Synthesis of Antioxidant Protein Material by Escherichia coli and Its Applications in Promoting Wound Healing

doi:10.13560/j.cnki.biotech.bull.1985.2026-0087

Abstract

Abstract:

Objective This study aimed to construct a self-assembling antioxidant protein material and to evaluate its effects on repairing oxidative cellular damage and promoting wound healing. Method We constructed recombinant plasmids by fusing a previously reported self-assembling elastin-like polypeptide scaffold with antioxidant amino acid sequences. We then transformed the constructs into Escherichia coli for fermentation and purification. We selected the protein with the optimal performance through free radical scavenging assays and further evaluated its application potential using cellular oxidative damage repair assays and rat wound healing experiments. Result The recombinant protein expressed and purified from E. coli self-assembled into a hydrogel at a concentration of 1%. After fusion with the antioxidant amino acid sequence, the free radical scavenging capacity of the protein was significantly enhanced. The scavenging rates of 80-2-MPH for 2,2-diphenyl-1-picrylhydrazyl (DPPH·), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS⁺·), and hydroxyl radical (·OH) reached 41.1%, 66.8%, and 57.2%, respectively, which were significantly higher than those of the original protein 80-2-RGD (20.4%, 50.0%, and 21.8%, respectively; P<0.000 1). The 80-2-MPH protein exhibited good biocompatibility, with a relative cell activity of 117.6% at a concentration of 1 g/L. In the cellular oxidative damage repair experiment, the cell activity in the 80-2-MPH repair group was 49.6%, significantly higher than that of the control group (28.0%), as well as the glutathione (36.7%) and vitamin C (40.9%) treatment groups. The intracellular reactive oxygen species (ROS) content in the 80-2-MPH group showed no significant difference compared with the glutathione group. In the rat full-thickness wound healing experiment, the 80-2-MPH hydrogel group showed a wound closure rate of 93.6% on day 10, which was significantly higher than that of the control group (74.8%) and the commercial hydrogel dressing group (84.5%). Histological staining results showed that the 80-2-MPH hydrogel effectively promoted collagen deposition and dermal repair. Conclusion An antioxidant protein material, 80-2-MPH, capable of self-assembling into a hydrogel, was successfully expressed in E. coli. This protein reduced intracellular ROS levels in oxidatively stressed cells and promoted the repair of oxidative damage. The hydrogel dressing formed by the self-assembly of this protein effectively accelerates wound healing in rats.

Key words: recombinant protein, functional protein materials, protein engineering, protein purification, antioxidant, hydrogel, oxidative damage repair

ZHAO Meng-die, XUE Wei-shi, WANG Jia-bang, LI Ling, YANG Ru-meng, WU Jun-jun. Synthesis of Antioxidant Protein Material by Escherichia coli and Its Applications in Promoting Wound Healing[J]. Biotechnology Bulletin, 2026, 42(9): 342-354.

Figures/Tables 9

Table 1 Amino acid and corresponding nucleotide sequences of the exposed active site of the protein, and primer sequences used in this study

氨基酸序列 AA sequences	名称 Name	碱基序列 Base sequences	引物序列 Primer sequences （5′-3′）	参考文献 References
AMRLTYNKPCLYGT	RP1	GCTATGAGGCTAACATATAATAAACCCTGTCTGTACGGTACC	F1： GCTATGAGGCTAACATATAATTGACTCGAGCACCACCACCA R1： ATTATATGTTAGCCTCATAGCACCGCTACCACCGCTACCCGGGGCGC F2： AAACCCTGTCTGTACGGTACCTGACTCGAGCACCACCACC R2：GGTACCGTACAGACAGGGTTTATTATATGTTAGCCTCATAGCACCGCTAC	［21］
LMGQW	MPH	CTGATGGGCCAGTGGTAA	F： CTGATGGGCCAGTGGTAATGACTCGAGCACCACCACC R： TTACCACTGGCCCATCAGACCGCTACCACCGCTACCCGGC	［22］
SDITRPGGNM	PP	AGCGATATTACCCGCCCGGGCGGCAACATGTAA	F：AGCGATATTACCCGCCCGGGCGGCAACATGTAATGACTCGAGCACCACCACC R：TTACATGTTGCCGCCCGGGCGGGTAATATCGCTACCGCTACCACCGCTACCCGGC	［23］
YYIVS	Ⅳ5C	TACTACATCGTGAGCTAA	F： TACTACATCGTGAGCTAATGACTCGAGCACCACCACC R：TTAGCTCACGATGTAGTAACCGCTACCACCGCTACCCGGC	［24］
SETYELRK	ABA	AGCGAAACCTATGAACTGCGCAAATAA	F： AGCGAAACCTATGAACTGCGCAAATAATGACTCGAGCACCACCACC R： TTATTTGCGCAGTTCATAGGTTTCGCTACCGCTACCACCGCTACCCGGC	［25］
GRGDSPG	RGD	GGCCGCGGCGATAGCCCGGGC	/	［19］

Fig. 1 Flowchart of the rat full-thickness wound healing experiment

Fig. 2 Construction of antioxidant proteins based on self-assembling elastin-like sequencesA: Schematic diagram of recombinant protein expression plasmid construction via inverse PCR. B: Schematic diagram of the primary structure of a single protein molecule and the self-assembled hydrogel structure. C: Structure diagram of the self-assembled protein predicted by AlphaFold 3. The yellow region represents the self-assembling sequence of one protein molecule, the orange region represents the elastin-like sequence, and the red region represents the exposed active site

Fig. 3 Protein expression and purification gel imagesA: Gel images of protein expression in E. coli before and after induction during protein fermentation. M: Marker; 1: before induction; 2: after induction. B-E: Gel images showing the purification of 80-2-MPH, 80-2-PP, 80-2-RP1, 80-2-Ⅳ5C, and 80-2-ABA, respectively. The molecular weight of each protein was approximately 27 kD. M: Marker; 1: cell lysate; 2: flow-through; 3: Ni-column eluted protein after washing; 4: desalted protein

Fig. 4 Chemical experiment characterizes the antioxidant properties of different proteinsA: DPPH· scavenging rate of different proteins. B: ABTS+· free radical scavenging rate. C: ·OH scavenging rate of different proteins. D: Free radical scavenging ability of different concentrations of 80-2-MPH. The photos show the effect of free radical scavenging. ****P<0.000 1, data are reported as mean ± SD (n=3), the same below

Fig. 5 Characterization of hydrogel gelation propertiesA: Different proteins were dissolved in physiological saline at a concentration of 1% (w/v), and no gelation occurred. From left to right: 80-2-Ⅳ5C, 80-2-PP, 80-2-RP1, 80-2-ABA, and 80-2-MPH. B: The protein self-assembled into a hydrogel after standing. C: After gelation at 4% (w/v), the protein hydrogel was applied to a porcine skin defect using a syringe to mimic its use as a wound dressing. D: Scanning electron microscopy images showing the network structure of the hydrogel at 5% (w/v). E: Scanning electron microscopy images showing the network structure of the hydrogel at 10% (w/v)

Fig. 6 Biological activity of 80-2-MPH proteinA: Verification of the biocompatibility of 80-2-MPH. Cells were cultured in complete medium containing different concentrations of 80-2-MPH, and cell viability was detected using the CCK-8 kit. B: Scanning electron microscopy images showing cell morphology in each group. ***P<0.001

Fig. 7 Effect of 80-2-MPH on the repair of cellular oxidative damage and the scavenging of intracellular reactive oxygen speciesA: Cell morphology at 12 h and 24 h after damage repair. B: Live/dead cell staining at 12 h after damage repair. C: Detection of reactive oxygen species in cells using DCFH-DA. D: Cell viability was measured by CCK-8 assay in each group at 12 h of repair. Data are reported as means ± SD (n=4). E: Detection of relative reactive oxygen species content in cells using flow cytometry. *P<0.05

Fig. 8 Closure of full-thickness wounds in ratsA: Wound healing in different groups of rats was recorded at regular intervals. B: Wound closure rate in different groups of rats. Data are reported as means ± SD (n=6). C: H&E staining and Masson staining of skin samples

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