A recombinant collagen, freeze-dried fiber and its preparation method

Abstract

This invention relates to recombinant collagen, lyophilized fibers, and their preparation method, belonging to the field of biomedical technology. The recombinant collagen has the amino acid sequence shown in SEQ ID NO. 8. Addressing the issue of endotoxin residue in recombinant collagen, this invention constructs an amino acid sequence with added His tags and enzyme digestion sequences from the source. This allows for effective separation of recombinant collagen and endotoxins during subsequent separation processes, and the added sequences can be removed by enzymatic digestion during subsequent purification, further improving protein purity. Through a multi-level endotoxin control strategy of "source burden reduction—core separation—deep removal," the endotoxin content of lyophilized collagen fibers can be reduced to injection standards.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a recombinant collagen, freeze-dried fiber, and its preparation method. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Collagen is the most widely distributed and abundant functional protein in animals. Its basic structural unit is highly conserved, consisting of three polypeptide chains with repeating Gly-XY sequences, wound together in a right-handed helical manner to form a stable triple helix structure. Collagen extracted from connective tissues such as skin, bones, and tendons of animals such as pigs, cattle, and sheep, as a material-derived extract, carries the risk of contamination by pathogens (such as viruses and prions), and its quality is inconsistent between batches.

[0004] Recombinant collagen prepared using engineered strains such as *E. coli* through high-density fermentation, separation, extraction, and purification processes overcomes the inherent limitations of animal-derived products in terms of quality uniformity and biosafety. However, in its transformation into high-end medical products (such as freeze-dried fiber), protein purity and endotoxin residues have become key bottlenecks restricting its application. Endotoxins are components of the cell walls of Gram-negative bacteria and are easily introduced along with the target product in expression systems using *E. coli* as the host. Even trace residues can induce fever, inflammatory responses, or even systemic toxicity, seriously threatening the biosafety of the product. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a recombinant collagen, lyophilized fibers, and a method for their preparation. Specific active sequences are selected from the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein, and tags and enzyme digestion sequences are added. By optimizing the host bacterial sequence and integrating multi-step chromatographic purification steps, the intracellular toxin content is reduced and the collagen purity is improved, resulting in lyophilized triple helix recombinant humanized collagen fibers that meet the stringent requirements for biomedical materials.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In a first aspect, there is a recombinant collagen having an amino acid sequence as shown in SEQ ID NO.8.

[0007] Secondly, a polynucleotide encoding the aforementioned recombinant collagen.

[0008] Thirdly, a recombinant expression vector having the aforementioned polynucleotides.

[0009] Fourthly, a host bacterium containing the above-mentioned recombinant expression vector, or integrating the above-mentioned polynucleotides, or expressing the above-mentioned recombinant collagen.

[0010] Fifthly, a freeze-dried fiber is prepared from the aforementioned recombinant collagen.

[0011] Sixthly, the method for preparing the above-mentioned freeze-dried fiber includes: culturing the above-mentioned host bacteria to express the recombinant collagen, sequentially performing separation and purification, protease cleavage to remove heterologous sequences and ultrafiltration concentration to obtain a protein aqueous solution, and freeze-drying the protein aqueous solution to obtain the freeze-dried fiber.

[0012] The above one or more technical solutions have the following beneficial effects: This invention addresses the issue of endotoxin residue in recombinant collagen by constructing an amino acid sequence with added His tags and enzyme digestion sequences at the source. This rationally constructs the recombinant collagen sequence, resulting in a protein with high expression levels and high purity. This allows for effective separation of recombinant collagen and endotoxins during subsequent isolation processes. Furthermore, the added sequences can be removed through enzymatic digestion during purification, further improving protein purity. Multi-stage purification techniques, such as nickel column + cation exchange for core separation and tag excision and ultrafiltration for deep removal, are then employed. This multi-stage endotoxin control strategy, based on the amino acid sequence of recombinant collagen, reduces the endotoxin content of freeze-dried collagen fibers to injectable standards, significantly reducing the risk of endotoxin-induced inflammatory reactions in clinical use, improving product safety, and expanding application areas. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 This is the electrophoretic pattern of recombinant collagen in the supernatant of Comparative Example 1 of the present invention.

[0015] Figure 2 The images shown are electrophoretic patterns of recombinant collagen before and after nickel column purification in Comparative Example 2 of this invention. In the images, A is the total bacterial protein sample, B is the sample before nickel column purification, C is the breakthrough buffer after nickel column purification, and D is the elution buffer after nickel column purification.

[0016] Figure 3The images shown are electrophoretic patterns of recombinant collagen before and after nickel column purification in Comparative Example 3 of this invention. In the images, A is the sample before nickel column purification, B is the permeate after nickel column purification, and C is the elution buffer after nickel column purification.

[0017] Figure 4 This is an electrophoretic pattern of recombinant collagen after enzyme digestion in Comparative Example 3 of the present invention, where A is the sample before enzyme digestion and B is the sample after enzyme digestion.

[0018] Figure 5 The images shown are electrophoretic patterns of recombinant collagen before and after nickel column purification in Example 1 of this invention. In the images, A is the total bacterial protein sample, B is the sample before nickel column purification, C is the breakthrough solution after nickel column purification, and D is the elution solution after nickel column purification.

[0019] Figure 6 This is an electrophoretic pattern of recombinant collagen before and after cation exchange chromatography in Example 1 of the present invention, wherein A is the sample before purification, B is the flow-through buffer after affinity chromatography purification, and C is the elution buffer after affinity chromatography purification.

[0020] Figure 7 This is an electrophoretic pattern of recombinant collagen after enzyme digestion in Example 1 of the present invention, where A is the sample before enzyme digestion and B is the sample after enzyme digestion.

[0021] Figure 8 This is a photograph of the freeze-dried fiber obtained in Example 1 of the present invention.

[0022] Figure 9 This is a high-performance liquid chromatography (HPLC) spectrum of the sample after enzyme digestion in Example 1 of the present invention.

[0023] Figure 10 This is the circular dichroism spectrum of the sample after enzyme digestion in Example 1 of the present invention.

[0024] Figure 11 Cell viability detection diagram of freeze-dried fibers in Example 1 of this invention. Detailed Implementation

[0025] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer.

[0026] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0027] One or more embodiments of the present invention provide a recombinant collagen having an amino acid sequence as shown in SEQ ID NO.8.

[0028] This is a recombinant humanized type III collagen obtained by selecting specific active sequences from the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein and adding tags and restriction enzyme sequences. The theoretical molecular weight is approximately 48 kDa. It contains a His tag and restriction enzyme sequences. The His tag is used to specifically capture the recombinant collagen and remove impurities and bound endotoxins. The restriction enzyme sequences provide sites for cleaving the His tag, achieving separation between the His tag and the recombinant collagen. This ensures that only pure recombinant collagen remains during subsequent purification, reducing the endotoxin content and improving protein purity.

[0029] One or more embodiments of the present invention provide a polynucleotide that encodes the above-described recombinant collagen.

[0030] Optionally, the polynucleotide has a nucleotide sequence as shown in SEQ ID NO.4.

[0031] Optionally, it includes a protein that encodes the same amino acid sequence as the nucleotide sequence shown in SEQ ID NO.4, but is different in sequence due to the degeneracy of the genetic code.

[0032] One or more embodiments of the present invention provide a recombinant expression vector having the above-described polynucleotides.

[0033] The recombinant expression vector is obtained by effectively linking the above-mentioned polynucleotide sequence to an expression vector, wherein the expression vector is a plasmid, specifically one of pET-28a, pET22a, and pET32a.

[0034] One or more embodiments of the present invention provide a host bacterium containing the above-mentioned recombinant expression vector, or integrating the above-mentioned polynucleotide, or expressing the above-mentioned recombinant collagen.

[0035] Optionally, the host bacterium is Escherichia coli.

[0036] Optionally, the host bacterium is Escherichia coli BL21(DE3).

[0037] One or more embodiments of the present invention provide a freeze-dried fiber prepared from the above-mentioned recombinant collagen.

[0038] The resulting freeze-dried fibers have the advantages of high purity, low immunogenicity, and high bioactivity, and can be used to prepare medical devices.

[0039] One or more embodiments of the present invention provide a method for preparing the above-mentioned freeze-dried fiber, comprising: culturing the above-mentioned host bacteria, causing the host bacteria to express the recombinant collagen, and then sequentially performing separation and purification, protease cleavage to remove heterologous sequences and ultrafiltration concentration to obtain a protein aqueous solution, and freeze-drying the protein aqueous solution to obtain the freeze-dried fiber.

[0040] In the above process, the tag and enzyme cleavage site themselves do not directly degrade / adsorb endotoxins; the His tag can indirectly reduce the endotoxin content of the final product by changing the protein purification behavior, optimizing chromatographic separation, and increasing the endotoxin removal step window; the enzyme cleavage site is used to provide the tag excision site, and the residual endotoxin adsorbed on the His tag is removed by secondary chromatography, while eliminating the electrostatic adsorption site of the tag itself, ensuring that the final tag-free recombinant collagen meets the endotoxin standard.

[0041] Optionally, the separation and purification method includes, in sequence, nickel column purification and cation exchange layer purification.

[0042] The method for excising heterologous sequences with protease includes treatment with TEV enzyme for 5.5 to 6.5 hours.

[0043] The present invention will be further described below with reference to specific embodiments and comparative examples.

[0044] Example 1 In the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein, a specific active sequence was selected and an 8×His tag and a TEV enzyme cleavage site were added to obtain a recombinant humanized type III collagen protein with an amino acid sequence as shown in SEQ ID NO.8, with a theoretical molecular weight of approximately 48 kDa.

[0045] The sequence encoding this protein was codon-optimized to obtain the nucleotide sequence shown in SEQ ID NO.4. After full-gene synthesis, it was ligated into the pET28a plasmid to construct the recombinant expression plasmid. The recombinant expression plasmid was transformed into E. coli BL21(DE3) competent cells, plated on LB solid medium containing kanamycin, and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking until OD. 600The concentration was 0.5, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 37°C for another 5 h, and the bacterial pellet was collected by high-speed centrifugation.

[0046] The bacterial cell precipitate was resuspended in Buffer A (50 mM Tris, 250 mM NaCl, pH 8.0), incubated on ice for 1 h, homogenized using a high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation. The supernatant was purified by nickel affinity chromatography: the column was equilibrated at 50 mM Tris, 250 mM NaCl, and pH 8.0, and the target protein was eluted at 50 mM Tris, 400 mM NaCl, and pH 8 to obtain the primary purified protein.

[0047] The solution of the primary purified protein was placed into a dialysis bag (8-14 KD, Beijing Solarbio Science & Technology Co., Ltd., dialysis bag MD25) and dialyzed to 50 mM Tris (pH 8.0), with the medium changed every 6 hours. After dialysis, further purification was performed using cation exchange chromatography: the chromatography column was equilibrated at 50 mM Tris, and contaminating proteins were washed with 50 mM Tris, 75 mM NaCl, and pH 8. The target protein was then eluted with 50 mM Tris, 400 mM NaCl, and pH 8, resulting in the secondary purified protein.

[0048] Then, TEV Plus Protease (His-tag) enzyme from Beyotime was added directly to the purified protein solution after elution, and the protein was digested at 30°C for 6 hours to completely remove the His tag and obtain the target protein after digestion.

[0049] The enzyme-digested protein solution was treated using a tangential flow ultrafiltration system (Hangzhou Kebote Filter Material Co., Ltd.) to remove small molecule impurities and enzyme fragments. Simultaneously, buffer replacement and sample concentration were performed to obtain a high-concentration pure protein aqueous solution. This pure protein aqueous solution was then freeze-dried to obtain the product shown below. Figure 8 The freeze-dried fiber shown is recombinant humanized type III collagen freeze-dried fiber.

[0050] The purification effect of recombinant collagen using nickel column electrophoresis was analyzed using SDS-PAGE. The electrophoresis results are shown below. Figure 5 As shown in the electrophoresis pattern, the main band of the purified sample in lane A is located at approximately 48 kDa, consistent with the theoretical molecular weight, indicating successful expression of the target protein. During nickel column affinity chromatography, the target band was effectively eluted, and... Figure 5 As shown in Figure C, there was almost no target protein residue in the permeate, indicating that the increase in the number of His tags further improved the binding ability of the nickel column. The target protein had a high binding efficiency with the nickel column, and the loss during the purification process was extremely low.

[0051] The electrophoretic pattern after further purification by cation exchange chromatography is as follows: Figure 6 As shown, Figure 6 The target protein was not detected in the permeate solution shown in B, indicating that the target protein was well bound to the cation column, there was no permeation loss during the loading process, and the purification effect was ideal.

[0052] Subsequent enzyme digestion results are as follows Figure 7 As shown, after treatment with TEV protease, the molecular weight of the target protein decreased from 48 kDa to approximately 42 kDa, which is consistent with the expected molecular weight after removing the 8×His tag and other heterologous sequences. This indicates that the His tag has been completely removed, and the obtained target protein after enzyme digestion is recombinant collagen without heterologous sequences.

[0053] The purity of the enzyme-digested samples was analyzed by high-performance liquid chromatography (HPLC). The chromatographic conditions were as follows: an SEC column (purchased from Waters; column model XBridge BEH125A SEC 3.5um) was used, with 50 mM phosphate buffer (containing 100 mM NaCl, pH 7.2) as the mobile phase, a flow rate of 1 mL / min, and a detection wavelength of 230 nm. The results are as follows. Figure 9 As shown, the target protein after enzymatic digestion exhibits a single symmetrical chromatographic peak with an integrated purity of 99%, indicating that the purification process can effectively remove host proteins and other impurities. The high-purity recombinant collagen lays a solid foundation for the product quality of subsequent freeze-dried fibers.

[0054] In addition, circular dichroism spectroscopy was used to detect the higher-order structures of the enzyme-digested samples. The specific test conditions were: bandwidth 1 nm, response time 1 s, and scan range 190°. 250 nm, scan rate 100 nm / min, solvent: deionized water; results are as follows Figure 10 As shown, the purified recombinant collagen exhibits typical collagen triple helix characteristic peaks in the near-ultraviolet region, indicating that it retains an active conformation similar to that of natural collagen. This structural integrity is of great significance for ensuring the biological function of freeze-dried fibers and the quality of the final product.

[0055] Simultaneously, the endotoxin content at each stage of recombinant collagen purification was detected. The endotoxin content was determined using the Limulus Amebocyte Lysate (LAL) microgel method, with the reagent being the Beyotime endotoxin detection kit. Endotoxin detection at each purification stage was performed according to the kit instructions, and the results are shown in Table 1. Tables 1, 2, and 3 represent three parallel experiments. The results show that after multi-stage purification, the endotoxin content of the final product consistently decreased to below 0.1 EU / mL, indicating that the preparation method has good process stability and endotoxin removal capability, and can repeatedly produce low-endotoxin lyophilized fibers that meet the requirements of high-end medical applications.

[0056] Table 1. Endotoxin test results at each stage

[0057] The cell viability of the obtained lyophilized fibers was evaluated by seeding mouse fibroblast L929 cells into 24-well culture plates at a density of 5 × 10⁶ cells per well. 4 Cells were cultured at 37°C for 24 hours, and then co-cultured with the recombinant humanized type III collagen lyophilized fibers prepared in this example and commercially available collagen. Cell adhesion rate was detected using the CCK-8 assay at 1, 3, and 5 days to assess cell OD. 450 absorbance in nm, results as follows Figure 11 As shown. Compared with commercially available recombinant collagen freeze-dried fibers, the recombinant humanized type III collagen freeze-dried fibers prepared in this embodiment exhibit significantly higher cell adhesion, indicating that they can effectively promote cell adhesion and spreading, and have good cell compatibility and biological activity.

[0058] The genetically engineered bacterial fermentation and recombinant expression system used in this embodiment eliminates the risk of contamination from animal-derived pathogens at the source and eliminates quality fluctuations caused by batch-to-batch differences in raw materials. The prepared recombinant humanized type III collagen has the outstanding advantages of 100% homology with human sequences and low immunogenicity. Combined with the precise control of its physicochemical properties in this embodiment, it becomes a stable, safe, and controllable "life material" that meets modern biomedical standards. During the purification and freeze-drying process, mild process conditions are used to maximize the protection of the triple helix conformation of the recombinant collagen composed of Gly-XY repeat sequences. This conformation is the basis for its biological functions such as cell recognition, adhesion, and signal transduction. At the same time, through multi-mode chromatography, this embodiment achieves precise separation of the target protein from host proteins, nucleic acids, pigments, and other impurities, significantly improving the protein purity of the obtained freeze-dried fibers and ensuring the quality uniformity and bioactivity of the final product. The prepared recombinant humanized collagen freeze-dried fibers have extremely low endotoxin content, which can meet the strict regulatory requirements of high-barrier medical devices such as injection filling and implant repair, significantly reducing the risk of fever and inflammatory reactions in clinical use, and removing key obstacles for the large-scale application of the product in the high-end medical field.

[0059] Comparative Example 1 In the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein, a specific active sequence was selected and a 6×His tag and a TEV enzyme cleavage site were added to obtain a recombinant humanized type III collagen protein with an amino acid sequence as shown in SEQ ID NO:5, with a theoretical molecular weight of approximately 48 kDa.

[0060] The protein-encoding sequence was codon-optimized to obtain the nucleotide sequence shown in SEQ ID NO.1. After completing the full gene synthesis, it was ligated into the pET28a plasmid to construct the recombinant expression plasmid. The recombinant expression plasmid was transformed into E. coli BL21(DE3) competent cells, plated on LB solid medium containing kanamycin, and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking until OD. 600 The concentration was 0.5, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 37 °C for 5 h, and the bacterial cells were collected by high-speed centrifugation. The bacterial cells were resuspended in Buffer A (50 mM Tris, 250 mM NaCl, pH 8.0), then homogenized by high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation.

[0061] SDS-PAGE was used to detect the expression of recombinant collagen in the supernatant to evaluate the induction effect. Results are as follows: Figure 1As shown, no obvious induced expression band was observed in the sample lanes after induction, near the expected protein molecular weight. This result indicates that the target protein failed to be successfully expressed under these conditions in this comparative example.

[0062] Comparative Example 2 In the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein, a specific active sequence was selected and a 6×His tag and a TEV enzyme cleavage site were added to obtain a recombinant humanized type III collagen protein with an amino acid sequence as shown in SEQ ID NO.6, with a theoretical molecular weight of approximately 48 kDa.

[0063] The sequence encoding this protein was codon-optimized to obtain the nucleotide sequence shown in SEQ ID NO.2. After full-gene synthesis, it was ligated into the pET28a plasmid to construct the recombinant expression plasmid. The recombinant expression plasmid was transformed into *E. coli* BL21(DE3) competent cells, plated on LB solid medium containing kanamycin, and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking until OD (dose elapsed). 600 The concentration was 0.5, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 37°C for 5 h, and the bacterial cells were collected by high-speed centrifugation. The bacterial cells were resuspended in Buffer A (50 mM Tris, 250 mM NaCl, pH 8.0), then homogenized by high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation.

[0064] The supernatant was purified by nickel column affinity chromatography: the column was equilibrated with 50 mM Tris, 250 mM NaCl, and pH 8.0, and the target protein was eluted with 50 mM Tris, 400 mM NaCl, and pH 8.

[0065] The apparent molecular weight of recombinant collagen was analyzed using SDS-PAGE, and the electrophoresis results are shown below. Figure 2 As shown in the electrophoresis pattern, the main band of the purified sample in lane A is located at approximately 48 kDa, consistent with the theoretical molecular weight, indicating successful expression of the target protein. However, during nickel column affinity chromatography, although the target band was successfully eluted in lane D, a large amount of target protein residue was detected in lane C of the permeate, indicating that severe permeation occurred during the loading process in this comparative example, resulting in a significant loss of the target protein and ultimately severely impacting the purification yield.

[0066] Comparative Example 3 In the full-length triple helix region of the Homo sapiens COL3A1 (NP_000081) protein, a specific active sequence was selected and a 6×His tag was added to obtain a recombinant humanized type III collagen protein with an amino acid sequence as shown in SEQ ID NO.7, with a theoretical molecular weight of approximately 48 kDa.

[0067] The sequence encoding this protein was codon-optimized to obtain the nucleotide sequence shown in SEQ ID NO.3. After full-gene synthesis, it was ligated into the pET28a plasmid to construct the recombinant expression plasmid. The recombinant expression plasmid was transformed into *E. coli* BL21(DE3) competent cells, plated on LB solid medium containing kanamycin, and incubated overnight at 37°C. Single colonies were picked and inoculated into LB liquid medium containing kanamycin, and cultured at 37°C with shaking until OD (out of control). 600 The concentration was 0.5, and IPTG was added to a final concentration of 0.5 mM. Expression was induced at 37°C for 5 h, and the bacterial cells were collected by high-speed centrifugation. The bacterial cells were resuspended in Buffer A (50 mM Tris, 250 mM NaCl, pH 8.0), then homogenized by high-pressure homogenizer, and the supernatant was collected by high-speed centrifugation.

[0068] The supernatant was purified by nickel affinity chromatography: the column was equilibrated with 50 mM Tris, 250 mM NaCl, and pH 8.0, and the target protein was eluted with 50 mM Tris, 400 mM NaCl, and pH 8. The eluted target protein solution was placed into a dialysis bag (8-14 KD, Beijing Solarbio Technology Co., Ltd., dialysis bag MD25) and dialyzed to 50 mM Tris, with the medium changed every 6 hours. After dialysis, further purification was performed using cation exchange chromatography: the column was equilibrated with 50 mM Tris, and contaminating proteins were washed with 50 mM Tris, 75 mM NaCl, and pH 8, followed by elution with 50 mM Tris, 400 mM NaCl, and pH 8. TEV enzyme was then added directly to the eluted protein solution, and the protein was digested at 30°C for 6 hours for complete removal of the His tag.

[0069] The purification effect of recombinant collagen using nickel column chromatography was analyzed by SDS-PAGE, and the results are as follows: Figure 3 As shown in the electrophoresis pattern, the main band of the purified sample in lane A is located at approximately 48 kDa, consistent with the theoretical molecular weight, indicating successful expression of the target protein. During nickel column affinity chromatography, the target band was effectively eluted, and only a very small amount of the target protein was detected in the permeate, indicating good purification effect and high binding efficiency of the target protein to the nickel column.

[0070] However, subsequent enzyme digestion results were as follows Figure 4The results showed that the molecular weight of the target protein did not change significantly after treatment with TEV protease, and the band positions were consistent before and after digestion, indicating that the His tag and other components were not effectively removed. This result indicates that the recombinant collagen in this comparative example cannot be digested to obtain the tag-free natural form.

[0071] The sequence listings involved in the above embodiments and comparative examples are shown below.

[0072] SEQ ID NO.1:

[0073] SEQ ID NO.2:

[0074] SEQ ID NO.3:

[0075] SEQ ID NO.4:

[0076] SEQ ID NO.5: MGSSHHHHHHGSSENLYFQgpagekgetgapglkgengapgpmgprgapgapgrpglpgaagargndgargsdgqpgppgppgtagfpgspgakgevgpagspghagaqgppgapglmgargppgpagangapglrggagepgkngakgprgapgepganglpgaagergapgfrgpagpngipgekgpagergprgepgrdgvpgspggpgsdgkpgpp gsqgesgppgapgknglpggpgpqgppgkngetgpqgppgdkgdtgppgapgipgppglaggagppgpeggkgaagppgppgaagtpglqgmpgergglgspgpkgdkgepggpgadgvpgkdgprgptgpagqpgdkgeggapglpgiagprgspgppgetgppgapgqnggkgergapgekgegppgvagppgkdgtsghpgpigppgprgnrger。

[0077] SEQ ID NO.6: MGSSHHHHHHGSSENLYFQgpagekgetgapglkgengapgpmgprgapgergrpglpgaagargndgargsdgqpgppgppgtagfpgspgakgevgpagspghagaqgppgapglmgargppgpagangapglrggagepgkngakgprgergepganglpgaagergapgfrgpagpngipgekgpagergprgepgrdgvpgspggpgsdgkpgpp gsqgesgppgapgkngerggpgpqgppgkngetgpqgppgdkgdtgppgapgergppglaggagppgpeggkgaagppgppgaagtpglqgmpgergglgspgpkgdkgepggpgadgvpgkdgprgptgpagqpgdkgeggapglpgiagprgspgergetgppgapgqnggkggergapgekgegppgvagppgkdgtsghpgpigppgprgnrger。

[0078] SEQ ID NO.7: MGSSHHHHHHHHHGSsgpagekgetgapglkgengapgpmgprgapgergrpglpgaagargndgargsdgqpgppgppgtagfpgspgakgevgpagspghagaqgppgapglmgargppgpagangapglrggagepgkngakgprgergepganglpgaagergapgfrgpagpngipgekgpagergprgepgrdgvpgspggpgsdgkpgppgs qgesgppgapgkngerggpgpqgppgkngetgpqgppgdkgdtgppgapgergppglaggagppgpeggkgaagppgppgaagtpglqgmpgergglgspgpkgdkgepggpgadgvpgkdgprgptgpagqpgdkgeggapglpgiagprgspgergetgppgapgqnggkggergapgekgeggppgvagppgkdgtsghpgpigppgprgnrger。

[0079] SEQ ID NO.8: MGSSHHHHHHHHGSSENLYFQgpagekgetgapglkgengapgpmgprgapgergrpglpgaagargndgargsdgqpgppgppgtagfpgspgakgevgpagspghagaqgppgapglmgargppgpagangapglrggagepgkngakgprgergepganglpgaagergapgfrgpagpngipgekgpagergprgepgrdgvpgspggpgsdgkgp pgsqgesgppgapgkngerggpgpqgppgkngetgpqgppgdkgdtgppgapgergppglaggagppgpeggkgaagppgppgaagtpglqgmpgergglgspgpkgdkgepggpgadgvpgkdgprgptgpagqpgdkgeggapglpgiagprgspgergetgppgapgqnggkggergapgekgegppgvagppgkdgtsghpgpigppgprgnrger。

[0080] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A recombinant collagen protein, characterized in that, It consists of the amino acid sequence shown in SEQ ID NO.

8.

2. A polynucleotide, characterized in that, The polynucleotide encodes the recombinant collagen as described in claim 1.

3. The polynucleotide as described in claim 2, characterized in that, It consists of the nucleotide sequence shown in SEQ ID NO.4; Alternatively, a nucleotide sequence that encodes the same amino acid sequence as the nucleotide sequence shown in SEQ ID NO.4, but is different in sequence due to the degeneracy of the genetic code.

4. A recombinant expression vector, characterized in that, The recombinant expression vector has the polynucleotide as described in claim 2 or 3.

5. A host bacterium, characterized in that, The host bacterium contains the recombinant expression vector as described in claim 4, or integrates the polynucleotide as described in claim 2 or 3, or expresses the recombinant collagen as described in claim 1.

6. The host bacterium as described in claim 5, characterized in that, The host bacterium is Escherichia coli.

7. The host bacterium as described in claim 6, characterized in that, The host bacterium is Escherichia coli BL21(DE3).

8. A freeze-dried fiber, characterized in that, It is prepared from the recombinant collagen as described in claim 1.

9. A method for preparing freeze-dried fibers, characterized in that, include: After culturing the host bacteria as described in any one of claims 6-7 and expressing the recombinant collagen, the host bacteria are sequentially separated and purified, derivatized by protease digestion to remove the heterologous sequence, and concentrated by ultrafiltration to obtain a protein aqueous solution. The protein aqueous solution is then freeze-dried to obtain the freeze-dried fiber.

10. The method for preparing freeze-dried fibers as described in claim 9, characterized in that, The separation and purification methods include, in sequence, nickel column purification and cation exchange layer purification; the method for protease excision of heterologous sequences includes, in sequence, treatment with TEV enzyme for 5.5-6.5 h.

Images