposed, but there are generally problems of slow response speed (in minutes) or small variation of modulus (< 20%). In recent years, hydrogels based on photo-activated flavin (PYP) have shown great potential due to their rapid light cycling characteristics on the order of milliseconds. In the prior art, studies (including applicant's previous work) have been performed to construct light responsive hydrogels using mutants at position 36 and 128 of PYP (PYP 36/128). However, in intensive mechanical biological studies, it was found that, although the PYP 36/128 hydrogel achieved reversible regulation, the magnitude of the stiffness change (i.e., the mechanical on-off ratio) before and after illumination was relatively limited (typically less than 50%). Conventional rubber elasticity theory tends to suggest that the longer the amino acid sequence spanning between mutation sites (e.g., 36/128 spanning 92 residues), the longer the chain length released by unfolding should theoretically produce a greater change in modulus. 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 (e.g., transdifferentiation of fibroblasts into myofibroblasts, induction of specific stem cell lineages), limiting its use in precision medical and disease models. This has led to a technical difficulty in the art that, how to break through the intuition of the traditional theory, constructing a photoresponsive hydrogel with a larger stiffness modulation amplitude by molecular design is not achieved by simple random mutation attempts, as most mutations can destroy protein folding stability and thus cannot gel. More importantly, a new molecular mechanism is needed to compensate for the limitation imposed by the mere reliance on "release chain length". The inventor finds that the change of macroscopic modulus not only depends on chain length change, but also depends on unfolding probability of protein under network tension through a large number of molecular biological screening and single molecular force spectrum researches. Based on the design, the invention provides a PYP 48/85 hydrogel system based on mechanical sensitivity rational design, and the wide-domain mechanical regulation characteristic is realized through specific topological structure design. Disclosure of Invention The invention mainly aims to solve the technical problem that the rigidity adjustment range of the existing photoresponsive hydrogel is limited, and provides the photoresponsive protein hydrogel with wide-range mechanical adjustment characteristics. In order to solve the problems, the scheme adopted by the invention is that the photoresponsive protein hydrogel with the wide-range mechanical regulation property is characterized in that the photoresponsive protein hydrogel with the wide-range mechanical regulation property is formed by polymerizing a photoactivated yellow protein PYP (48/85) double mutant and a multi-arm polyethylene glycol derivative with a maleimide group modified at the tail end, wherein the multi-arm polyethylene glycol derivative with the maleimide group modified at the tail end is preferably 8-arm-maleimide-polyethylene glycol (8-armed PEG-Mal,20 kDa). The PYP 48/85 double mutant is obtained by mutating the 48 th and 85 th amino acid residues in a wild type Photoactivated Yellow Protein (PYP) amino acid sequence into cysteine by a genetic engineering means. These two sites are located at specific locations on the protein surface, creating a unique mechanical linkage geometry. The scientific principle of the invention