High-stability non-hydroxylase modified recombinant humanized collagen as well as preparation method and application thereof

Abstract

The invention belongs to the field of protein engineering. The invention provides high-stability non-hydroxylase modified recombinant humanized collagen as well as a preparation method and application thereof. The amino acid sequence of the high-stability non-hydroxylase modified recombinant humanized collagen comprises one of amino acid sequences shown as SEQ ID NO.1 or SEQ ID NO.2. The invention further provides a preparation method of the high-stability non-hydroxylase modified recombinant humanized collagen. Under the condition that the recombinant humanized collagen does not need to be subjected to hydroxylation modification, the triple helix stability is remarkably improved, a streptococcus Scl2 collagen-like protein triple helix stable structural domain is excavated through molecular dynamics simulation to be embedded with a natural collagen sequence, the natural sequence is reserved, the biological activity of the collagen is ensured, and the recombinant humanized collagen has the advantages of being simple in structure, convenient to use and the like. The dependence of a traditional recombinant expression technology on hydroxylation modification of proline is also avoided.

Description

Technical Field

[0001] The present invention belongs to the field of protein engineering, and in particular relates to a recombinant humanized collagen protein and a preparation method and application thereof. Background Art

[0002] Collagen is the most abundant protein in the human body, accounting for approximately 30% of the total. It plays an important role in maintaining tissue structure, mechanical strength, and physiological function, and is widely used in biomedicine, medical cosmetology, and other fields. The high-level structure of collagen is the structural basis for its unique biochemical properties and biological functions. Among them, the triple helix conformation, as the characteristic structure of collagen, not only effectively resists degradation by proteases, but also gives the molecule excellent stability. At the same time, it provides the necessary spatial structure for functional sites, which is the core element for maintaining collagen function. However, its industrial application still faces two major technical bottlenecks: first, traditional animal-derived collagen has the risk of immunogenicity. Second, recombinant collagen produced by microbial expression systems often lacks a complete post-translational modification mechanism, which often leads to poor stability of its triple helix structure.

[0003] Although co-expression of hydroxylase can partially improve the structural stability problem, this strategy faces industrial barriers such as unstable enzyme activity and complex processes, and poses major challenges in industrial production.

[0004] Therefore, it is necessary to develop new stabilization technologies that do not rely on traditional hydroxylation modifications. Summary of the Invention

[0005] In response to the above technical problems, the present invention provides a highly stable non-hydroxylase-modified recombinant humanized collagen protein, as well as its preparation method and application. Compared with existing collagen proteins, the recombinant humanized collagen protein has significantly improved triple helix stability without the need for hydroxylation modification.

[0006] The object of the present invention is to provide a highly stable non-hydroxylase-modified recombinant humanized collagen, the amino acid sequence of which includes one of the amino acid sequences shown in SEQ ID NO.1 or SEQ ID NO.2.

[0007] The present invention also aims to provide a gene sequence encoding the above amino acid sequence.

[0008] The present invention also aims to provide a recombinant vector or recombinant engineered bacteria carrying a gene sequence encoding the above amino acid sequence.

[0009] The present invention also aims to provide a method for preparing highly stable non-hydroxylase-modified recombinant humanized collagen, comprising the following steps: synthesizing a gene sequence encoding the above-mentioned amino acid sequence, connecting the gene sequence to a vector, transforming it into an engineering strain, and constructing a recombinant genetically engineered bacterium; expressing the constructed recombinant genetically engineered bacterium, collecting the bacteria, collecting the supernatant by centrifugation, and obtaining the recombinant humanized collagen after purification.

[0010] The present invention also aims to provide a method for constructing a highly stable non-hydroxylase-modified recombinant humanized collagen, comprising the following steps:

[0011] (1) Through modeling and molecular dynamics simulation of the Streptococcus Scl2 collagen-like protein, residues 82-151 were identified as the stable core region, and two key stabilizing modules were further screened;

[0012] (2) Using 1 / 4ND (containing the N-terminal peptide) as a template, the two key stabilizing modules and the charged GPP triplet sequence screened in step (1) were integrated into the N-terminus and C-terminus of 1 / 4ND, respectively, and the recombinant peptide was obtained after expression and purification verification;

[0013] (3) Circular dichroism, variable temperature circular dichroism, and cryo-electron microscopy were performed to verify the stability of the triple helical structure of collagen formed by the recombinant peptide.

[0014] The present invention selected a partial fragment of the type I collagen α1 chain, attached to its N-terminus with a Scl2 triple-helical domain identified as thermally stable through molecular dynamics simulations. Four charged GPP repeats (GPPGPPGPPGPP) were introduced to its C-terminus, forming a synergistic stabilization strategy of "Scl2 stabilizing domain + GPP modification." Testing confirmed that this partial fragment of the type I collagen α1 chain significantly improved its thermal stability. The recombinant peptide of the present invention exhibits significantly enhanced triple-helical stability without the need for hydroxylase modification.

[0015] Application of the above-mentioned highly stable non-hydroxylase-modified recombinant humanized collagen in the preparation of biomaterials.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] (1) Molecular dynamics simulations identified residues 82-151 of the Scl2 triple-helical domain with good thermal stability, and further screened out two key stabilizing modules whose energy fluctuations were significantly lower than those of the thermally stable fragments in other regions;

[0018] (2) Using a 1 / 4ND fragment (including the N-terminal peptide) as a modification template, the key stabilizing module of Scl2 was integrated into its N-terminus, and four charged GPP repeats, GPPGPPGPPGPP, were introduced into the C-terminus, forming a synergistic stabilization strategy of Scl2 stabilization domain + GPP modification. The modified 1-1 / 4ND and 2-1 / 4ND chimeras exhibited significantly enhanced triple helical structural characteristics: the positive ellipticity values ​​at 225nm increased from 10 to 20 and 30, respectively (an increase of 100% and 200%), and the thermal denaturation temperatures increased to 22.6℃ and 23.0℃, which were 30.6% and 33.0% higher than the original 1 / 4ND (17.3℃). Cryo-electron microscopy observations showed a reduction in the proportion of short fragments, indicating that the stability of the triple helical conformation was significantly improved, among which the structural stability of the 2-1 / 4ND variant was particularly significant.

[0019] In summary, the present invention obtained two recombinant humanized type I collagen proteins through semi-rational design. The partial fragment 1 / 4ND fragment of type I collagen (including N-terminal peptide) was used as the modification template, and the triple-helix stable domain of Streptococcus Scl2 collagen-like protein was mined through molecular dynamics simulation to chimerize it with the natural collagen sequence. This not only retains the natural sequence to ensure its biological activity, but also avoids the dependence of traditional recombinant expression technology on proline hydroxylation modification, providing a new technical route for the development of highly stable, low-cost, and industrialized recombinant collagen, which has important scientific value and market potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the design scheme for the transformation of natural collagen fragments of the present invention;

[0021] Figure 2 The colony PCR images of the two transformed strains at different G418 concentrations after transformation;

[0022] Figure 3 Denaturing polyacrylamide gel electrophoresis diagram of the original recombinant collagen expression;

[0023] Figure 4 Denaturing polyacrylamide gel electrophoresis diagram of the expression of recombinant collagen after transformation;

[0024] Figure 5 This is the elution curve of the original collagen fragment purified by HiTrap HP SP cation exchange chromatography;

[0025] Figure 6 SDS-PAGE analysis of the purification process of the original collagen fragment;

[0026] Figure 7 This is the elution curve of the modified 1-1 / 4ND HiTrap HP SP cation exchange chromatography purification;

[0027] Figure 8 SDS-PAGE analysis of the purification process of 1-1 / 4 ND after transformation;

[0028] Figure 9 This is the elution curve of the modified 2-1 / 4ND HiTrap HP SP cation exchange chromatography purification;

[0029] Figure 10 SDS-PAGE analysis of the purification process of 2-1 / 4 ND after transformation;

[0030] Figure 11 is the CD spectrum of the original collagen fragment;

[0031] Figure 12 Comparison of CD spectra of two modified recombinant collagens and original collagen fragments;

[0032] Figure 13 is the temperature-dependent CD spectrum curve of the original collagen fragment;

[0033] Figure 14 The temperature-dependent CD spectra curves of the two recombinant collagens after modification are shown;

[0034] Figure 15 is the denaturation temperature of the original collagen fragment;

[0035] Figure 16 Comparison of the denaturation temperatures of the two modified recombinant collagens and the original collagen fragments;

[0036] Figure 17 This is a cryo-electron microscopy image of the original collagen fragment;

[0037] Figure 18 These are cryo-electron microscopy images of two types of recombinant collagen. DETAILED DESCRIPTION

[0038] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Based on the human type I collagen α1 chain coding sequence (NM_000088.4) in the GenBank database, we selected the key functional region encompassing the complete triple-helical domain (the sequence from the N-terminal propeptide to the C-terminal propeptide). This sequence was systematically optimized based on the codon usage preference of Pichia pastoris, and the optimized gene sequence was commissioned for full gene synthesis by Nanjing GenScript Biotechnology Co., Ltd.

[0040] A quarter triple-helix fragment containing an N-terminal peptide was named 1 / 4ND, and primers were designed for plasmid construction. Through molecular dynamics simulations and computational analysis, two non-GPO sequences capable of forming a stable triple helix were identified from the Streptococcus Scl2 collagen-like protein (which forms a highly thermostable triple helix without the need for proline hydroxylase modification). These sequences were then integrated into the N-terminus of native collagen, and four GPPs were integrated into the C-terminus.

[0041] The present invention selects a partial fragment of type I collagen α1 chain 1 / 4 ND fragment (its amino acid sequence is shown in SEQ ID NO.3) as the core template for design and modification. In order to enhance its structural stability, the following modification strategies are adopted: (1) two functional sequences screened from Streptococcus Scl2 collagen-like protein are inserted into the N-terminus; (2) four groups of charged GPP repeating units are introduced into the C-terminus to enhance the charge interaction. The structural design is as follows: Figure 1 As shown, the two amino acid sequences obtained are shown as SEQ ID NO.1 or SEQ ID NO.2, wherein the amino acid sequence SEQ ID NO.1 is called 1-1 / 4ND, and the amino acid sequence SEQ ID NO.1 is called 2-1 / 4ND.

[0042] Example 1

[0043] 1. Transform the plasmid pPIC9k containing the target gene with the amino acid sequence shown in SEQ ID NO. 1 or SEQ ID NO. 2 into competent E. coli cells, spread the plasmid onto LB plates containing 100 μg / mL Amp, and culture at 37°C overnight. Select positive clones and send them to RuiMian for sequencing verification. Finally, select the colonies with correct sequencing results to expand the culture and extract the plasmid.

[0044] 2. Electroporation transformation of Pichia pastoris strain GS115

[0045] The plasmid pPIC9k containing the target gene was linearized by digestion with SacI and incubated in a water bath at 37°C for 15 min. The linearization reaction system is shown in Table 1.

[0046] Table 1 Linearization reaction system

[0047]

[0048] After linearization with SacI, the plasmid was transformed into Pichia pastoris GS115 competent cells by electroporation. The transformation products were spread onto MD solid medium containing 200 μg / mL and 1 mg / mL G418, respectively, and incubated inverted at 30°C for 3 days. Yeast colonies observed after incubation exhibited typical morphological characteristics: milky white, smooth, and approximately 1-2 mm in diameter.

[0049] 3. Colony PCR

[0050] To identify positive clones, five single colonies with good morphology were selected from each pPIC9k-transformed GS115 plate containing 1 mg / mL and 200 μg / mL G418 for colony PCR verification.

[0051] The results of KOD enzyme amplification are as follows Figure 2 As shown in the figure, M represents DNA marker. Lanes 1-5 show PCR products from colonies screened on a 1 mg / mL G418 plate, and lanes 6-10 show PCR products from colonies screened on a 200 μg / mL G418 plate. The results show that all samples contained a single, bright amplified band of the expected size, indicating successful transformation.

[0052] 4. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE)

[0053] By comprehensively analyzing antibiotic resistance and colony PCR band intensities, the best positive transformants were screened for expansion. The specific process is as follows: First, the selected strain was inoculated into 200 mL of BMGY liquid medium. When the OD600 reached 2-6, the strain was transferred to 200 mL of BMMY medium for induction with 1% methanol. Samples were collected at 24, 48, 72, and 96 hours after induction and analyzed by SDS-PAGE.

[0054] The expression of original natural collagen fragments is as follows Figure 3 As shown in Figure 2, it can be seen that obvious degradation bands appeared after 2 days of induction. The induced expression results of the two engineered strains after transformation are shown in Figure 2. Figure 4 As shown, SDS-PAGE electrophoresis analysis detected significant target protein bands in both samples. (A) shows the 1-1 / 4ND amplification result, and (B) shows the 2-1 / 4ND amplification result. M: prestained protein marker, 0: supernatant of uninduced strain, 1: supernatant of strain induced for 1 day, 2: supernatant of strain induced for 2 days, 3: supernatant of strain induced for 3 days, and 4: supernatant of strain induced for 4 days. Analysis shows that the 1-1 / 4ND and 2-1 / 4ND variants exhibit significant protein accumulation and significantly improved degradation. This result confirms that the structural modification strategy can effectively alleviate the degradation problem of the 1 / 4ND fragment.

[0055] 5. Sequence analysis

[0056] First, the amino acid sequence of the target collagen protein (FASTA format) was obtained from the NCBI Protein database (https: / / www.ncbi.nlm.nih.gov / protein). The FASTA sequence was then input into the online tool ExPASy ProtParam (https: / / web.expasy.org / protparam / ) to calculate the predicted isoelectric point (pI) and molecular weight. The predicted pI and molecular weight are shown in Table 2.

[0057] Table 2 Sequence isoelectric point and molecular weight prediction results

[0058]

[0059] 6. Protein purification

[0060] According to the prediction results of the protein isoelectric point, purification was carried out using a buffer with a pH of 6.5.

[0061] Ion exchange column treatment: First, according to the predicted isoelectric point of the target protein, an anion exchange strain (Histrap QHP 5ml) was used to remove most of the nucleic acids and impurities, and then a cation exchange column (Histrap SP HP 1ml) was used to separate and purify the target protein.

[0062] SDS-PAGE verification analysis: The unpurified fermentation supernatant sample and the collected purified protein sample were subjected to SDS-PAGE comparative analysis. The specific method is as follows: Figure 5 As shown, the gel image of the purification process is as follows Figure 6 M: Prestained protein marker; 0: Uninduced yeast supernatant; NQ: Supernatant sample before membrane pack concentration; NS: Supernatant sample after membrane pack concentration; NC: Reconstituted sample after membrane pack concentration; TS: Retained supernatant sample after dialysis after concentration; TC: Reconstituted sample after dialysis of the precipitate; YS: Flow-through of anion exchange column (loaded on cation exchange column); YC: Breakthrough of cation exchange column; 6-9: Elution samples from cation exchange column (different NaCl gradient elution).

[0063] (1) Purification of 1-1 / 4ND after transformation

[0064] The results of the HiTrap HP SP cation exchange chromatography purification of recombinant collagen 1-1 / 4ND are as follows Figure 7As shown in the figure, the elution curve shows that a main characteristic peak (absorbance of approximately 220mAU) appears in the elution volume range of 2-6mL. SDS-PAGE analysis confirms that tubes 7, 8, and 9 corresponding to this peak contain 1-1 / 4ND of the target protein at high concentration but with degradation. This unique bimodal elution feature indicates the possible existence of two different binding states or conformations. This purification method successfully achieves efficient enrichment and purification of the target protein.

[0065] The recombinant protein purification process was analyzed by SDS-PAGE. Figure 8 As shown in the figure, 6-9 are cation exchange column elution samples (different NaCl gradient elution). As can be seen from the figure, the target protein content in the initial sample (NQ) is low, and it is enriched after membrane concentration (NS), but it is not detected in the concentrated precipitate (NC) or dialyzed precipitate (TC), indicating that the protein has good solubility. The results of cation exchange chromatography showed that the target protein was specifically enriched in elution tubes 7-9 and was not detected at all in the penetration component (YC), indicating a significant purification effect. A clear and single target band was observed at approximately 35kDa, which is consistent with the theoretical molecular weight higher than 1-1 / 4ND, which is related to the excessively high content of proline. It was confirmed that this method successfully obtained a high-purity protein.

[0066] (2) Purification of 2-1 / 4ND

[0067] The results of the HiTrap HP SP cation exchange chromatography purification of recombinant collagen 2-1 / 4ND are as follows Figure 9 The elution curve shows two characteristic peaks within the 2-10 mL elution volume range: a narrow front peak with high absorbance (approximately 70 mAU) and a broad rear peak with low absorbance (approximately 45 mAU). SDS-PAGE analysis confirmed that tubes 9, 10, and 11, corresponding to the second major peak, contained 2-1 / 4 ND of the target protein at high purity and concentration. This result demonstrates that the purification protocol effectively isolates the target protein.

[0068] The recombinant protein purification process was analyzed by SDS-PAGE. Figure 10 As shown. Figures 8-11 represent cation exchange column elution samples (different NaCl gradient elution). As can be seen from the figure, the target protein content in the initial sample (NQ) is low, and it is enriched after membrane concentration (NS), but it is not detected in the concentrated precipitate (NC) or dialyzed precipitate (TC), indicating that the protein has good solubility. The results of cation exchange chromatography showed that the target protein was specifically enriched in elution tubes 9-11, and was not detected at all in the penetration component (YC), indicating a significant purification effect. A clear and single target band was observed at approximately 35 kDa, which is consistent with a theoretical molecular weight higher than 2-1 / 4 ND, which is related to the excessively high content of proline. It was confirmed that this method successfully obtained a high-purity protein.

[0069] Example 2 Characterization of recombinant protein properties

[0070] First, circular dichroism (CD) spectroscopy was used to examine the characteristic spectra within the 190-250nm wavelength range to analyze the formation of the triple helical structure. Denaturing CD experiments were then used to assess the thermal stability of the protein by monitoring the changes in characteristic peaks with temperature. Cryo-electron microscopy was also used to observe the assembly morphologies of different humanized collagens and their chimeras.

[0071] 1. Circular dichroism (CD) test

[0072] Before the experiment, the purified protein sample was kept at 0°C overnight to ensure that the protein was correctly folded. A Jasco J-1500 circular dichroism spectrometer was used for detection, and a quartz cuvette with a 1mm optical path was selected to scan in the wavelength range of 190-250nm. Focus on monitoring the characteristic peak signal at 225nm. The change in ellipticity at this wavelength can reflect the presence or absence of a triple helix structure. Each sample was scanned 3 times and the average value was taken to improve data reliability. Data processing: Origin 2021 software was used to process and analyze circular dichroism data. First, the raw data output by the instrument was imported into the workbook, and the fast Fourier transform (FFT) filter was used for data smoothing. The window width was set to 20 data points to retain the effective spectral features, and the smoothed ellipticity data (unit: mdeg) was baseline corrected.

[0073] The CD spectrum of native collagen typically exhibits a negative absorption peak at approximately 205 nm, due to the unique random coil structure within the collagen molecule. A positive absorption peak at 225 nm is attributed to the presence of polyproline secondary structures, which correlates with the level of triple helix formation in the collagen. During collagen denaturation, the absorption peak decreases with increasing denaturation, eventually falling below zero. To further verify whether the expressed collagen fragment can self-assemble into a triple superhelical structure, circular dichroism spectroscopy was performed on its aqueous solution.

[0074] The circular dichroism analysis results of the original recombinant collagen before modification are as follows Figure 11 The circular dichroism analysis results of the modified chimeric collagen fragment and the original fragment are shown in Figure 12 As shown, (A) CD spectra comparison of the modified 1-1 / 4ND and original 1 / 4ND; (B) CD spectra comparison of the modified 2-1 / 4ND and original 1 / 4ND. The results show that the triple helical structure characteristics of the modified 1-1 / 4ND and 2-1 / 4ND chimeras are significantly enhanced, with the positive ellipticity values ​​at 225nm increasing from 10 to 20 and 30, respectively (increases of 100% and 200%), indicating a significant improvement in triple helical conformational stability. The 2-1 / 4ND variant, in particular, exhibits superior structural stability.

[0075] This difference in structural stability is highly consistent with its biological properties: the 1-1 / 4ND and 2-1 / 4ND variants showed significantly enhanced resistance to protease degradation. These results confirm a direct positive correlation between the robustness of the triple helical conformation and its biological stability, demonstrating that a stable triple helical structure effectively resists protease attack.

[0076] 2. Variable temperature CD

[0077] Before the experiment, the purified protein sample was kept at 0°C overnight to ensure structural integrity. During the test, a continuous scan was performed over the 0-50°C temperature range at a heating rate of 0.5°C / min. Data was collected after 1 minute of equilibration at each temperature point, focusing on changes in ellipticity at 205nm (a peak characteristic of secondary structure) and 225nm (a peak characteristic of triple helix structure). 10mM PBS buffer (pH 6.5) was used as the solvent system throughout the test.

[0078] Data processing: This study used variable temperature circular dichroism spectroscopy to analyze protein conformational transitions. First, the raw data were fitted with a Boltzmann nonlinear equation: y = A2 + (A1-A2) / [1+exp((TT m ) / B)], where A1 and A2 represent the baseline values ​​after denaturation and native state, respectively, T m The fitting process uses the nonlinear curve fitting tool of Origin software to optimize the parameters by the least square method to ensure the goodness of fit R 2 >0.95. The first-order derivative (dθ / dT) was then calculated based on the fitting curve using the Savitzky-Golay differential method (window number of 5 points) to reduce noise interference. The minimum value of the derivative curve corresponds to the inflection point temperature (T m ), which is consistent with the T obtained by Boltzmann fitting. m Parameters are mutually validated. By analyzing the extreme position and peak shape characteristics of the derivative curve, the thermal stability of the protein and the cooperativity of its conformational transitions can be accurately determined. All data processing is completed on the Origin 2021 software platform to ensure the repeatability and accuracy of the analysis process.

[0079] To investigate the effect of temperature on the triple helix structure of collagen, the circular dichroism (CD) signal changes at characteristic wavelengths (225 nm and 205 nm) of the collagen fragments were monitored in real time using a temperature control device (temperature range: 0–50°C, heating rate: 0.5°C / min). The results showed that as the temperature increased, the positive ellipticity at 225 nm decreased significantly, indicating unwinding of the triple helix structure; while the negative peak at 205 nm showed an increasing trend, further confirming the conformational transition. This result is consistent with the typical thermal denaturation characteristics of collagen and confirms the temperature-dependent differences in conformational stability.

[0080] (1) Absorption at 225 nm changes with temperature

[0081] The thermal denaturation curve of the original collagen fragment at 225nm is as follows Figure 13 As shown in Figure 2, the thermal denaturation curve of the modified chimeric collagen fragment at 225 nm is as follows: Figure 14 (A) shows the thermal denaturation curve of chimera 1-1 / 4 ND at 225 nm, and (B) shows the thermal denaturation curve of chimera 2-1 / 4 ND at 225 nm. The results show that the triple helical characteristic peak at 225 nm for all tested sequences exhibits a typical steep unfolding curve as the temperature increases, and their phase transition behavior is highly consistent with the thermal denaturation characteristics of native collagen.

[0082] (2) Absorption first derivative at 225 nm

[0083] By performing nonlinear curve fitting and derivative analysis on the curve of the characteristic peak at 225nm changing with temperature, the temperature corresponding to the minimum point of the derivative curve is the triple helix denaturation temperature (Tm). This parameter represents the critical temperature at which the triple helix structure is completely unwound. Only collagen fragments with the ability to independently form a triple helix structure will exhibit a significant Tm value.

[0084] The first-order derivative of thermal denaturation of the original recombinant collagen fragment at 225 nm is as follows Figure 15 As shown, the thermal denaturation temperature of 1 / 4 ND was 17.3°C.

[0085] The first derivative of thermal denaturation of the modified chimeric collagen fragment at 225 nm is as follows: Figure 16As shown, (A) Comparison of the first-order derivative curves of thermal denaturation at 225 nm for the modified 1-1 / 4ND and the original 1 / 4ND (B) Comparison of the first-order derivative curves of thermal denaturation at 225 nm for the modified 2-1 / 4ND and the original 1 / 4ND. Analysis shows that 1-1 / 4ND and 2-1 / 4ND exhibit significantly improved triple-helix thermal stability, with Tm values ​​reaching 22.6°C and 23.0°C, respectively, which are 30.6% and 33.0% higher than the original 1 / 4ND (17.3°C). This result validates the effectiveness of the "Scl2 stabilizing domain + GPP modification" synergistic strategy, indicating that the stabilizing function of hydroxyproline can be partially replaced by introducing the highly charged interaction network of bacterial collagen.

[0086] 3. Cryo-electron microscopy

[0087] The morphology of the three collagen fragments was observed using cryo-electron microscopy. The specific method is as follows: the purified collagen sample was concentrated by centrifugation through an ultrafiltration tube and then equilibrated at 0°C for 24 hours to ensure the formation of the triple helical structure.

[0088] The grid used was a Quantifoil (R1.2 / 1.3, Au, 200 mesh) gold grid. Before preparing cryo-EM samples, the grid carrying the sample needed to be hydrophilized. Glow discharge was performed using a mixed gas of different components as a medium to make the grid surface carry an electric charge and promote the full spreading of the sample solution. The hydrophilic treatment conditions were: 25 mA, 60 s.

[0089] Take 2.5 μL of sample and drop it vertically onto the grid. After standing for 1 minute, move to Vitrobot Mark IV for freezing sample preparation. Set the sample preparation conditions as follows: humidity: 100%, temperature: 20°C, Blot Force: 0, Blot Time: 3.5s, Wait Time: 1s, Drain Time: 0.5s, Blot Total: 1. Transfer the prepared sample to a liquid nitrogen tank and freeze it for later use.

[0090] Electron microscopy observations were performed using a 200 kV cryo-transmission electron microscope (Glacios) (Analytical Testing Center, Shanghai Jiao Tong University) with an operating voltage of 200 kV and low-dose mode. Images were acquired at a magnification of SA73,000×, using a Gatan 626 cryostage (-170°C) to maintain the sample frozen. Images were acquired using a FEI Eagle 4k×4k CCD camera with a 1-second exposure time. Contrast was optimized by manually adjusting the defocus value (1.5-2.5 μm). At least five different areas of each sample were imaged to ensure representative data.

[0091] Cryo-electron microscopy images of the original collagen fragments before transformation Figure 17 As shown, the cryo-electron microscopy image of the modified chimeric collagen fragment is shown in Figure 18 As shown, (A) 1-1 / 4ND cryo-EM image, (B) 2-1 / 4ND cryo-EM image. The results show that 1 / 4ND, 1-1 / 4ND and 2-1 / 4ND all formed obvious filamentous structures.

[0092] Compared to pre-modification, the 1-1 / 4ND and 2-1 / 4ND filaments were more uniform and longer, with fewer short fragments, indicating improved protein degradation and enhanced self-assembly. The improved thermal stability is speculated to be due to the addition of salt bridges (such as lysine-glutamate pairs) and hydration networks in the chimera. These interactions compensate for the lack of hydroxylation modifications in microbial expression systems, providing new ideas for the rational design of collagen.

[0093] Using dynamic simulations to screen for highly stable fragments and integrate them into the native sequence, characterization was performed using circular dichroism spectroscopy fitting analysis, first-order derivative analysis of variable-temperature experiments, and rapid cryo-electron microscopy sample preparation. This confirmed that the chimeric design based on the Scl2 stabilization module effectively improved the conformational stability and degradation resistance of the collagen fragment. The recombinant protein described in this invention does not require hydroxylase modification, thus eliminating the hydroxylation modification step. This rational design not only fully preserves the structural characteristics of native collagen, but also significantly improves the thermal stability of the triple helical structure, providing a new molecular design strategy for the development of high-performance collagen materials.

[0094] The above embodiments are only used to illustrate the present invention, rather than to limit the present invention. Those skilled in the art may make various changes or modifications without departing from the spirit and scope of the present invention. Therefore, all equivalent technical solutions should also fall within the scope of the present invention and should be defined by the claims.

Claims

1. A highly stable non-hydroxylase-modified recombinant humanized collagen, characterized in that: The amino acid sequence thereof includes one of the amino acid sequences shown in SEQ ID NO.1 or SEQ ID NO.

2.

2. A gene sequence encoding the amino acid sequence according to claim 1.

3. A recombinant vector or recombinant engineered bacterium carrying a gene sequence encoding the amino acid sequence according to claim 1.

4. A method for preparing the highly stable non-hydroxylase-modified recombinant humanized collagen according to claim 1, characterized in that: The method comprises the following steps: synthesizing a gene sequence encoding the amino acid sequence according to claim 1, connecting the gene sequence to a vector, transforming the gene sequence into an engineering strain, and constructing a recombinant genetically engineered bacterium; The recombinant genetic engineering bacteria constructed by expression are collected, the supernatant is collected by centrifugation, and the non-hydroxylase modified recombinant humanized collagen is obtained after purification.

5. Use of the highly stable non-hydroxylase-modified recombinant humanized collagen according to claim 1 in the preparation of biomaterials.

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