A method for maintaining the triple helix structure during the preparation of methacrylated recombinant collagen hydrogels
By controlling the dropping rate of methacrylic anhydride and adjusting the pH, a methacrylated recombinant collagen hydrogel with a triple helix structure was prepared, solving the problem of triple helix unwinding caused by rapid addition and improving the stability and mechanical properties of the hydrogel.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- JIANGNAN UNIV
- Filing Date
- 2024-12-31
- Publication Date
- 2026-06-30
AI Technical Summary
In the preparation of methacrylated recombinant collagen hydrogels, the addition rate of methacrylic anhydride is too fast, which leads to the unwinding of the triple helix structure and affects the stability and mechanical properties of the hydrogel.
Methacrylic anhydride was added at a constant dropping rate, and the pH of the reaction system was monitored in real time and controlled within the range of 7.0 to 7.4 to prepare methacrylated recombinant collagen, which was then crosslinked with ultraviolet light to prepare a hydrogel.
The triple helix structure of recombinant collagen was maintained, which improved the mechanical properties and controllability of the hydrogel and ensured its stability and softness.
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Figure CN122302034A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for maintaining the triple helix structure during the preparation of methacrylated recombinant collagen hydrogels, belonging to the field of genetic engineering technology. Background Technology
[0002] As a major component of the extracellular matrix (ECM) and the most abundant protein in vertebrates, collagen possesses all the biological cues required for vascular cell adhesion and proliferation. It supports cell growth, maintains the stability and structural integrity of tissues and organs, and exhibits unique biological characteristics such as low immunogenicity, biocompatibility, and biodegradability. Therefore, collagen has been widely used in the biopharmaceutical, cosmetic, and food industries.
[0003] Compared to other proteins, collagen has three significant characteristics. First, the primary sequence of collagen is a (Gly-Xaa-Yaa)n repeating sequence, requiring a glycine (Gly) residue for every three amino acids in its peptide chain. Second, the most common amino acids in Xaa and Yaa are proline (Pro) and hydroxyproline (Hyp), with hydroxyproline being a collagen-specific amino acid produced intracellularly by hydroxylase catalysis. Both of these amino acids participate in the formation of hydrogen bonds between polypeptide chains, playing a crucial role in collagen stability. Finally, collagen possesses a triple helix structure, consisting of three left-handed polypeptide chains (often called α chains) tightly wound in a right-handed helix, with a precisely staggered residue between adjacent chains. Collagen molecules self-assemble within tissues to form their final functional state; the most characteristic and common form of collagen is the D-period fibrillary. After folding, thousands of triple helices self-assemble to form crystalline supramolecular fibers, providing the foundation for the extracellular matrix.
[0004] Hydrogels prepared from collagen can form a highly resilient matrix capable of withstanding low loads generated by cells. Therefore, hydrogels are commonly used as scaffold materials in tissue engineering, providing a stable matrix for cell growth, remodeling, and regeneration, and supporting functional tissues. However, the supply of collagen extracted from animal tissues is insufficient, and its complex composition, susceptibility to pathogens, and difficulty in monomer separation increase the difficulty of obtaining animal-derived collagen. Furthermore, the structure and mechanical properties of hydrogels vary significantly depending on the tissue source and animal age, resulting in poor stability of the prepared hydrogels. Therefore, in the fields of biomaterials and biomedicine, recombinant collagen prepared using gene recombination technology has become an attractive alternative to animal-derived collagen materials, offering advantages such as low heterogeneity and low risk of zoonotic diseases.
[0005] Hydrogels are typically prepared through physical crosslinking, chemical crosslinking, and biological crosslinking. However, hydrogels prepared by physical and biological crosslinking are highly susceptible to environmental influences, have poor tunability, and ultimately exhibit weak mechanical properties, failing to meet the demands of the biomedical and materials fields. Therefore, photocrosslinking, a method within chemical crosslinking, is employed to prepare hydrogels. Photocrosslinking allows for the preparation of hydrogels with enhanced mechanical properties while minimizing the introduction of toxic reagents.
[0006] Patent CN110790950A discloses a photocrosslinked recombinant human collagen hydrogel, its preparation method, and its application in 3D printing. The method involves adding methacrylic anhydride to a phosphate solution of recombinant human collagen, stirring to obtain modified recombinant collagen, dissolving it in a culture medium, adding a photoinitiator, mixing thoroughly, and then photocrosslinking and curing to obtain the photocrosslinked recombinant collagen hydrogel.
[0007] In this invention, methacrylic anhydride (MA) is added in a manner of 600 μL within 2 minutes. This rapid addition of a large amount of MA leads to a significant increase in the polymerization rate, potentially causing instantaneous heating of the reaction. This could result in the unwinding of the triple helix of environmentally sensitive collagen, ultimately leading to poor hydrogel performance. Furthermore, the rapid addition of MA may invert the concentration distribution in the reaction mixture, potentially resulting in different degrees of crosslinking in certain regions of the hydrogel, leading to differences in physical and chemical properties.
[0008] In his paper "Design of synthetic collagens that assemble into supramolecular banded fibers as a functional biomaterial testbed", Hu Jinyuan mentioned that recombinant collagen was tested for folding and assembly at pH levels between 3 and 11. The results showed little difference, thus ruling out the influence of pH changes on collagen folding.
[0009] Currently, most patents and literature use a specific dropping rate to add methacrylic anhydride, mostly between 0.5 mL / min and 1 mL / min, with a few using a single-drop addition method. However, at most addition rates of 0.5 mL / min to 1 mL / min, methacrylic anhydride (MA) reacts rapidly with the amino acid side chains in collagen, potentially causing collagen chain distortion or irregular folding, ultimately affecting the stability and function of the prepared hydrogel. Furthermore, excessively rapid addition can lead to an increase in reaction temperature, further impacting collagen stability. Since the formation and maintenance of the triple helix typically require relatively mild conditions, rapid polymerization may cause collagen to lose its native conformation at high temperatures or high viscosity. Rapid polymerization can result in a high cross-linking density; this dense network structure may negatively impact the triple helix stability of collagen, making it more prone to denaturation. While a high cross-linking density may enhance the mechanical properties of the hydrogel, it may also lead to a decrease in the biological function and biocompatibility of collagen.
[0010] Therefore, developing a method for adding methacrylic anhydride to ensure that recombinant collagen can still maintain its triple helix after chemical cross-linking to prepare hydrogels has extremely high practical and economic value. Summary of the Invention
[0011] To address the shortcomings of the aforementioned technologies, the present invention aims to provide a method for controlling the addition rate of MA so that the triple helix of the prepared methacrylated collagen can still maintain a high degree of folding, thereby solving the problem of low or non-existent triple helix folding during the preparation of hydrogels in chemical crosslinking.
[0012] This invention utilizes the reaction of methacrylic anhydride (MA) with the amino groups of collagen side chains to prepare hydrogels by crosslinking under ultraviolet light with the addition of a photocrosslinking agent; the addition of methacrylic anhydride (MA) may have a significant impact on the triple helix structure of collagen, especially its addition rate.
[0013] To avoid localized triple helix unwinding during MA addition, this invention employs a constant dropping rate and monitors the pH changes of the reaction system in real time for adjustment. Hydrogels prepared using this reaction method typically exhibit excellent water retention, softness, and controllability. The prepared hydrogels can withstand significant forces and their structure and properties can be precisely controlled.
[0014] The first objective of this invention is to provide a method for preparing methacrylated recombinant collagen with a triple helix structure, the method comprising:
[0015] Methacrylic anhydride was added to the recombinant collagen solution at a rate of 0.5–500 μL / min to obtain methacrylated recombinant collagen with a triple helix structure.
[0016] The molar ratio of methacrylic anhydride to recombinant collagen is 1–10:1–3.
[0017] Optionally, the addition rate of methacrylic anhydride is 0.5–4 μL / min;
[0018] Preferably, the addition rate of methacrylic anhydride is 1–3 μL / min;
[0019] More preferably, the addition rate of methacrylic anhydride is 2 μL / min.
[0020] Optionally, the molar ratio of methacrylic anhydride to recombinant collagen is 1:1 to 2;
[0021] Preferably, the molar ratio of methacrylic anhydride to recombinant collagen is 1:2.
[0022] In one embodiment, the recombinant collagen has the following structure:
[0023] From the N-terminus to the C-terminus, the sequence is: first repeat sequence, collagen domain, and second repeat sequence.
[0024] Optionally, the amino acid sequences of the first repeat sequence and the second repeat sequence are as shown in SEQ ID NO.3;
[0025] Optionally, the amino acid sequence of the collagen domain is shown in SEQ ID NO.3.
[0026] In one embodiment, the N-terminus of the recombinant collagen is also connected to a folding domain;
[0027] The amino acid sequence of the folded domain is shown in SEQ ID NO.1;
[0028] Optionally, there is an enzyme cleavage site between the folded domain and the first repeat sequence, and the amino acid sequence of the enzyme cleavage site is LVPRGSP.
[0029] In one embodiment, the reaction temperature is 35–40°C, the rotation speed is 200–220 rpm, and the pH is 7.0–7.4. The reaction ends when the pH no longer changes and stabilizes at 7.0–7.4.
[0030] Specifically, during the reaction, the pH value is monitored every hour. If the pH value is lower than 7.0, the pH needs to be adjusted to 7.0-7.4 and the reaction continues until the pH value is maintained within the range of 7.0-7.4, at which point the reaction is considered complete.
[0031] A second object of the present invention is to provide methacrylated recombinant collagen with a triple helix structure prepared by any of the above methods.
[0032] A third objective of this invention is to provide a recombinant collagen hydrogel prepared from the aforementioned methacrylated recombinant collagen with a triple helix structure.
[0033] Optionally, the recombinant collagen hydrogel is prepared from methacrylated recombinant collagen with a triple helix structure and a photocrosslinking agent.
[0034] In one embodiment, the photocrosslinking agent used is LAP (phenyl-2,4,6-trimethylbenzoyl lithium phosphine) at a working concentration of 10 mg / mL.
[0035] In one embodiment, the ultraviolet lamp has a wavelength of 365nm and a power of 360mW.
[0036] A fourth object of the present invention is to provide the application of any of the above-described methods or the above-described methacrylated recombinant collagen having a triple helix structure or the above-described recombinant collagen hydrogel in the fields of biology, food, chemical industry, medicine, biomaterials, tissue engineering or cosmetics.
[0037] In one embodiment, the application includes: using methacrylated recombinant collagen with a triple helix structure to prepare drug delivery materials, three-dimensional scaffold materials, hemostatic materials, tissue filler materials, and biosensors.
[0038] A fifth object of the present invention is to provide a recombinant collagen product containing the above-mentioned methacrylated recombinant collagen having a triple helix structure.
[0039] In one embodiment, the collagen product also contains vitamins, minerals, hyaluronic acid, natural polysaccharides, essential oils, polyphenols, natural plant extracts, etc.
[0040] In one embodiment, the collagen product is a cosmetic, food, or pharmaceutical.
[0041] In one embodiment, the collagen product may be a drug carrier, a medical product, or a cosmetic product.
[0042] The sixth objective of this invention is to provide a method for improving the mechanical properties of recombinant collagen hydrogels while maintaining the triple helix structure of recombinant collagen, comprising: preparing hydrogels using methacrylated recombinant collagen, including:
[0043] Methacrylic anhydride was added to the recombinant collagen solution at a rate of 0.5–4 μL / min to obtain methacrylated recombinant collagen with a triple helix structure.
[0044] The molar ratio of methacrylic anhydride to recombinant collagen is 1:1 to 3.
[0045] Optionally, the addition rate of methacrylic anhydride is 1–3 μL / min;
[0046] Preferably, the addition rate of methacrylic anhydride is 2 μL / min.
[0047] Optionally, the molar ratio of methacrylic anhydride to recombinant collagen is 1:1 to 2.
[0048] Preferably, the molar ratio of methacrylic anhydride to recombinant collagen is 1:2.
[0049] In one embodiment, the recombinant collagen has the following structure:
[0050] From the N-terminus to the C-terminus, the sequence is: first repeat sequence, collagen domain, and second repeat sequence.
[0051] Optionally, the amino acid sequences of the first repeat sequence and the second repeat sequence are as shown in SEQ ID NO.3;
[0052] Optionally, the amino acid sequence of the collagen domain is shown in SEQ ID NO.3.
[0053] In one embodiment, the N-terminus of the recombinant collagen is also connected to a folding domain;
[0054] The amino acid sequence of the folded domain is shown in SEQ ID NO.1;
[0055] Optionally, there is an enzyme cleavage site between the folded domain and the first repeat sequence, and the amino acid sequence of the enzyme cleavage site is LVPRGSP.
[0056] Beneficial effects:
[0057] This invention improves the parameters in the process of preparing hydrogels from recombinant collagen. The addition of methacrylic anhydride is done by titration. The degree of folding of collagen at different titration rates is detected and compared by circular dichroism spectroscopy. It was found that when the addition rate of methacrylic anhydride is 2 μL / min and the molar ratio of methacrylic anhydride to recombinant collagen is 1:2, the triple helix of recombinant collagen can be maintained to a great extent.
[0058] The repeating sequences and methods of this invention are applicable to various collagen domains and provide stronger mechanical properties than hydrogels prepared by conventional chemical crosslinking. Attached Figure Description
[0059] Figure 1 This is a schematic diagram of the collagen structure.
[0060] Figure 2 This is a schematic diagram of the collagen structure after the folded regions have been removed.
[0061] Figure 3 This is an SDS-PAGE image of the supernatant collected after cell lysis and centrifugation; in the image, T and S correspond to the V-P5BP5 sequence in whole cells and supernatant, respectively, and the arrows represent the target band; M: protein marker.
[0062] Figure 4 SDS-PAGE images of the target protein eluted at 50mM and 400mM imidazole concentrations are shown. In the figure, 1 and 2 correspond to the target band of sequence V-P5BP5. 1 is V-P5BP5 eluted at 50mM imidazole concentration, and 2 is V-P5BP5 eluted at 400mM imidazole concentration; M: protein marker.
[0063] Figure 5 The image shows an SDS-PAGE of collagen with V-domain after trypsin digestion. The sequences V-P5BP5 and P5BP5 correspond to 0h to 12h in the image. The arrows represent the target bands. M: protein marker.
[0064] Figure 6 The purpose is to verify, via MALDI-TOF, whether this is the molecular weight of the target protein P5BP5.
[0065] Figure 7 The full wavelength spectrum, thermochromic curve, and first derivative plot of the thermochromic curve of the designed type I collagen are shown.
[0066] Figure 8 The Rpn (ratio of the positive peak at 220 nm to the negative peak at 198 nm) and MRE plots of collagen at MA drop rates of 0.5 μL / min, 2 μL / min, 4 μL / min, and 500 μL / min are shown.
[0067] Figure 9 The full wavelength spectra are for the MA:col (molecular ratio) of the MA-col process at 10:1 and 1:2, and at reaction temperatures of 35℃ and 40℃.
[0068] Figure 10 The 1H NMR spectrum results were used to verify the success of MA conversion.
[0069] Figure 11 To prepare a sample with a concentration of 2M, it was dissolved in 10mM sodium phosphate buffer and photographed before and after photocrosslinking.
[0070] Figure 12 The strength of hydrogels prepared by microfluidic varistometry using P5BP5 at different MA addition rates is shown; where A, B, C, and D are the strength results of hydrogels prepared at MA addition rates of 0.5 μL / min, 2 μL / min, 4 μL / min, and 500 μL / min, respectively. Detailed Implementation
[0071] Culture medium:
[0072] LB medium (g / L): tryptone 10, yeast extract 5, NaCl 10, pH 7.0;
[0073] TB medium (g / L): tryptone 12, yeast extract 24, glycerol 4 mL, KH2PO4 2.31, K2HPO4 12.54
[0074] Cultivation method (shake flask fermentation):
[0075] Aspirate 50 μL of bacterial culture from the glycerol tube containing the target gene into 5 mL of LB (Amp resistant) and incubate overnight at 37°C and 200 rpm. Transfer 1% of the culture to 100 mL of TB fermentation broth (Amp resistant) and incubate at 37°C and 200 rpm for 24 h. Add IPTG to a final concentration of 1 mmol / L and ferment at 25°C and 200 rpm for 10 h, then transfer to 15°C for another 14 h.
[0076] Protein purification methods:
[0077] After fermentation, the bacterial culture was collected, centrifuged at 10,000 rpm for 5 min at 4°C, the supernatant was discarded, and the bacterial precipitate was collected. After disruption, the precipitate was centrifuged at 10,000 rpm for 20 min at 4°C and filtered through a 0.45 μm aqueous filter membrane. Then, it was filtered using a His Trap filter. TM HP affinity purification was performed using 5 mL of buffer A (20 mmol / L Na₂HPO₄, 20 mmol / L NaH₂PO₄, 500 mmol / L NaCl, 10 mmol / L Iminazole, pH 7.4), followed by loading at a flow rate of 3 mL / min. After loading, a gradient elution was performed using elution buffer B (20 mmol / L Na₂HPO₄, 20 mmol / L NaH₂PO₄, 500 mmol / L NaCl, 500 mmol / L Iminazole, pH 7.4) to obtain the target protein. The purification status was analyzed using SDS-PAGE.
[0078] Trypsin digestion:
[0079] The purified collagen was dissolved in water to a concentration of 0.5 mg / mL, and trypsin at a concentration of 2.5 g / L was added at a molar ratio of 8:1. The mixture was digested in a shaker at 25°C for 24 h, and the purity was verified by SDS-PAGE analysis.
[0080] Desalination freeze-drying method:
[0081] The enzyme digestion reaction product was desalted using HiTrap Desalting with ultrapure water as the mobile phase at a flow rate of 5 mL / min. The peak sample was collected, verified by SDS-PAGE, and then freeze-dried under vacuum at -50 °C.
[0082] Identification of the triple helix structure and stability of the samples:
[0083] Circular dichroism (CD) chromatography was used for identification. The specific steps were as follows: the lyophilized sample was dissolved in 10 mmol / L, pH 7.4 sodium phosphate buffer to a concentration of 1 mg / mL, equilibrated at 4°C for 24 h, and then subjected to CD chromatography. The CD spectrum was measured at 190-260 nm at 4°C with 1 nm intervals, and the average scan time was 5 s. The thermal curve was obtained by monitoring the CD signal at 223 nm, increasing the temperature from 10°C to 80°C at a rate of 10°C / h, equilibrating for 8 s at each temperature, and determining the melting temperature (T0). m The stability of the sample is obtained by taking the median absorbance values of the fitted thermal curve at 10℃ and 80℃.
[0084] Methods for determining molecular weight using MALDI-TOF-MS (ultrafleXtreme) mass spectrometry:
[0085] The lyophilized sample was dissolved in water to a concentration of 1 mg / mL, and the molecular weight was determined using a MALDI-TOF-MS (ultrafleXtreme) mass spectrometer. The matrix used was DHAP (2-acetylresorcinol, combined with ethanol and diammonium hydrogen citrate), and the operation was performed in linear mode.
[0086] MA-type reaction process:
[0087] The recombinant collagen with the correct test results was dissolved in PB at 2 mM. Methacrylic anhydride (95%) was added according to a certain molecular molar ratio. During the reaction, the pH was adjusted with sodium hydroxide to keep the reaction system at 7.0-7.4. After the reaction was completed, the mixture was diluted with ten times the volume of the reaction system and dialyzed. After dialyzing for 48 hours, the impurities and unreacted MA were removed and then lyophilized. The lyophilized powder was stored at -80℃ for later use.
[0088] Determination of MA residue:
[0089] High performance liquid chromatography (HPLC) was used to detect and analyze the residual amount of MA in the reaction system. The mobile phase used was acetonitrile and 0.1% trifluoroacetic acid. The method used acetonitrile to perform gradient elution of the reaction system to detect the peak. The detector wavelength was selected as 220 nm, and the reaction temperature was controlled at 35 °C.
[0090] Indicators of successful MA conversion:
[0091] The obtained reaction system was analyzed using proton nuclear magnetic resonance (NMR) spectroscopy. The NMR spectrometer used was an AVANCE NEO 600MHz, the reaction temperature was room temperature, and the test results were processed and analyzed using the MestReNova software.
[0092] Photocrosslinking process:
[0093] After MA conversion, collagen was dissolved in PB, and a photocrosslinking agent (LAP) was added to it at a working concentration of 10 mg / mL. The hydrogel was prepared by UV crosslinking at room temperature.
[0094] Microfluidic variation of hydrogel strength:
[0095] To quantify the strength of the hydrogel, the storage modulus G' and dissipation modulus G' of the prepared hydrogel were measured using microrheology. A solution or hydrogel mixed with fluorescent polypropylene beads was injected into the capillary space between two glass slides, and the motion trajectory of the fluorescent beads was tracked and recorded using a high-speed camera. The storage modulus and dissipation modulus of the sample were then derived from the mean square displacement.
[0096] Example 1: Design of collagen sequences
[0097] Design as Figure 1 The collagen amino acid sequence shown is illustrated below, and the collagen structure after enzyme digestion is shown below. Figure 2 As shown.
[0098] Among them, the introduction of the first and second repeat sequences (abbreviated as P5, amino acid sequence as shown in SEQ ID NO.3); the folding domain (abbreviated as V, amino acid sequence as shown in SEQ ID NO.1) in the collagen sequence can assist collagen folding to form a triple helix structure; the introduction of the collagen domain (abbreviated as B, amino acid sequence as shown in SEQ ID NO.2) is achieved by analyzing the α1 chain of type I collagen through protein computational analysis and thermostability prediction, and selecting the predicted T m Sequence B, with a high tendency for triple helix at temperatures close to 37°C, was selected. An LVPRGSP restriction site was added between the folded region and the first repeat sequence to facilitate subsequent restriction enzyme cleavage to remove the folded region. Considering that a proline (P) residue at the end of the sequence might be detrimental to protein expression, an additional glycine (G) residue was added to the end of the collagen amino acid sequence.
[0099] Example 2: Construction of recombinant plasmids and recombinant bacteria
[0100] (1) Construction of recombinant plasmids
[0101] Starting from the amino acid sequence of the recombinant collagen sequence V-P5BP5 obtained in Example 1, when synthesizing the nucleotide sequence of the protein single chain, the base GC was introduced at the 5' flanking end, and Nco I and Bam HI restriction sites were introduced at the 5' and 3' ends, respectively. The nucleotide sequence of the Nco I restriction site is CCATGG, and the nucleotide sequence of the Bam HI restriction site is GGATCC, thus obtaining the nucleotide sequence of the recombinant collagen sequence V-P5BP5, as shown in SEQ ID NO.5.
[0102] The synthesized nucleotide sequence of V-P5BP5 was inserted between Nco I and BamHI of the pColdIII plasmid (purchased from Genewiz), and sequencing was performed to obtain a plasmid expressing recombinant collagen protein.
[0103] (2) Construction of recombinant strains
[0104] The recombinant plasmids that were correctly sequenced in step (1) were transformed into E. coli BL21(DE3) competent cells, plated on LB plates containing ampicillin, cultured and screened, and preserved in glycerol tubes to obtain recombinant bacteria containing recombinant collagen, which were named E. coli BL21(DE3)-V-P5BP5.
[0105] Example 3: Expression, purification, and enzyme digestion of collagen sequences
[0106] The recombinant E. coli BL21(DE3)-V-P5BP5 obtained in Example 2 was cultured in shake flasks. After cell collection, disruption, and centrifugation, the SDS-PAGE results of whole cells and supernatant are as follows: Figure 3 As shown. The supernatant is retrieved using His Trap. TM HP 5 mL was used for affinity purification, and samples were collected at imidazole concentrations of 50 mmol / L and 400 mmol / L. The samples were verified as the target protein by SDS-PAGE, and the results are as follows. Figure 4 As shown.
[0107] The purified collagen was dissolved in water to a concentration of 0.5 mg / mL and digested with trypsin (purchased from Shanghai Titan Technology Co., Ltd.) at a molar ratio of 8:1 and a concentration of 2.5 g / L to remove the folded domains and obtain the pure collagen domain structure.
[0108] Under the action of trypsin, the V-domain is digested into multiple short peptides containing 2-20 amino acid residues. If the collagen domain correctly folds into a rigid triple helix structure under the action of the V-domain, it will not be digested by trypsin in a short time. After enzymatic digestion, desalting is performed to remove the short peptides, finally yielding collagen P5BP5. The results were verified by SDS-PAGE as follows... Figure 5 Lane 12h indicates collagen after enzyme digestion for 12 hours, with a purity greater than 90%.
[0109] The validated protein was desalted, freeze-dried, and weighed to obtain its yield. The yield of recombinant collagen P5BP5 was 55.02 mg / L.
[0110] Example 4: MALDI-TOF Identification
[0111] The collagen P5BP5 protein obtained after enzymatic digestion and desalting in Example 3 was lyophilized. The lyophilized samples were then dissolved in water to a concentration of 1 mg / mL, and the molecular weight was determined using a MALDI-TOF-MS (ultrafleXtreme) mass spectrometer. The matrix used was DHAP (2-acetylresorcinol, combined with ethanol and diammonium citrate), and the operation was performed in linear mode. The results confirmed that the obtained collagen molecular weight matched the theoretical value. Figure 6 As shown.
[0112] Example 5: Circular dichroism characterization of collagen and its sequence structure
[0113] To confirm the secondary structure of the collagen domain, the lyophilized collagen P5BP5 sample after enzymatic digestion and desalting in Example 3 was prepared into a 1 mg / mL solution using 10 mmol / L sodium phosphate buffer and equilibrated at 4°C for 24 h. After equilibration, full-wavelength scanning was performed using circular dichroism spectroscopy.
[0114] The results are as follows Figure 7 As shown, P5BP5 exhibits a characteristic positive absorption peak at 223 nm, indicating that collagen P5BP5 correctly folds into a triple helix structure with the assistance of the V-domain. The thermochromatograms at 223 nm from 4℃ to 80℃ were monitored using circular dichroism spectroscopy, and the Tg of collagen was obtained by taking the first derivative of the thermochromatograms. m Value, T of P5BP5 m The value is 44℃.
[0115] The above results demonstrate that the collagen sequence designed in this invention can be correctly folded to form a triple helix structure.
[0116] Example 6: Preparation of methacrylated recombinant collagen by titration rate as a variable in the MA process
[0117] The recombinant collagen P5BP5 identified in Examples 4 and 5 was prepared into a lyophilized powder. The lyophilized P5BP5 was dissolved in PB. The initial reaction concentration of the recombinant collagen was 2 mM. The amount of methacrylic anhydride (MA) added was 1:2 in the MA:col molar ratio (i.e., MA was 1 mM). The addition rates of methacrylic anhydride (MA) were 0.5 μL / min, 2 μL / min, 4 μL / min, and 500 μL / min, respectively.
[0118] During the reaction at 35℃, the pH of the system was adjusted using 200mM NaOH to maintain the reaction system at 7.0–7.4 (this range does not affect the results) until the pH of the reaction system no longer changed, at which point the reaction was considered complete (the pH value was monitored every hour during the reaction process; if it was lower than pH 7.0, the pH was adjusted to 7.0–7.4, and the reaction continued until the pH was maintained within the range of 7.0–7.4, at which point the reaction was considered complete).
[0119] After the reaction, the buffer was replaced with 10 times its volume. Samples of the resulting system were then subjected to circular dichroism spectroscopy to verify whether the MA conversion process maintained the existence of the recombinant collagen triple helix. The triple helix results and the ratio of positive to negative peaks are shown below. Figure 8 As shown.
[0120] like Figure 8 As shown in the left figure, compared with the un-MA-treated P5BP5 triple helix structure, the triple helix results are best maintained at an MA titration rate of 2 μL / min; the triple helix still exists at titration rates of 0.5 μL / min and 4 μL / min, but the values are low; while at a titration rate of 500 μL / min, the triple helix is completely destroyed.
[0121] Collagen Rpn (representing the ratio of a positive peak at 220 nm to a negative peak at 198 nm) can help determine the triple helix folding. Figure 8 The left figure shows the full-wavelength spectrum of collagen triple helix formation under different MA drop acceleration rates; Figure 8 In the right figure, the horizontal axis represents the MA addition rate, and the vertical axis represents the values of Rpn and MRE at the corresponding MA addition rates. The right figure clearly shows that Rpn and MRE are highest at a MA addition rate of 2 μL / min, indicating that under these conditions, the triple helix result is best maintained at a MA addition rate of 2 μL / min.
[0122] Example 6: Preparation of methacrylated recombinant collagen by MA addition amount and reaction temperature as variables.
[0123] Methacrylic anhydride (MA) and recombinant collagen were added at MA:col molar ratios of 10:1 and 1:2, respectively. The reaction shaker was set at 200 rpm, and two temperature gradients were set at 35℃ and 40℃, respectively. The MA addition rate was 2 μL / min, and the addition was completed in about 3 hours.
[0124] The triple helix of the prepared methacrylated recombinant collagen is as follows: Figure 9 As shown in the figure, the triple helix still exists after the MA reaction, but the triple helix with a MA:col molar ratio of 1:2 is better than that with a ratio of 10:1; and the triple helix is better maintained at 35℃ than at 40℃.
[0125] The system samples were sampled and the MA residue was determined by high performance liquid chromatography. The results showed that after 10-fold volume replacement, the MA residue was 0.008%, which confirmed that any unreacted MA in the system had been completely replaced and there was no residue. This can avoid the presence of unreacted MA in the system affecting the subsequent gelation results.
[0126] Example 7: Successful MA conversion verified by 1H NMR spectroscopy
[0127] To quantify the degree of replacement of recombinant collagen MA, the following was performed. 1 ¹H NMR (Bruker Avance III 600) determination. P5BP5 and P5BP5-MA were dissolved in deuterium oxide and the determination was performed at room temperature. 1 1H NMR analysis was performed, and the chemical shift of each sample was recorded on the spectrum over 1 hour. The results were analyzed using MestReNova software.
[0128] The results are as follows Figure 10 As shown, under alkaline conditions, photoactive type I methacrylated recombinant collagen (P5BP5-MA) was prepared by nucleophilic addition between the e-amino group of lysine residues on the collagen side chain and methacrylic anhydride. Compared with unmodified type I collagen (P5BP5), P5BP5-MA exhibits... 1 The presence of methacrylamide proton peaks (methylene, d = 5.4 and 5.6 ppm) in the H-NMR spectrum indicates that methacrylic anhydride was successfully introduced into the recombinant collagen side chain, thereby generating a new hydrogen shift under the new chemical shift.
[0129] Example 8: Preparation of hydrogel by photocrosslinking of methacrylated recombinant collagen
[0130] To verify the successful MA-mediated crosslinking of recombinant collagen, a photocrosslinking agent (LAP) was added at a working concentration of 10 mg / mL, and crosslinking was performed at room temperature under light irradiation at 365 nm. The gelation process is as follows: Figure 11As shown in the image, the left image represents the solution before light exposure, while the right image represents the solution after light exposure. It is clear from the images that the solutions changed from clear and transparent to slightly yellow after light exposure, indicating that the reaction occurred.
[0131] Example 9: Verification of the mechanical strength of the prepared hydrogel
[0132] Hydrogels prepared at different MA addition rates were subjected to microrheological measurements to obtain their respective storage modulus and dissipation modulus. The mechanical strength of the hydrogels was determined by analyzing the obtained spectra.
[0133] The results are as follows Figure 12 As shown, the results indicate that at a gelling concentration of 2M, the solutions at MA titration rates of 0.5 μL / min, 4 μL / min, and 500 μL / min are all viscous solutions with G' < G” (i.e., non-gelling); while at an addition rate of 2 μL / min, P5BP5 forms a soft gel at 0.454 rad·s -1 At the scanning frequency, it reaches its gel point (G'=G”).
[0134] Therefore, the results show that when the dropping rate of MA is 2 μL / min and the concentration of P5BP5 is 2 M, the sol-gel transition point is reached, with a modulus of 3.11164E-5 Pa, and higher mechanical strength.
[0135] The sequence involved in this invention:
[0136] SEQ ID NO.1: Amino acid sequence of V-domain
[0137] ADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD
[0138] SEQ ID NO.2: Amino acid sequence of collagen domain B
[0139] GPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPA
[0140] SEQ ID NO.3: Amino acid sequence of (GPP)5
[0141] GPPGPPGPPGPPGPP
[0142] SEQ ID NO.4: Amino acid sequence of V-P5BP5
[0143] HHHHHHADEQEEKAKVRTELIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALDLVPRGSPGPPGPPGPPGPPGPPGPRGEQGPQGLPGKDGEAGAQGPAGPMGPAGFPGERGEKGEPGTQGAKGDRGETGPVGPRGERGEAGPAGKDGERGPVGPAGPPGPPGPPGPPGPPG
[0144] SEQ ID NO.5: Nucleotide sequence of V-P5BP5
[0145] CATCATCATCACCATCACGCAGACGAACAAGAAGAAAAGGCCAAAGTGCGCACCGAACTGATTCAAGAATTAGCCCAAGGTTTAGGTGGCATCGAGAAGAAAAACTTTCCGACTTTAGCGATGAGGATCTGGACCACACCTACATGACC AAGCTGCTGACCTATTTACAAGAACGCGAACAAGCTGAAAATAGCTGGCGCAAACGTTTACTGAAGGGTATTCAAGATCACGCTTTAGATCTGGTTCCGCGTGGCTCCCCCGGCCCTCCGGGTCCCCCCGGTCCCCCCGGTCCGCCCGGT CCTCCCGGTCCTCGCGGTGAACAAGGCCCGCAAGGTTTACCGGGTAAAGACGGTGAAGCCGGTGCACAAGGTCCGGCTGGTCCGATGGGCCCGGCTGGTTTCCCGGGCGAGCGTGGTGAGAAAGGTGAGCCGGGCACCCAAGGTGCTAAA GGTGACCGTGGTGAAACCGGTCCCGTTGGTCCTCGTGGCGAGCCGTGAAGCTGGTCCCGCTGGTAAAGACGGCGAGCGCGGTCCCGTTGGTCCGGCCGGCCCCCCGGGCCCGCCCGGTCCGCCGGGCCCCCCCGGTCCCCCCGGTTAA
[0146] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.
Claims
1. A method for preparing a methylacrylated recombinant collagen having a triple helix structure, characterized by, The method includes: Methacrylic anhydride was added to the recombinant collagen solution at a rate of 0.5–4 μL / min to obtain methacrylated recombinant collagen with a triple helix structure. The molar ratio of methacrylic anhydride to recombinant collagen is 1:1 to 3.
2. The method of claim 1, wherein, Recombinant collagen has the following structure: From the N-terminus to the C-terminus, the sequence is: first repeat sequence, collagen domain, and second repeat sequence. Optionally, the amino acid sequences of the first repeat sequence and the second repeat sequence are as shown in SEQ ID NO.3; Optionally, the amino acid sequence of the collagen domain is shown in SEQ ID NO.
3.
3. The method according to any one of claims 1 to 2, characterized in that, The N-terminus of recombinant collagen also has a folding domain attached; The amino acid sequence of the folded domain is shown in SEQ ID NO.1; Optionally, there is an enzyme cleavage site between the folded domain and the first repeat sequence, and the amino acid sequence of the enzyme cleavage site is LVPRGSP.
4. The method according to any one of claims 1 to 3, characterized in that, The reaction temperature was 35–40℃, the rotation speed was 200–220 rpm, and the pH was 7.0–7.
4.
5. The methacrylated recombinant collagen with a triple helix structure prepared by the method according to any one of claims 1 to 4.
6. A recombinant collagen hydrogel, characterized in that, It was prepared from the methacrylated recombinant collagen with a triple helix structure as described in claim 5; Optionally, the recombinant collagen hydrogel is prepared from methacrylated recombinant collagen with a triple helix structure and a photocrosslinking agent.
7. The application of the method according to any one of claims 1 to 4, the methacrylated recombinant collagen with a triple helix structure according to claim 5, or the recombinant collagen hydrogel according to claim 6 in the fields of biology, food, chemical industry, medicine, biomaterials, tissue engineering, or cosmetics.
8. The application according to claim 7, characterized in that, The applications include: using methacrylated recombinant collagen with a triple helix structure to prepare drug delivery materials, three-dimensional scaffold materials, hemostatic materials, tissue filler materials, and biosensors.
9. A recombinant collagen product, characterized in that, The product contains the methacrylated recombinant collagen with a triple helix structure as described in claim 5.
10. A method for improving the mechanical properties of recombinant collagen hydrogels while maintaining the triple helix structure of recombinant collagen, characterized in that, Hydrogels were prepared using methacrylated recombinant collagen, including: Methacrylic anhydride was added to the recombinant collagen solution at a rate of 0.5–4 μL / min to obtain methacrylated recombinant collagen with a triple helix structure. The molar ratio of methacrylic anhydride to recombinant collagen is 1:1 to 3.