Development and application of a new protein hair dye

Abstract

This application relates to the development and application of a novel protein hair dye. This invention discloses a biological hair dyeing system involving three color proteins, mussel adhesive protein, and split intein, and its application, belonging to the field of biotechnology. This invention uses Escherichia coli as the chassis cell to construct and express a recombinant protein functional module mediated by split intein: three color proteins (red, yellow, and blue) connected to the C-terminus of the split intein, and a mussel adhesive protein connected to the N-terminus of the split intein. The key points of its technical solution include: (1) using red, yellow, and blue primary color proteins to achieve multi-tone and full-spectrum color coverage through precise compounding in different proportions; (2) using the excellent underwater interfacial adhesion ability of mussel adhesive protein to firmly anchor the color-developing module to the surface of hair fibers; (3) using the specific interaction between split inteins and protein trans-splicing reaction to achieve covalent connection between the color-developing module and the adhesive module at the molecular level. This invention effectively overcomes the technical bottlenecks of existing microbial pigments, such as monotonous colors and incomplete color gamut. It has the advantages of rich and controllable color development, mild hair dyeing conditions, excellent color fixing performance, and high biological safety, and has broad application prospects in the field of green cosmetics.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and cosmetics technology, specifically relating to a novel protein hair dye constructed using recombinant DNA technology, based on tri-pigment proteins (red, yellow, and blue) and mussel adhesive proteins, formed through spontaneous splicing mediated by the breaking of internal peptides, as well as its preparation method and application. Background Technology

[0002] Hair dye, as a cosmetic, is primarily used to change hair color for aesthetic, fashionable, or gray-covering purposes. Since the introduction of the first synthetic dye, aniline violet, in 1856, the modern hair dyeing market has developed for over a century, becoming a relatively mature industry. With the progress of the times, the diversification of social aesthetic concepts, and the continuous improvement of personal image awareness, hair dyeing has gradually extended from specific groups to the general public.

[0003] Most hair dyes currently on the market are traditional chemical hair dyes. Paraphenylene diamine (PPD) in traditional chemical hair dyes is a strong allergen that can cause skin allergies, edema, anemia, asthma, and other symptoms. Compared to traditional chemical hair dyes, biomass hair dyes have better biocompatibility and a significantly reduced risk of sensitization (Abd-ElZaher MA (2012) Some toxicological health hazards associated with subchronic dermal exposure to paraphenylene-diamine (PPD): An experimental study. Egypt. J. ForensicSci. 2 (3), 105–11). The development of biomass hair dyes mainly includes the development and utilization of plant-based hair dyes, microbial hair dyes, and biomimetic hair dyes.

[0004] Plant-based hair dyes primarily utilize natural pigments found in the roots, stems, leaves, flowers, and fruits of plants to create richer colors and are safer. However, they suffer from drawbacks such as unsatisfactory coloring effects, poor stability, susceptibility to temperature, pH, and light (ultraviolet radiation), low wash fastness, and easy fading (Cheng Mengqi (2022) Research on Plant-Based Hair Dyes [J]. Applied Chemical Industry, 2022, 51(S2): 227-230). Furthermore, the extraction process of plant pigments is complex, resulting in higher production costs. Biomimetic hair dyes, on the other hand, mimic the formation and color development of melanin in hair. The most common biomimetic hair dyeing method uses substances such as dopamine to synthesize melanin in vitro. During the melanin synthesis process, it spontaneously aggregates to form micron-sized spheres and rods. Due to its large size, it does not penetrate into the hair matrix. Therefore, the synthesized melanin is usually deposited on the hair surface to form a coating, which can also protect the hair from external damage (Im KM (2017) Metal-Chelation-Assisted Deposition of Polydopamine on Human Hair: A Ready-to-Use Eumelanin-Based Hair Dyeing Methodology. ACS Biomater. Sci. Eng. 3(4):628-636). Biomimetic hair dyes are greener and safer, but have poor color richness (Deng Xinxin (2025) Research Status and Prospect of Hair Dyes [J]. Chemical Bulletin (Chinese and English). 88(06):643-650). Microbial pigments are a secondary metabolite of microorganisms, and there are many types of pigment colors. Compared with other natural pigments, microbial pigments have a shorter production cycle, lower cost, are not affected by weather, and are easier to produce industrially. Furthermore, due to their advantages such as being environmentally friendly, having mature fermentation processes, and good biocompatibility, they will be one of the main methods for synthesizing natural pigments in the future. However, limited by the singularity of microbial metabolic pathways, natural microbial pigments generally face technical bottlenecks such as incomplete chromatographic distribution and difficulty in multi-color compounding. At the same time, due to the low affinity of these pigment molecules for hair fibers, their colorfastness after washing is poor; and the chromophores are unstable under light, easily degrading and fading, and their lightfastness cannot meet the long-lasting requirements of commercial coloring (Zhang Xinyao (2023) Research progress of green and safe hair dyes [J]. Chemical Bulletin, 86(06):699-709). Therefore, developing a microbial hair dye with rich colors, good biocompatibility, and strong adhesion has important application prospects.

[0005] To further broaden the sources of natural pigments, chromoproteins have emerged as a novel type of biological dye. Highly homologous to fluorescent proteins, and benefiting from their extremely high visible light absorption coefficients, chromoproteins exhibit vibrant colors and minimal background fluorescence under natural light, possessing great potential to replace traditional dyes. However, as biomacromolecules, chromoproteins often have difficulty penetrating the substrate structure due to their large molecular size, and their complex surface charge distribution results in weak binding to the fiber matrix. This instability of interfacial bonding directly leads to the problem of fixation after coloring, making them easily lost during washing or friction. Therefore, how to achieve long-term anchoring and effective fixation of chromoproteins on the substrate is a key technical bottleneck that urgently needs to be solved (Dedecker P (2013) Fluorescent proteins: shine on, you crazy diamond. J. Am.Chem. Soc. 135:2387–402).

[0006] Mussels are marine organisms with strong adhesive properties, secreting mussel adhesion protein (Mfp). The most important characteristic of Mfp is its high content of 3,4-dihydroxyphenylalanine (DOPA) groups and lysine. DOPA plays a crucial role in the adhesion of mussel byssal proteins. DOPA enables mussel adhesion proteins to adhere through aryl coupling, thiol addition, and Fe... 3+ The cross-linking of mussel foot protein is achieved through the coordination of multiple ligands and the chemisorption of surface metal oxides (Ni P (2024) Mussel Foot Protein-Inspired Adhesive Tapes with Tunable Underwater Adhesion. ACS Appl. Mater. Interfaces 16(34):45550-45562). Mussel foot protein can strongly adhere to various interfaces such as metal, glass and plastic through the synergistic effect of DOPA-mediated metal complexation, hydrogen bonding and oxidative cross-linking, and can maintain extremely high adhesion strength and chemical stability in humid environments. The dopa in mussel adhesive protein is derived from the post-modification of tyrosine residues. Tyrosine residues in the protein are catalyzed by tyrosinase to form dopa residues, thus possessing adhesive properties. The research on mussel adhesive protein by materials scientists has provided ideas for the development of hair dye in this project.

[0007] Based on research on chromoproteins and mussel adhesive proteins, this invention aims to develop a protein hair dye with rich colors, good biocompatibility, and high adhesion stability in complex environments. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of the prior art and provide a novel bio-hair dye based on the principle of three primary colors and mussel adhesive protein. Through modular connection technology mediated by splitting internal peptides, the efficient combination of chromoprotein and adhesive protein is achieved, resulting in a novel hair dye product with adjustable color, strong adhesion, and high safety.

[0009] To achieve the above objectives, the specific implementation method is as follows: S1: Design and construction of a splitting in-peptide-chromoprotein module Based on known chromoproteins such as red chromoproteins eforRed and meffRed, yellow chromoproteins amilGFP, fwYellow, and amajLime, and blue chromoproteins aeBlue and meffBlue (more chromoproteins can be listed, such as which ones are red, which ones are green, and which ones are blue, etc.), and the base sequences of splitting integrins such as VidaL, Cat, and SspDnaB, the AlphaFold3 technique is used to predict the effect of the connection between splitting integrins and chromoproteins on the spatial structure and color effect of chromoproteins. Furthermore, SOPMA, PSIPRED, and GOR4 are used to predict the secondary structure information of the chromoprotein-splitting integrin module. Based on this, a module in which splitting integrins are connected to chromoproteins through flexible linker peptides is designed.

[0010] S2: Design and Construction of Mussel Adhesive Protein-Fragmented Intrin Module Based on the base sequences of mussel adhesive protein and fragmented integrins, the spatial structure of the connection between the fragmented integrins and mussel adhesive protein was predicted using AlphaFold3 technology. Furthermore, bioinformatics technology was used to predict the secondary structure information of the Mfp-fragmented integrin module, thereby designing a module in which mussel adhesive protein is connected to the fragmented integrins via flexible linker peptides.

[0011] S3: Splitting intron-mediated module linkage The fragmented intrapeptide VidaL consists of two independent segments, an N-terminus and a C-terminus, which spatially recognize each other and spontaneously undergo protein splicing to form natural peptide bonds. This invention utilizes this characteristic to fuse chromoprotein and mussel adhesive protein, achieving covalent linkage between the two after in vitro mixing. This avoids the use of chemical cross-linking agents, ensuring the biocompatibility and safety of the product.

[0012] S4: Construction of the Recombinant Expression System To achieve efficient expression of three color protein modules and one adhesion module, this invention constructed four orthogonal expression systems, each employing different inducible promoters and replicons, ensuring independent and controllable expression of each module in *E. coli*: pBAD vector (arabinose-induced): expresses eforRed-Int C pACYC vector (IPTG induced): expresses amilGFP-Int C pTet vector (tetracycline-induced): expresses aeBlue-Int C pET28a(+) vector (IPTG induced): expressing Mfp-5-Int N Four sets of plasmids were co-transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain that could express all modules simultaneously.

[0013] S5: Expression and purification of the target protein The recombinant strains obtained in S4 were inoculated into LB liquid medium containing the corresponding antibiotics (such as ampicillin, kanamycin, chloramphenicol, and tetracycline, the specific type and concentration of which were determined based on the resistance markers of each plasmid) and cultured overnight at 37°C with shaking. Then, 1%–2% of the inoculum was transferred to fresh selective YT medium and cultured at 37°C and 200–250 rpm until OD500 was reached. 600 The target protein concentration (Pc) was 0.6–0.8. Inducers corresponding to the four expression systems were added, and the cells were cultured at an appropriate temperature (e.g., 16–37°C) for 16–20 hours to induce gene expression in the four plasmids. After induction, the bacterial cells were collected and resuspended in pre-chilled lysis buffer. The cells were disrupted by sonication, and the supernatant (soluble protein) and precipitate (inclusion bodies) were then separated. Affinity chromatography columns were used for purification based on the tags on the target proteins. The purified proteins were validated by SDS-PAGE and Coomassie Brilliant Blue staining to confirm the expression and co-purification of each module. The purified proteins were aliquoted and stored at -80°C for subsequent studies.

[0014] S6: Protein Hair Dye Test This invention constructs a highly efficient and stable bio-protein hair dyeing complex by molecularly coupling and precisely modulating the hue of the target protein obtained in step S5. Subsequently, under specific buffer conditions, the complex is used to perform an in-situ binding reaction with pretreated bleached hair samples. Interface dyeing is achieved through the bioaffinity between the protein and hair fibers, and the sensory properties and color development characteristics after dyeing are initially observed. To further verify its application performance, this invention conducts cyclic washing experiments, color durability tracking, and color difference distribution analysis on dyed hair samples. Quantitative evaluations are performed from multiple dimensions, including wash fastness, environmental aging resistance, and spatial color uniformity, aiming to systematically evaluate the color-fixing efficiency, dyeing depth, and color consistency of this protein-based hair dye at the bio-fiber interface.

[0015] Beneficial effects: Compared with the prior art, the design solution provided by the present invention has the following significant advantages: This invention combines chromoproteins with mussel adhesive protein, utilizing fragmented intima-integrated peptides for flexible linkage. By adjusting the ratio of the three chromoproteins, a variety of colors can be formulated, greatly enriching the color spectrum of microbial pigments. Furthermore, compared to traditional hair dyes, this invention utilizes the adhesive properties of mussel adhesive protein to fix the chromoproteins to the hair. Mussel adhesive protein has a certain protective effect on hair, reducing damage caused by dyeing. The chromoproteins and mussel adhesive protein have good biocompatibility, making it a safer and less polluting hair dye. Attached Figure Description

[0016] Picture 1 Design ideas for protein-based bio-hair dyes; Detailed Implementation The technical solution of the protein hair dye of the present invention and its uses is further illustrated below through specific embodiments.

[0017] Example 1: Plasmid construction, the specific steps are as follows: In a specific embodiment of the present invention, a chromogenic module based on split intein and a mussel adhesive protein anchoring module were constructed in detail. First, the amino acid sequences of the target proteins were retrieved and obtained through the iGEM Registry of Standard Biological Parts, specifically including: red chromogen eforRed, yellow chromogen amilGFP, blue chromogen aeBlue, and the C-terminal (VidaL-C) and N-terminal (VidaL-N) sequences of the highly orthogonal split intein VidaL.

[0018] In terms of molecular structure design, the VidaL-C-terminal sequence was intercalated and cloned with three chromoprotein sequences via flexible linkers to construct a "fragmented inteptide C-terminus-chromoprotein" fusion expression module; simultaneously, the VidaL-N-terminal sequence was fused with a mussel adhesive protein sequence to construct a "fragmented inteptide N-terminus-mussel adhesive protein" anchoring module. To enhance the expression level of heterologous proteins in chakra cells, targeting *Escherichia coli* (… E. coli Codon bias was used to optimize the whole genome sequence of all the above fusion sequences, and a 6×His-tag was added to the end of each module sequence according to the subsequent purification requirements.

[0019] In terms of expression system construction, to achieve multi-vector compatibility and precise control of different colors, the optimized eforRed, amilGFP, and aeBlue chromogenic modules were cloned into pBAD, pACYCDuet, and pTet expression vectors with different inducible promoters and replicon initiation sites, respectively. Simultaneously, the mussel adhesive protein anchoring module was cloned into the high-level expression vector pET28a(+). Finally, the constructed recombinant plasmids were transformed into E. coli (BL21) competent cells, and stable recombinant genetically engineered strains were obtained through antibiotic resistance screening for subsequent protein induction expression and bio-dyeing applications.

[0020] Example 2: The four recombinant plasmids obtained in Example 1 (the colorimetric and anchoring modules based on pBAD, pACYCDuet, pTet, and pET28a vectors, respectively) were introduced into Escherichia coli. E. coli BL21(DE3) competent cells.

[0021] S1: Combine the four constructed recombinant plasmids with... E. coli BL21(DE3) competent cells were mixed, incubated on ice for 30 min, and then heat-shocked at 42℃ for 90 s. LB culture was added and the cells were thawed at 37℃ for 1 h, then plated onto selective plates containing the corresponding antibiotics and incubated upside down at 37℃ for 12–16 h. Positive clones with correct genotypes were screened and preserved for subsequent operations by colony PCR and plasmid sequencing.

[0022] S2: Positive strains were inoculated into LB medium and cultured until OD600 reached 0.6–0.8. Depending on the vector type, 0.5% (w / v) L-arabinose, 1 mM IPTG, or 0.5 μg / mL tetracycline hydrochloride were added, and the mixture was induced at 16°C for 16–20 h. The bacterial cells were collected by centrifugation and resuspended in lysis buffer (pH 8.0). The cells were then sonicated on ice and centrifuged to obtain the supernatant. The target protein component was purified by Ni-NTA affinity chromatography. Finally, the mixture was dialyzed continuously in PBS buffer (pH 7.4) for at least 24 h to obtain the biological protein dye raw material.

[0023] Example 3: The induction and purification of the target protein were strictly carried out in accordance with the following steps: S1: Verified positive monoclonal antibodies were inoculated into YT liquid medium (containing 8 g / L tryptone, 8 g / L yeast extract, 2.5 g / L NaCl, and antibiotics) and cultured at 37°C and 220 rpm until the OD600 reached 0.6–0.8. Inducing agents were added for different vector systems: 0.5% (w / v) L-arabinose was added to the pBAD vector; 1 mM IPTG was added to the pACYCDuet and pET28a vectors; and 0.5 μg / mL tetracycline hydrochloride was added to the pTet vector. The culture was then induced at 16°C and 220 rpm for 16–20 h to obtain highly soluble heterologous proteins.

[0024] S2: The fermentation broth was centrifuged at 4℃ and 9000rpm for 5 min to collect the cells. The cells were resuspended in lysis buffer (containing 20mM Tris-HCl, 500mM NaCl, 20mM imidazole, pH 8.0) and sonicated on ice. The supernatant was collected and loaded onto a Ni-NTA affinity chromatography column. A 6×His-tag was used for capture, and each target protein fraction was collected by elution using a gradient of different imidazole concentrations. The eluent was transferred to a dialysis bag and dialyzed continuously with PBS or Tris-HCl buffer (pH 7.4) for at least 24 h to remove imidazole and high salt content, yielding the biological protein dye raw material.

[0025] Implementation Case 4: By leveraging the highly specific molecular recognition capabilities and biocatalytic activity of split inteins, precise covalent assembly of the chromoprotein module and the mussel adhesive protein anchoring module at the molecular level was achieved. The specific steps are as follows: S1: First, the colored protein modules (each carrying the VidaL-C fragment) obtained in Example 3 were mixed with the mussel adhesion protein module (Mfp-Int-N, carrying the VidaL-N fragment) at a preset stoichiometric ratio. The mixing ratio was adjusted according to the color development requirements, preferably in the range of 1:1 to 1:3, to ensure that each component reacted fully. The mixed protein was placed in a specially prepared splicing buffer, which consisted of 100 mM phosphate, 150 mM NaCl, 1 mM EDTA, and 10% glycerol by mass. The pH of the system was adjusted to a physiologically compatible 7.2 to simulate the optimal microenvironment for protein folding and interaction.

[0026] S2: To prevent the oxidation of key sites (such as cysteine ​​residues) in the splicing reaction active center, a potent reducing agent, tris(2-carboxyethyl)phosphine (TCEP), was added to the above system at a final concentration of 1 mM to maintain the reduced state of the protein active sites and enhance reaction efficiency. The reaction mixture was placed in a 30°C constant temperature water bath in the dark for 30-60 min. During this process, the N-terminal and C-terminal fragments of the splitting integrins distributed at the ends of the two types of functional proteins complete recognition and topological folding through highly precise intermolecular affinity. Subsequently, a transpeptide bond rearrangement mechanism was autonomously induced, and finally, by removing the integrin sequence fragments, stable peptide bonds (covalent bonds) were formed between the various colored proteins and the mussel adhesive protein molecules, thereby constructing a complete protein complex with strong adhesion ability.

[0027] S3: After the reaction, an appropriate amount of the reaction product was taken, and an equal volume of 2×SDS loading buffer was added. The mixture was then denatured at 100℃ for 5 min, followed by inoculation onto a 12% polyacrylamide gel for SDS-PAGE electrophoresis analysis. By observing changes in band migration, if a newly formed high-molecular-weight band appeared at the corresponding molecular weight position on the gel, and the original substrate protein band weakened or disappeared, the successful occurrence of the splicing reaction was confirmed at the molecular level. Image grayscale scanning analysis was used to quantitatively evaluate the splicing yield and conversion efficiency, providing data support for subsequent development of bio-based hair dye formulations.

[0028] Example 5: This embodiment aims to verify the compatibility of different color protein modules and their color development characteristics in the visible light range. The specific operating steps are as follows: S1: Take the red protein eforRed-Mfp5, yellow protein amilGFP-Mfp5, and blue protein aeBlue-Mfp5 prepared and purified in Example 4. To eliminate the influence of concentration deviation on the color adjustment ratio, use protein preservation buffer (e.g., 20 mM Tris-HCl, pH 7.0) to precisely adjust the molar concentration of the three color protein modules to be consistent (preferably the same concentration in the range of 50 μM to 200 μM).

[0029] S2: Based on the principles of subtractive color mixing and primary color mixing, three series of gradient mixing experiments are designed to achieve continuous adjustment of the color space: Series A (Orange-Red to Yellow-Orange Series): eforRed-Mfp5 (red) and amilGFP-Mfp5 (yellow) were cross-mixed at volume ratios of 3:1, 2:1, 1:1, 1:2, and 1:3 to obtain 5 groups of red to orange-yellow composite systems with color gradients. Series B (Yellow-Green to Blue-Green Series): amilGFP-Mfp5 (yellow) and aeBlue-Mfp5 (blue) were mixed at volume ratios of 3:1, 2:1, 1:1, 1:2, and 1:3, respectively, to obtain 5 groups of yellow-green to blue-green composite systems with color gradients. Series C (purple-blue-violet series): eforRed-Mfp5 (red) and aeBlue-Mfp5 (blue) were compounded at volume ratios of 3:1, 2:1, 1:1, 1:2, and 1:3 respectively to obtain 5 groups of purple-red to blue-violet composite systems with color gradients.

[0030] S3: Place the above mixtures on a vortex mixer and gently shake for 30 seconds at room temperature to allow the three chromoprotein molecules to move and distribute evenly in the buffer solution. Then, place the mixture flat and let it stand for 10-15 minutes to allow the intermolecular interactions to stabilize. Observe the macroscopic color development of the mixture to confirm that no precipitation occurs and the color transition is uniform.

[0031] S4: Add 100 μL of each compound sample to a microvolume chromogenic dish or a 96-well microplate, and perform wavelength scanning using a UV-Vis spectrophotometer. The scanning range is set to 500 nm to 700 nm, with a sampling step of 1 nm. Obtain the full-spectrum scanning curves of each compound system, extract and record the maximum absorption wavelength (λmax) and corresponding absorbance peak for each system. Experimental results show that by adjusting the volume ratio, the absorption intensity of each compound system exhibits a significant linear migration trend, and the chromatographic distribution topology conforms to the preset proportional weights, thus verifying the additive color development and precise color adjustment efficiency of the protein-based hair dyeing module at the molecular level.

[0032] Implementation Case 6: The dyeing effect, wash resistance, and impact on hair quality of the constructed recombinant protein hair dyeing system based on fragmented peptide-mediated "chromoprotein-mussel adhesive protein" (hereinafter referred to as protein hair dye) were systematically evaluated. The specific operating steps and testing methods are as follows: S1: Natural human hair bundles (approximately 15 cm in length and 2 g in weight) that have undergone standardized bleaching were selected as the dyeing matrix. The hair bundles to be tested were completely immersed in the protein hair dye solutions of various colors prepared in Example 5, with the liquor ratio (solid-to-liquid ratio) controlled at 1:10 (g:mL). The dyeing system was incubated in a 37°C constant temperature shaking water bath for 60 min, with gentle physical shaking promoting the penetration and adsorption of protein molecules on the hair surface. After the reaction, the samples were removed, rinsed with deionized water to remove unbound free protein, and then air-dried at room temperature (25±2°C).

[0033] S2: To verify the advanced nature of the biological covalent anchoring technology, the following parallel control group was established: (1) Blank control group: Untreated raw bleached hair strands were used; (2) Positive control group: Dyeing was performed using commercially available oxidative chemical hair dye according to the instructions; (3) Negative control group: Equal amounts of a physical mixture of “chromoprotein module and mussel adhesive protein module” that has not undergone splicing reaction were treated under the same conditions.

[0034] S3: Using a high-precision spectrophotometer and based on the International Commission on Illumination (CIE) standard color space system, the lightness axis values, red-green axis coordinates, and yellow-blue axis coordinate data of each group of hair strand samples were accurately collected. By analyzing the offset of each coordinate relative to the blank control sample and calculating the arithmetic square root of the sum of the squares of the differences in the above three dimensions, the total color difference value of the sample was obtained. This was used to quantitatively evaluate the initial colorimetric rate and color richness of each color protein module under bio-anchoring, thereby establishing a quantitative colorimetric standard for biological protein dyes.

[0035] S4: To simulate extreme washing conditions, the dyed hair strands were placed in a surfactant solution containing 1% (w / v) sodium dodecyl sulfate (SDS) and subjected to 1, 3, 5, 10, and 20 cycles of agitation washing at 37°C. After each washing cycle, the color difference values ​​were remeasured to assess the color retention rate. Simultaneously, strictly following the requirements of the national standard GB / T 3920-2008 "Textiles - Tests for Color Fastness to Rubbing", the dry and wet rubbing fastness of the hair strands was evaluated using a rubbing fastness meter, and the staining grade of the standard white cotton fabric was recorded and observed.

[0036] S5: Microscopic morphology and structural characterization: (1) Surface micromorphology: The integrity of the hair cuticle and the uniformity of the protein coating on the surface of the hair were observed at different magnifications using a scanning electron microscope (SEM). (2) Protein distribution detection: Based on the autofluorescence properties of the chromoproteins themselves, the distribution depth and continuity of each chromoprotein on the hair surface and the edge of the cortex are observed using a fluorescence microscope or a laser confocal microscope.

[0037] S6: The mechanical properties of hair strands before and after dyeing were characterized using a single-fiber tensile tester, and their breaking strength and elongation at break were measured. By comparing the data differences between each experimental group and the control group, the mechanical structure of the hair fiber was evaluated, and its bio-safety and hair care performance were verified.

[0038] Example 7: To assess the safety of the constructed protein hair dye in biological applications, the in vitro cytotoxicity of the protein hair dye was quantitatively evaluated using the MTT assay, strictly following the requirements of ISO 10993-5 "Medical devices - Biological evaluation - Part 5: In vitro cytotoxicity tests". The specific steps are as follows: S1: L929 mouse fibroblasts, as recommended by standardization, were selected as the toxicity evaluation model. L929 cells in the logarithmic growth phase and in good growth condition were collected, and the cell suspension concentration was adjusted using DMEM complete medium containing 10% (v / v) fetal bovine serum. Cells were distributed at a concentration of 1 × 10⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well cell culture plates and incubated at 37°C in a humidified incubator containing 5% CO2 for 24 hours. Subsequent drug treatments were only performed after the cells had completely adhered to the plate and exhibited a normal spindle-shaped morphology.

[0039] S2: The stock solutions of various protein hair dyes prepared in Example 4 were serially diluted using serum-free DMEM medium. Multiple concentration gradient groups were set up in the experiment: 10, 20, 50, 100, and 200 μM (based on protein concentration). At the same time, DMEM medium without dye (containing 0 μM protein) was set up as a solvent control.

[0040] Two sets of standard comparisons were added simultaneously: (1) Negative control group: Add an equal volume of DMEM complete medium containing 10% fetal bovine serum as a reference for normal cell growth; (2) Positive control group: A phenol solution with a final concentration of 0.1% (w / v) was added to verify the sensitivity of cells to toxic substances.

[0041] Three parallel standard replicates were set up for each concentration group and the control group to reduce experimental error. After drug addition, the culture was continued for 24 h under the above-mentioned culture conditions.

[0042] S3: After incubation, aspirate the old culture medium from each well and add 20 μL of 5 mg / mL MTT solution (thiazolyl blue) to each well in the dark. Incubate the plate again for 4 h, allowing succinate dehydrogenase in the mitochondria of live cells to reduce MTT to blue-purple formazan crystals. After incubation, carefully discard the supernatant from each well and precisely add 150 μL of dimethyl sulfoxide (DMSO) to each well as a substrate dissolution medium. Shake gently for 10 min on a shaker to fully dissolve the formazan crystals and form a homogeneous purple solution.

[0043] S4: The absorbance of each well was measured using a multi-functional microplate reader at the characteristic absorption wavelength of 490 nm. (OD value). The relative cell viability at each concentration was calculated by comparing the OD values ​​of each experimental group with those of the negative control group. Simultaneously, the microscopic morphology of each group of cells was observed using an inverted fluorescence microscope and photographed under high magnification to assess cell density, morphology (e.g., whether they became rounded, detached, or developed a granular appearance), and degree of confluence. If the calculation results showed that the cell viability was still significantly higher than 70% under high concentration treatment, and there was no obvious ectopic deformation of the morphology, then the protein hair dye of this invention was confirmed from a cell biology perspective to have good biocompatibility and clinical application safety potential.

Claims

1. A protein hair dye, characterized by comprising: Its raw material components include: color protein module, mussel adhesive protein module, and splitting peptide module.

2. The protein hair dye according to claim 1, characterized in that: The chromoprotein module is fused with the C-terminal fragment of the splitting in-peptide to form the first fusion protein; The mussel adhesive protein module is fused with the N-terminal fragment of the splitting inner peptide to form a second fusion protein; The color protein module and the C-terminal fragment of the splitting peptide are connected by flexible linker peptides, as are the mussel adhesive protein module and the N-terminal fragment of the splitting peptide.

3. The protein hair dye according to claim 2, characterized in that: The color protein module and the mussel adhesive protein module spontaneously form a covalently linked protein complex through the protein trans-splicing reaction mediated by the splitting intini.

4. The protein hair dye according to claim 1, characterized in that: The chromoprotein module is selected from at least one of eforRed, amilGFP, and aeBlue, which are red, yellow, and blue, respectively.

5. The protein hair dye according to claim 1, characterized in that: The mussel adhesive protein module is a recombinantly expressed mussel adhesive protein.

6. A recombinant expression vector system, characterized in that, The system includes at least one of the following expression vectors: a) pBAD vector, used to express fusion proteins containing eforRed and splitting inteptide fragments; b) pACYC vector, used to express fusion proteins containing amilGFP and splitting inteptide fragments; c) pTet vector, used to express fusion proteins containing aeBlue and splitting inteptide fragments; d) pET28a(+) vector for expressing a fusion protein containing recombinant mussel adhesive protein and splitting intima-intima peptide fragments.

7. The use of the recombinant expression vector system according to claim 6 in the preparation of the protein hair dye according to claim 1.

8. A method for producing a protein hair dye, characterized in that, Includes the following steps: (1) Transform the expression vector described in claim 6 into Escherichia coli to obtain recombinant engineered bacteria; (2) Using the recombinant engineered bacteria as the fermentation strain and glucose as the carbon source substrate, fermentation culture was carried out; (3) Inducing and expressing the fusion protein of the color protein module and the splitting inner peptide, and the fusion protein of the mussel adhesive protein module and the splitting inner peptide.

9. Use of the protein hair dye according to any one of claims 1 to 5 in hair dyeing.

10. The use according to claim 9, characterized in that, The staining steps include: The color protein module and the mussel adhesive protein module are applied to the hair surface; By utilizing trans-splicing mediated by splitting intein, chromogens are covalently anchored to the hair surface via mussel adhesive protein modules.

Images