Preparation method of culture medium for improving hydroxylation rate of recombinant collagen and application thereof
By optimizing the culture medium composition, the problem of low hydroxylation rate of recombinant collagen was solved, achieving a balance between efficient hydroxylation modification and cell growth, and providing an efficient and simple recombinant collagen production solution.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- 中原食品实验室
- Filing Date
- 2026-03-26
- Publication Date
- 2026-05-29
AI Technical Summary
Existing recombinant collagen production systems lack effective hydroxylation modification, resulting in low hydroxylation rates. Furthermore, high concentrations of NaCl inhibit bacterial growth, affecting protein yield and making it difficult to balance hydroxylation rates with bacterial growth.
An optimized culture medium containing tryptone, yeast extract, sodium chloride, hydroxyproline, sorbitol, ammonium sulfate, magnesium chloride, and calcium chloride was used. By adjusting the concentration of the components, the hydroxylation rate of recombinant collagen was significantly improved, while the cell density and viable cell count were increased.
Without affecting bacterial growth and protein yield, the hydroxylation rate of recombinant collagen is significantly increased to over 90%, with hydroxyproline accounting for about 20% of the protein's amino acid sequence, exceeding the hydroxylation level of natural collagen.
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Figure CN122104547A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing a culture medium that improves the hydroxylation rate of recombinant collagen and its application. Background Technology
[0002] Collagen is a major structural protein of the extracellular matrix in the human body, playing a crucial role in protecting tissue structure and regulating the cellular physiological environment. Collagen and its derivatives are widely used in cosmetics, pharmaceuticals, and tissue engineering due to their low immunogenicity and excellent cell compatibility. However, the functional properties of collagen are highly dependent on its unique triple helix structure, and the stability and function of this structure hinge on the hydroxylation modification of proline residues. Recombinant collagen refers to the use of genetic engineering techniques to insert gene fragments of human collagen into specific host cells (such as E. coli, yeast, and mammalian cells), allowing these cells to produce collagen fragments or peptides in large quantities, much like a "factory." Currently, expression systems mainly include E. coli and yeast cells, but efficient expression systems in E. coli and yeast lack the post-translational modification of collagen in animal cells, necessitating the introduction of corresponding hydroxylases. Therefore, researchers have adopted a method of co-expressing proline hydroxylase with collagen to address this issue. While exogenous introduction of hydroxylase genes can achieve hydroxylation modification in vivo, enabling recombinant collagen to reach the level of natural collagen, this approach is not feasible. However, multi-gene co-expression systems also have some problems. First, the technical difficulty is far greater than expressing only collagen genes, and they are prone to instability during long-term culture. Second, the catalytic efficiency is not ideal; the microenvironment within microbial cells (such as pH and cofactor concentration) may not be the optimal conditions for hydroxylases, leading to incomplete hydroxylation or poor site specificity. Moreover, forced expression of multiple exogenous proteins places a huge metabolic burden on the host cell, potentially causing slow cell growth and decreased protein yield. Studies have shown that hydroxyproline (Hyp) can be introduced into recombinant mammalian collagen sequences expressed in *E. coli* through co-translational incorporation of the proline (Pro) codon during fermentation. Adding NaCl can increase the uptake and utilization efficiency of Hyp in the culture medium by cells through hypertonic stimulation, thereby improving the hydroxylation rate. However, high concentrations of NaCl (>200 mM) severely inhibit bacterial growth activity, leading to a sharp drop in protein yield. At lower NaCl concentrations, the hydroxylation rate decreases significantly. To address the technical bottlenecks in existing recombinant collagen production systems, such as expression systems in Escherichia coli and yeast, which lack the ability to hydroxylate collagen through post-translational modification in animal cells, have limitations in existing hydroxylase co-expression strategies, and struggle to balance hydroxylation rate with cell growth, this invention provides a method for preparing a culture medium that improves the hydroxylation rate of recombinant collagen and its application, achieving synergistic optimization of high yield and high hydroxylation rate of recombinant collagen.
[0003] Technical problem to be solved: In view of the above-mentioned technical problems, the purpose of this invention is to provide a method for preparing a culture medium that improves the hydroxylation rate of recombinant collagen and its application. The culture medium can significantly improve the hydroxylation rate of recombinant collagen without causing toxic side effects to the bacteria, while promoting the increase of bacterial density and viable bacteria count, and without significantly affecting the collagen yield.
[0004] Technical solution: A culture medium for improving the hydroxylation rate of recombinant collagen, comprising the following components at the following concentrations: tryptone 10-15 g / L, yeast extract 5-8 g / L, sodium chloride 10-13 g / L, hydroxyproline 20-30 g / L, sorbitol 0.9-2 g / L, ammonium sulfate 1-2 g / L, magnesium chloride 1-2 g / L, and calcium chloride 1-2.5 g / L.
[0005] The method for preparing the above-mentioned culture medium for improving the hydroxylation rate of recombinant collagen includes the following steps:
[0006] S1. Weigh out tryptone, yeast powder, sodium chloride, sorbitol, ammonium sulfate, magnesium chloride and calcium chloride, dissolve them in ultrapure water and sterilize;
[0007] S2. Dissolve hydroxyproline in a small amount of ultrapure water, filter to remove bacteria, add to the sterilized culture medium, mix well to obtain the culture medium.
[0008] The above-mentioned culture medium for increasing the hydroxylation rate of recombinant collagen is applied in the preparation of recombinant collagen with a high hydroxylation rate.
[0009] The preparation method of the above-mentioned high-hydroxylation-rate recombinant collagen includes the following steps:
[0010] (1) After activating the engineered bacteria, inoculate them into the culture medium prepared according to claim 2 and culture them until OD. 600 When the concentration reaches 0.6~0.8, IPTG solution is added to induce expression, bacterial cells are collected, resuspended in PBS, sonicated and the supernatant is collected by centrifugation;
[0011] (2) The supernatant was filtered, purified by column chromatography, desalted and concentrated to obtain recombinant collagen with high hydroxylation rate.
[0012] Preferably, the engineered bacteria in step (1) are any genetically engineered bacteria capable of expressing recombinant collagen.
[0013] Preferably, the amount of engineered bacteria in the culture medium in step (1) is 1-5%.
[0014] Preferably, the final concentration of the IPTG solution in the culture medium in step (1) is 0.1~1 mol / L.
[0015] Beneficial effects:
[0016] 1. This invention, based on the addition of hydroxyproline to LB medium, further introduces sorbitol, ammonium sulfate, magnesium chloride, and calcium chloride to form an optimized culture medium formulation. This medium exhibits no toxic side effects on bacterial growth when culturing recombinant collagen-producing engineered bacteria, significantly increasing bacterial density and viable cell count. Simultaneously, the yield of the target protein remains essentially the same as that of conventional media, without any yield reduction due to hyperosmolar stimulation.
[0017] 2. By using the culture medium provided by this invention to ferment engineered bacteria, highly efficient hydroxylation modification of recombinant collagen was directly achieved. The hydroxylation rate of recombinant collagen stably reached over 90%, and hydroxyproline accounted for about 20% of the protein's amino acid sequence, significantly exceeding the hydroxylation level of natural collagen (approximately 12%).
[0018] 3. The culture medium provided by this invention has simple components, low cost, and is easy to prepare. It can be used by simply replacing the components when culturing engineered strains without the need for additional complex operating steps or genetic engineering modifications. It can stably produce recombinant collagen with a hydroxylation rate exceeding the natural level, providing an efficient and universal culture solution for the industrial production of high-quality recombinant collagen. Attached Figure Description
[0019] Figure 1 Here is the liquid chromatogram of recombinant collagen from Example 1;
[0020] Figure 2 The liquid chromatogram of recombinant collagen from Comparative Example 1 is shown.
[0021] Figure 3 The liquid chromatogram of recombinant collagen in Comparative Example 2 is shown below.
[0022] Figure 4 This is the liquid chromatogram of recombinant collagen from Comparative Example 3. Detailed Implementation
[0023] The present invention will be further described below with reference to the embodiments. The following embodiments are explanations of the present invention and the present invention is not limited to the following embodiments.
[0024] Example 1
[0025] This embodiment describes a method for constructing the engineered bacterium BL21(DE3)-pET-N-His-PreScission-SUMO-COL3A1, which can express recombinant human type III collagen, including the following steps:
[0026] S1. Select the human type III collagen sequence and obtain the gene sequence of the human type III collagen α1 chain (GenBank: AB384877.1) from the NCBI database. In this invention, the N-terminal domain and the collagen triplet repeat region connected to it (amino acids 155-377) are selected and optimized according to the codon preference of E. coli to synthesize the required recombinant human type III collagen DNA sequence.
[0027] S2. The synthesized DNA fragment sequence was ligated into the PMV261 vector. The ligation system is shown in Table 1. The ligation conditions were: 4℃ for 12 h to obtain the recombinant plasmid.
[0028] S3. The recombinant plasmid was transformed into E. coli DH5α competent cells, and a single clone plasmid was extracted. The single clone plasmid was double-digested with two restriction endonucleases, BamHI and XhoHI. The target gene was recovered by gel excision after verification by 1% agarose gel electrophoresis.
[0029] S4. The target gene was ligated into the pET-N-His-PreScission-SUMO E. coli expression vector. The ligation system is shown in Table 1. The ligation conditions were: 4℃ for 12 h to obtain the recombinant expression plasmid of human type III collagen. The recombinant expression plasmid was introduced into E. coli BL21(DE3) competent cells, plated on LB plates containing 50 μg / mL kana resistance, and cultured at 37℃ for 12 h until colonies appeared. Positive clones were screened, and 5 positive clones were picked and inoculated into LB liquid medium. After culturing at 37℃ and 200 rpm for 12 h, the bacterial solution was taken and PCR amplified according to Tables 2-4. The results were verified by 1% agarose gel electrophoresis and DNA sequencing to obtain the engineered strain BL21(DE3)-pET-N-His-PreScission-SUMO-COL3A1.
[0030] Table 1 Connection System
[0031]
[0032] Table 2 PCR primer sequences
[0033]
[0034] Table 3 PCR amplification system
[0035]
[0036] Table 4 PCR Amplification Program
[0037]
[0038] Example 2
[0039] This embodiment describes the construction method of the engineered bacterium Rosseta(DE3)-pET-N-His-PreScission-SUMO-COL3A1, which can express recombinant human type III collagen. The difference from Example 1 is that the E. coli BL21(DE3) competent cells in step S5 are replaced with E. coli Rosseta(DE3) competent cells, and the remaining steps are the same as in Example 1.
[0040] Example 3
[0041] This embodiment describes a method for preparing recombinant human type III collagen with high hydroxylation rate using the engineered bacterium BL21(DE3)-pET-N-His-PreScission-SUMO-COL3A1 constructed in Example 1, including the following steps:
[0042] S1. Weigh 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride, 1.82 g of sorbitol, 1.3 g of ammonium sulfate, 2 g of magnesium chloride, and 1.11 g of calcium chloride. Add them to 950 mL of ultrapure water and sterilize at 121℃ for 20 min. Dissolve 26.22 g of hydroxyproline in 50 mL of ultrapure water, filter and sterilize, add it to the sterilized culture medium, mix well, and obtain LH culture medium.
[0043] S2. The engineered strain BL21(DE3)-pET-N-His-PreScission-SUMO-COL3A1 constructed in Example 1 was inoculated into LB medium to prepare a seed culture. The seed culture was inoculated into LH medium at a 1% inoculation rate and cultured at 37°C and 200 rpm until OD... 600 When the concentration reached between 0.6 and 0.8, IPTG solution with a final concentration of 0.1 mol / L was added, and expression was induced at 16℃ and 200 rpm. After 24 h, the OD of different treatment groups was measured. 600 Count the viable bacteria, collect the bacterial cells, resuspend them in PBS, sonicate for 30 min, and centrifuge to collect the supernatant.
[0044] S3. Filter the supernatant and transfer it to a pre-equilibrated Ni Sepharose solution. TM Recombinant human collagen was eluted from the column using 250 mol / L imidazole elution buffer in an HP chromatography column. The purified sample was then desalted and concentrated using an ultrafiltration tube. The protein concentration of the sample was determined, and the content of Hyp and Pro in the sample was detected by high performance liquid chromatography.
[0045] Example 4
[0046] The difference between this embodiment and embodiment 3 is that the amount of sorbitol added to the LH culture medium in this embodiment is 0.91 g, and the other steps are the same as in embodiment 3.
[0047] Example 5
[0048] The difference between this embodiment and embodiment 3 is that the amount of calcium chloride added to the LH culture medium in this embodiment is 2.22 g, and the other steps are the same as in embodiment 3.
[0049] Example 6
[0050] The difference between this embodiment and embodiment 3 is that the amount of magnesium chloride added to the LH culture medium in this embodiment is 1 g, and the other steps are the same as in embodiment 3.
[0051] Example 7
[0052] The difference between this embodiment and embodiment 3 is that in this embodiment, the engineered bacteria BL21(DE3)-pET-N-His-PreScission-SUMO-COL3A1 is replaced with the engineered bacteria Rosseta(DE3)-pET-N-His-PreScission-SUMO-COL3A1 prepared in embodiment 2, and the remaining steps are the same as in embodiment 3.
[0053] Example 8
[0054] The difference between this embodiment and embodiment 8 is that the amount of sorbitol added to the LH culture medium in this embodiment is 0.91 g, and the other steps are the same as in embodiment 8.
[0055] Example 9
[0056] The difference between this embodiment and embodiment 8 is that the amount of calcium chloride added to the LH culture medium in this embodiment is 2.22 g, and the other steps are the same as in embodiment 8.
[0057] Example 10
[0058] The difference between this embodiment and embodiment 8 is that the amount of magnesium chloride added to the LH culture medium in this embodiment is 1 g, and the other steps are the same as in embodiment 8.
[0059] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:
[0060] Comparative Example 1
[0061] The difference between this comparative example and Example 3 is that LH medium is replaced with LB medium in this comparative example. The preparation method of LB medium is as follows: weigh 10 g of tryptone, 5 g of yeast powder, 10 g of sodium chloride, add 1 L of ultrapure water, sterilize at 121℃ for 20 min to obtain LB medium, and the remaining steps are the same as in Example 3.
[0062] Comparative Example 2
[0063] The difference between this comparative example and Example 3 is that the LH medium is replaced with LB medium with added hydroxyproline in this comparative example. The preparation method of LB medium is the same as that of comparative example 1, and the amount of hydroxyproline added is 26.22 g. The remaining steps are the same as those of Example 3.
[0064] Comparative Example 3
[0065] The difference between this comparative example and Example 3 is that the LH medium is replaced with LB medium supplemented with hydroxyproline and sorbitol. The preparation method of LB medium is the same as that of Comparative Example 1. The amount of hydroxyproline added is 26.22 g and the amount of sorbitol added is 1.82 g. The remaining steps are the same as those of Example 3.
[0066] Comparative Example 4
[0067] The difference between this comparative example and Example 3 is that the LH medium is replaced with LB medium containing hydroxyproline and ammonium sulfate. The preparation method of LB medium is the same as that of Comparative Example 1. The amount of hydroxyproline added is 26.22 g, and the amount of ammonium sulfate added is 1.3 g. The remaining steps are the same as those of Example 3.
[0068] Comparative Example 5
[0069] The difference between this comparative example and Example 3 is that the LH medium is replaced with LB medium supplemented with hydroxyproline and magnesium chloride. The preparation method of LB medium is the same as that of Comparative Example 1, the amount of hydroxyproline added is 26.22 g, the amount of magnesium chloride added is 2 g, and the remaining steps are the same as those of Example 3.
[0070] Comparative Example 6
[0071] The difference between this comparative example and Example 3 is that the LH medium is replaced with LB medium containing hydroxyproline and calcium chloride. The preparation method of LB medium is the same as that of Comparative Example 1. The amount of hydroxyproline added is 26.22 g, and the amount of calcium chloride added is 1.11 g. The remaining steps are the same as those of Example 3.
[0072] Comparative Example 7
[0073] The difference between this comparative example and Example 3 is that the LH medium is replaced with LB medium containing hydroxyproline, sorbitol and ammonium sulfate. The preparation method of LB medium is the same as that of Comparative Example 1. The amount of hydroxyproline added is 26.22 g, the amount of sorbitol added is 1.82 g, and the amount of ammonium sulfate added is 1.3 g. The remaining steps are the same as those of Example 3.
[0074] Comparative Example 8
[0075] The difference between this comparative example and Example 3 is that in this comparative example, LH medium is replaced with LB medium supplemented with hydroxyproline, sorbitol and magnesium chloride. The preparation method of LB medium is the same as that of comparative example 1. The amount of hydroxyproline added is 26.22 g, the amount of sorbitol added is 1.82 g, and the amount of magnesium chloride added is 2 g. The remaining steps are the same as those of Example 3.
[0076] Comparative Example 9
[0077] The difference between this comparative example and Example 3 is that in this comparative example, LH medium is replaced with LB medium supplemented with hydroxyproline, sorbitol and calcium chloride. The preparation method of LB medium is the same as that of comparative example 1. The amount of hydroxyproline added is 26.22 g, the amount of sorbitol added is 1.82 g, and the amount of calcium chloride added is 1.11 g. The remaining steps are the same as those of Example 3.
[0078] Table 5 Indicator Detection Results
[0079]
[0080] As shown in Table 1, the Hyp / Pro ratio, collagen hydroxylation rate, and viable cell count in the fermentation broth of Examples 3-10 of the present invention were significantly better than those of the comparative examples. Specifically, Example 1, compared to Comparative Example 1 (LB medium), showed a significantly higher OD ratio. 600 The value increased by 18%, the number of viable bacteria increased by 160%, the yield of the target protein remained basically the same, and the hydroxylation rate was >90%, with hydroxyproline accounting for about 20% of the protein's amino acid sequence, exceeding the hydroxylation level of natural collagen (12%).
[0081] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.
Claims
1. A culture medium for improving the hydroxylation rate of recombinant collagen, characterized in that, It includes the following components at the following concentrations: tryptone 10-15 g / L, yeast extract 5-8 g / L, sodium chloride 10-13 g / L, hydroxyproline 20-30 g / L, sorbitol 0.9-2 g / L, ammonium sulfate 1-2 g / L, magnesium chloride 1-2 g / L, and calcium chloride 1-2.5 g / L.
2. The method for preparing a culture medium for improving the hydroxylation rate of recombinant collagen according to claim 1, characterized in that, Includes the following steps: S1. Weigh out tryptone, yeast powder, sodium chloride, sorbitol, ammonium sulfate, magnesium chloride and calcium chloride, dissolve them in ultrapure water and sterilize; S2. Dissolve hydroxyproline in a small amount of ultrapure water, filter to remove bacteria, add to the sterilized culture medium, mix well to obtain the culture medium.
3. The application of the culture medium for improving the hydroxylation rate of recombinant collagen according to claim 1 in the preparation of recombinant collagen with a high hydroxylation rate.
4. The application according to claim 3, characterized in that, The method for preparing the high-hydroxylation-rate recombinant collagen includes the following steps: (1) After activating the engineered bacteria, inoculate them into the culture medium prepared according to claim 2 and culture them until OD. 600 When the concentration reaches 0.6~0.8, IPTG solution is added to induce expression, bacterial cells are collected, resuspended in PBS, sonicated and the supernatant is collected by centrifugation; (2) The supernatant was filtered, purified by column chromatography, desalted and concentrated to obtain recombinant collagen with high hydroxylation rate.
5. The application according to claim 4, characterized in that, In step (1), the engineered bacteria can be any kind of genetically engineered bacteria that can express recombinant collagen.
6. The application according to claim 4, characterized in that, In step (1), the amount of engineered bacteria inoculated into the culture medium is 1-5%.
7. The application according to claim 4, characterized in that, In step (1), the final concentration of the IPTG solution in the culture medium is 0.1~1 mol / L.