Method for improving hydroxylation rate of recombinant human III-type collagen
By optimizing the nucleotide sequence and altering cell membrane permeability, the problem of low hydroxylation rate of recombinant human type III collagen in the E. coli expression system was solved, achieving a highly efficient hydroxylation effect and enhancing the application potential of recombinant human type III collagen.
Patent Information
- Application Number
- CN202511135042.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-14
- Publication Date
- 2025-11-28
AI Technical Summary
In existing technologies, the E. coli expression system cannot effectively hydroxylate recombinant human type III collagen, resulting in low hydroxyproline content and limiting its application.
By optimizing the nucleotide sequence of recombinant human type III collagen, a recombinant expression vector was constructed and expressed in Escherichia coli. The hydroxylation rate was increased by combining exogenous addition of hydroxyproline and alteration of cell membrane permeability.
A high hydroxylation rate was achieved in recombinant human type III collagen, with hydroxyproline accounting for 60.62%, which is close to the level of natural human type III collagen, exhibiting good biological activity and stability.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application belongs to the field of biotechnology, and particularly relates to a method for improving the hydroxylation rate of recombinant human collagen type III. BACKGROUND
[0002] Collagen, as an important structural protein in animals, is one of the main components of extracellular matrix, and is widely distributed in connective tissues such as skin, cartilage, tendon, blood vessels, etc. This protein not only provides mechanical support for tissues, but also plays a key role in tissue repair, wound healing and organ development by regulating biological processes such as cell adhesion, migration, differentiation and proliferation. With its excellent biocompatibility and degradability, collagen has been widely used in many fields such as biomedical materials, tissue engineering scaffolds, cosmetic fillers, functional food additives and drug delivery systems.
[0003] From the molecular structure, collagen is composed of a characteristic (Gly-X-Y) n repeat sequence to form a triple helix structure, in which glycine (Gly) occupies the first position of each triplet, and X and Y sites are usually occupied by proline (Pro) and hydroxyproline (Hyp). It is worth noting that hydroxyproline at the Y site is not directly encoded by the gene, but is formed by hydroxylation of specific proline residues through a post-translational modification process catalyzed by prolyl-4-hydroxylase (P4H). This unique hydroxylation modification is crucial for maintaining the thermal stability of collagen. Studies have shown that when the content of hydroxyproline in the core domain is less than 15%, the collagen molecule is difficult to form a stable triple helix conformation at 37℃ physiological environment.
[0004] The three-dimensional helical structure of natural collagen is closely related to its biological function. Compared with non-hydroxylated or low-hydroxylated recombinant collagen, collagen molecules containing sufficient hydroxyproline not only exhibit higher thermal denaturation temperature (Tm value can be increased by 10-15℃), but also have higher molecular flexibility, protease resistance and spatial conformation of cell recognition sites, which are closer to endogenous collagen in the human body. This structural biomimetic property makes it exhibit significant advantages in inducing directional migration of cells, promoting tissue regeneration, etc., and becomes an ideal tissue engineering scaffold material.
[0005] In current industrial production, E. coli expression system has become the mainstream platform for the production of recombinant collagen due to its clear genetic background, low culture cost, and short fermentation cycle (usually 18-24 h to high-density culture). However, this system has significant limitations: the prokaryotic expression system lacks the unique proline hydroxylase system of eukaryotes, resulting in the complete absence of hydroxyproline in the expression product. Existing improvement strategies mostly use the engineering scheme of co-expressing hydroxylase genes, including dual-plasmid systems (such as pETDuet series) or single-plasmid multi-cistronic design. However, these schemes have the following bottlenecks: (1) The expression of exogenous hydroxylase increases the metabolic burden of the host, resulting in a 30-40% decrease in bacterial growth rate; (2) The genetic instability of multi-gene expression systems results in a high plasmid loss rate of 15-20% after continuous passage; (3) The hydroxylation efficiency obtained by existing technologies is still not ideal, with the proportion of hydroxyproline in the product generally below 5%, far from the 12-14% hydroxylation level of natural collagen. These technical difficulties have seriously restricted the application expansion of recombinant human collagen. SUMMARY
[0006] The technical problem to be solved: In view of the problem that collagen cannot be hydroxylated in the E. coli expression system, the present application provides a method for improving the hydroxylation rate of recombinant human type III collagen.
[0007] Technical scheme: A recombinant human type III collagen, whose amino acid sequence is shown in SEQ ID No. 1, and the nucleotide sequence encoding the recombinant human type III collagen is shown in SEQ ID No. 2.
[0008] A recombinant expression vector containing the above-mentioned amino acid sequence.
[0009] As a preferred, the recombinant expression vector is an expression vector with T7 promoter / lac operator in the vector framework.
[0010] Further, the recombinant expression vector is pET-N-His-PreScission-SUMO, which is a prokaryotic expression vector for expressing a target protein with His and SUMO double tags at the N terminus.
[0011] A recombinant genetically engineered bacterium containing the above-mentioned nucleotide sequence or recombinant expression vector.
[0012] As a preferred, the recombinant genetically engineered bacterium is E. coli.
[0013] Further, the recombinant genetically engineered bacterium is BL21 (DE3) strain.
[0014] A method for improving the hydroxylation rate of recombinant human type III collagen, characterized in that it comprises the following steps:
[0015] S1. Selecting a nucleotide sequence encoding full-length human collagen type III without N short end peptide, optimizing according to the codon bias of E. coli, synthesizing the required recombinant human collagen type III DNA sequence, and connecting to the E. coli expression vector to construct a recombinant human collagen type III expression vector, which is transformed into E. coli;
[0016] S2. Adding Hyp to induce the expression of recombinant human collagen type III, and detecting the content of proline and hydroxyproline in the recombinant human collagen type III by liquid phase method;
[0017] S3. Further improving the hydroxylation rate of recombinant human collagen type III by changing the cell membrane permeability, purifying the recombinant human collagen type III, and detecting the content of proline and hydroxyproline by liquid phase method.
[0018] As preferred, the nucleotide sequence encoding full-length human collagen type III without N short end peptide in step S1 is shown in SEQ ID No. 3.
[0019] As preferred, IPTG needs to be added in the induction process in step S2, and the concentration of IPTG is 0.1-0.5 mmol / L.
[0020] As preferred, the concentration of Hyp in step S2 is 50-200 mmol / L, and it has no obvious negative effect on the growth of the strain.
[0021] As preferred, the method for changing the cell membrane permeability in step S2 is adding inorganic salt or ultrasonic treatment.
[0022] Further, the concentration of the inorganic salt is 100-150 mmol / L, and the inorganic salt is CaCl2 or MgSO4.
[0023] Further, the concentration of CaCl2 is 150 mmol / L, and the concentration of MgSO4 is 100 mmol / L,
[0024] Beneficial effects:
[0025] 1. The present application constructs an E. coli BL21(DE3)-pET-N-His-PreScission-SUMO-col3A recombinant human collagen type III expression strain, and realizes the expression of high-hydroxylated recombinant human collagen type III by controlling the addition amount of hydroxyproline (Hyp).
[0026] 2. In the prior art, the method of co-expressing collagen and hydroxylase genes is often used to improve the hydroxylation rate, but the method is complex in operation, poor in stability, and the expression system of double plasmids or single plasmid double genes has a certain negative effect on the growth of the strain. The recombinant human collagen obtained by the method has a low proportion of hydroxyproline, and the method provided by the application is simple in operation, high in feasibility, high in hydroxylation rate and has a promoting effect on the growth of the strain. The application uses exogenous addition of hydroxyproline (Hyp) and improves the permeability of the cell membrane to obtain recombinant human type III collagen with a hydroxylation rate of proline of 60.62%, and the proportion of hydroxyproline in the total amino acid sequence is 13.32%, close to the natural human type III collagen hydroxylation rate.
[0027] 3. The high-hydroxylation recombinant human type III collagen produced by the method of the application can tolerate trypsin digestion and has good biological activity and stability. BRIEF DESCRIPTION OF DRAWINGS
[0028] Figure 1 pET-N-His-PreScission-SUMO-col3A1 recombinant expression vector;
[0029] Figure 2 hydroxyproline kit test results of the hydroxylation rate of the recombinant human type III collagen of Example 1;
[0030] Figure 3 hydroxyproline kit test results of the hydroxylation rate of the recombinant human type III collagen of Example 1;
[0031] Figure 4 hydroxyproline kit test results of the hydroxylation rate of the recombinant human type III collagen of Example 2;
[0032] Figure 5 hydroxyproline kit test results of the hydroxylation rate of the recombinant human type III collagen of Example 3;
[0033] Figure 6 hydroxyproline kit test results of the hydroxylation rate of the recombinant human type III collagen of Example 4;
[0034] Figure 7 hydroxyproline kit test results of the hydroxylation rate of the recombinant human type III collagen of Example 2. DETAILED DESCRIPTION
[0035] The application will be further described below in conjunction with examples, and the following examples are an explanation of the application, and the application is not limited to the following examples:
[0036] Example 1
[0037] This embodiment is the preparation of high-hydroxylation recombinant human type III collagen, which comprises the following steps:
[0038] S1. Selecting human collagen type III sequence, obtaining the gene sequence of human collagen type III alpha 1 chain (GenBank: AB384877.1) in NCBI database, selecting the fragment sequence of N-terminal domain and collagen triad repeat region connected thereto (155~377th amino acid) in the application, optimizing according to the codon bias of E. coli, synthesizing the required recombinant human collagen DNA sequence.
[0039] S2. Connecting the synthesized DNA fragment sequence to PMV261 vector to form a recombinant plasmid, transforming the recombinant plasmid into DH5α for cloning, extracting a single colony plasmid, double enzyme cutting with two restriction endonucleases BamH I and Xho I, using 1% agarose gel electrophoresis, cutting the gel to recover the target gene, connecting the target gene to the pET-N-His-PreScission-SUMO E. coli expression vector to form a recombinant expression plasmid of human collagen type III;
[0040] S3. Introducing the recombinant expression plasmid into E. coli BL21, coating on an LB plate containing 50 μg / mL kana resistance, culturing until colonies appear, screening out positive clones, picking up 5 positive clones and inoculating in LB liquid medium, culturing at 37℃, 200 rpm for 12~16 h, then taking the bacterial liquid to perform PCR verification according to Tables 1~3, after verifying the correct band by nucleic acid electrophoresis, performing sequencing verification;
[0041] Table 1 Primer sequence required for PCR
[0042]
[0043] Table 2 PCR amplification system
[0044]
[0045] Table 3 PCR amplification program
[0046]
[0047] S4. Making seed liquid of the strain with correct sequencing result, inoculating 1% in liquid LB medium, culturing at 37℃, 200 rpm, when OD 600 reaches 0.6~0.8, adding 0.1 mol / L IPTG solution, simultaneously adding 0, 50, 100, 150, 200 mmol / L Hyp, inducing expression at 16℃, 200 rpm for 24 h, centrifuging to collect bacterial cells, resuspending with PBS, ultrasonic breaking for 30 min, centrifuging to collect supernatant;
[0048] S5. The supernatant was filtered and transferred to the equilibrated Ni Sepharose TM The recombinant human collagen type III was eluted from the column with 250 mol / L imidazole elution solution in the HP chromatography column, and the purity of the recombinant human collagen type III was 95% after SDS-PAGE analysis;
[0049] S6. The purified sample was desalted and concentrated by using an ultrafiltration tube, and the Hyp content under different Hyp addition amounts was detected by using a Hyp detection kit. After qualitative determination, the highest result was selected, and the contents of Hyp and Pro were quantitatively determined by using a high-performance liquid chromatography method.
[0050] Example 2
[0051] The difference between this example and example 1 is that 150 mmol / L CaCl2 solution is additionally added in this example. The preparation of the recombinant human collagen type III in this example includes the following steps:
[0052] S1. The strain with correct sequencing results in example 1 was used to prepare a seed solution, 1% of which was inoculated into a liquid LB culture medium and cultured at 37°C and 200 rpm. When the OD 600 was between 0.6 and 0.8, 0.1 mol / L IPTG solution, 200 mmol / L Hyp and 150 mmol / L CaCl2 were added, and expression was induced at 16°C and 200 rpm for 24 h. The bacterial cells were collected by centrifugation, resuspended with PBS, and then ultrasonically broken for 30 min. The supernatant was collected by centrifugation;
[0053] S2. The supernatant was filtered and transferred to the equilibrated Ni Sepharose TM The recombinant human collagen type III was eluted from the column with 250 mol / L imidazole elution solution in the HP chromatography column, and the purity of the recombinant human collagen type III was 95% after SDS-PAGE analysis;
[0054] Example 3
[0055] The difference between this example and example 1 is that 100 mmol / L MgSO4 solution is additionally added in this example. The preparation of the recombinant human collagen type III in this example includes the following steps:
[0056] S1. The strain with correct sequencing results in example 1 was used to prepare a seed solution, 1% of which was inoculated into a liquid LB culture medium and cultured at 37°C and 200 rpm. When the OD 600When reaching 0.6-0.8, 0.1 mol / L IPTG solution, 200 mmol / L Hyp and 100 mmol / L MgSO4 are added, expression is induced at 16℃ and 200 rpm for 24 h, bacterial bodies are collected by centrifugation, and after resuspension with PBS, ultrasonic crushing is performed for 30 min, and the supernatant is collected by centrifugation;
[0057] S2. The supernatant is filtered and transferred to a balanced Ni Sepharose TM HP chromatography column, and the recombinant human collagen type III is eluted from the column with 250 mol / L imidazole eluent, and the purity of the recombinant human collagen type III obtained by SDS-PAGE analysis is 95%.
[0058] Example 4
[0059] The difference between this example and Example 1 is that in this example, the bacterial solution is ultrasonically treated before the addition of IPTG and Hyp, the ultrasonic frequency is 25 kHz, the treatment time is 1 min, the duty cycle is 80%, and the ultrasonic power is 30%, and the preparation of the recombinant human collagen type III of this example includes the following steps:
[0060] S1. Seed liquid is prepared from a strain with correct sequencing results in Example 1, 1% of which is inoculated into liquid LB medium, and cultured at 37℃ and 200 rpm, and when the OD 600 When reaching 0.6-0.8, the bacterial solution is ultrasonically treated at an ultrasonic frequency of 25 kHz for 1 min at a duty cycle of 80% and an ultrasonic power of 30%, and then 0.1 mol / L IPTG solution and 200 mmol / L Hyp are added, expression is induced at 16℃ and 200 rpm for 24 h, bacterial bodies are collected by centrifugation, and after resuspension with PBS, ultrasonic crushing is performed for 30 min, and the supernatant is collected by centrifugation;
[0061] S2. The supernatant is filtered and transferred to a balanced Ni Sepharose TM HP chromatography column, and the recombinant human collagen type III is eluted from the column with 250 mol / L imidazole eluent, and the purity of the recombinant human collagen type III obtained by SDS-PAGE analysis is 95%.
[0062] To further illustrate the technical effects of the present application, the present application also provides a comparative example, which is as follows:
[0063] Comparative Example 1
[0064] The difference between this comparative example and Example 1 is that in this comparative example, 100 mmol / L NaCl solution is additionally added, and the preparation of the recombinant human collagen type III of this comparative example includes the following steps:
[0065] S1. The strain with correct sequencing results in Example 1 was used to prepare a seed solution, which was inoculated into liquid LB medium at 1%, and cultured at 37°C and 200 rpm. When the OD 600 When the OD reached 0.6-0.8, 0.1 mol / L IPTG solution, 200 mmol / L Hyp and 100 mmol / L NaCl were added, and expression was induced at 16°C and 200 rpm for 24 h. The bacterial cells were collected by centrifugation, resuspended in PBS and ultrasonically broken for 30 min. The supernatant was collected by centrifugation.
[0066] S2. The supernatant was filtered and transferred to a balanced Ni Sepharose TM HP column, and the recombinant human collagen type III was eluted from the column with 250 mol / L imidazole eluent. SDS-PAGE analysis showed that the purity of the recombinant human collagen type III was 95%.
[0067] Comparative Example 2
[0068] The difference between this comparative example and Example 1 is that only 0.1 mol / L IPTG and 100 mmol / L ascorbic acid were added in this comparative example. The preparation of recombinant human collagen type III in this comparative example included the following steps:
[0069] S1. The strain with correct sequencing results in Example 1 was used to prepare a seed solution, which was inoculated into liquid LB medium at 1%, and cultured at 37°C and 200 rpm. When the OD 600 When the OD reached 0.6-0.8, 0.1 mol / L IPTG solution and 100 mmol / L ascorbic acid were added, and expression was induced at 16°C and 200 rpm for 24 h. The bacterial cells were collected by centrifugation, resuspended in PBS and ultrasonically broken for 30 min. The supernatant was collected by centrifugation.
[0070] S2. The supernatant was filtered and transferred to a balanced Ni Sepharose TM HP column, and the recombinant human collagen type III was eluted from the column with 250 mol / L imidazole eluent. SDS-PAGE analysis showed that the purity of the recombinant human collagen type III was 95%.
[0071] Index detection:
[0072] 1. Test of the hydroxylation rate of recombinant human collagen type III:
[0073] (1) Prepare the relevant reagents:
[0074] Standard: 0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 1.0 mmol / L 4-hydroxy-L-proline (Hyp) and L-proline (Pro) standard were prepared with ddH2O;
[0075] Derivatization reagent: 40 mg of fluorenylmethyl chloroformate (FMOC-Cl, RWAN) was weighed and dissolved in 8 mL of acetonitrile;
[0076] H3BO3 buffer: 0.4 mol / L H3BO3 buffer was prepared, and the pH was adjusted to 10.5 with NaOH;
[0077] HCl solution: 6 mol / L HCl solution was prepared;
[0078] (2) Sample treatment: 0.5 mL of liquid sample was weighed and dissolved with 0.5 mL of 6 mol / L HCl, and then was placed in a glass tube with a plug, and was sealed and placed in a high temperature oven for acidolysis at 105°C for 22 h. The hydrolyzate was adjusted to pH 6.0-8.0 with NaOH, 100 μL of the hydrolyzate was taken, 200 μL of 0.4 mol / L H3BO3 buffer was added, and was shaken and mixed, then 100 μL of FMOC-Cl was added, and was shaken for 30 s, and was ultrasonically treated for 10 min, and was filtered with a 0.22 μm filter membrane and was loaded;
[0079] (3) Detection by high performance liquid chromatography, and the specific detection conditions were as follows:
[0080] Chromatographic column: Agilent ZORBAX Eclipse Plus C18 column (4.6 x 150 mm, 5 μm); mobile phase A: acetonitrile, mobile phase B: 0.1% acetic acid; gradient elution: 0-10 min, A phase from 40% to 80%, 10-12 min, A phase from 80% to 98%, 12-16 min, A phase maintained at 98%, 16-19 min, A phase from 98% to 40%, 19-25 min, A phase maintained at 40%; PDA detector detection wavelength: 262 nm; injection volume: 20 μL; flow rate: 1 mL / min; column temperature: 30°C; the retention time of Pro was 10.4 min, the retention time of Hyp was 7.6 min, and the retention time of derivatization reagent FMOC-Cl was 9.9 min.
[0081] (4) Result calculation: the standard curve was drawn with the standard concentration of Hyp and Pro as the abscissa and the liquid phase peak area as the ordinate, the content of Hyp and Pro was calculated according to the corresponding liquid phase peak area of the sample, and the proportion of Hyp in total proline was calculated.
[0082] 2. Live bacteria detection:
[0083] The bacteria solution after induction for 24 h was gradient diluted with PBS, 100 μL of 10 -6 ,10 -7 The gradient diluted bacteria solution was coated on LB plate, and after 12 h culture at 37℃, colony counting was performed. The number of live bacteria after induction with IPTG only was used as control.
[0084] 3. Trypsin degradation test:
[0085] A 2.5 mg / L trypsin solution was prepared, 80 μL of collagen was taken, and the collagen without trypsin treatment was used as control, 10 μL PBS and 10 μL trypsin were added, and the sample was digested at 30℃ for 1 h, and then sampled for SDS-PAGE analysis of protein degradation results.
[0086] As shown in Table 4, the liquid chromatography detection results of 200 mmol / L Hyp addition amount in Example 1 are shown. Figure 1
[0087] As shown in Table 4, the liquid chromatography detection results of 200 mmol / L Hyp addition amount in Example 1 are shown. Figure 2 Table 4 Liquid chromatography detection results of 200 mmol / L Hyp addition amount in Example 1
[0088]
[0089] As shown in Table 4, the liquid chromatography detection results of 200 mmol / L Hyp addition amount in Example 1 are shown.
[0090] Figure 3 and Table 4, the liquid chromatography detection results of 200 mmol / L Hyp addition amount in Example 1 are shown, and the results show that the hydroxylation rate reaches 32.05%.
[0091] Table 5 Test results of indicators of Examples 1-4 and Comparative Examples 1-3
[0092]
[0093] As shown in Table 5, the proportion of Hyp in total proline in the recombinant human collagen type III prepared in Examples 1-4 is 30.05%, 60.62%, 36.58% and 41.05% respectively, indicating that the present application successfully constructs a recombinant human collagen type III E. coli expression strain, and also invents a feasible and simple experimental method for improving the hydroxylation of recombinant human collagen type III. By adding CaCl2 (Example 2) or MgSO4 (Example 3) or ultrasonic treatment (Example 4) to change the cell membrane permeability, the hydroxylation rate of the recombinant human collagen type III can be improved. Compared with Examples 1-2 and Comparative Example 1, although the addition of NaCl can also improve the hydroxylation rate of the recombinant human collagen type III, it will affect the normal growth of the strain and reduce the protein expression amount of the strain. The hydroxylation rate of Example 2 is the highest, and it has a promoting effect on the growth of the strain and the best functional performance. The hydroxylation rate of Comparative Example 2 is 0, resulting in the loss of protein function.
[0094] In summary, the method provided by the present application improves the hydroxylation rate of proline in the recombinant human collagen type III, and the hydroxylation proportion reaches 60.62% (Example 2), and the hydroxyproline proportion in the total amino acid sequence reaches 13.32%. It is close to the hydroxylation rate of natural human collagen type III.
[0095] The above is only a preferred embodiment of the present application, and does not limit the present application in any form. Any person skilled in the art can make many possible changes and modifications to the technical solutions of the present application, or modify equivalent embodiments, without departing from the spirit and technical solutions of the present application. Therefore, any simple modification, equivalent replacement, equivalent change and modification of the above embodiments according to the technical essence of the present application, which does not deviate from the technical solutions of the present application, still belongs to the protection scope of the technical solutions of the present application.
Claims
1. A recombinant human type III collagen, characterized in that: The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID No.
1.
2. The recombinant human type III collagen according to claim 1, characterized in that: The nucleotide sequence encoding the recombinant human type III collagen is shown in SEQ ID No.
2.
3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the amino acid sequence of claim 1, and the recombinant expression vector is an expression vector with a vector framework containing a T7 promoter / lac operon.
4. A recombinant genetically engineered bacterium, characterized in that: The recombinant genetically engineered bacteria contain the nucleotide sequence described in claim 2 or the recombinant expression vector described in claim 3.
5. A method for increasing the hydroxylation rate of recombinant human type III collagen, characterized in that, Includes the following steps: S1. Select a full-length human type III collagen nucleotide sequence that encodes a protein without an N-terminal peptide, optimize it according to the codon preference of E. coli, synthesize the required recombinant human type III collagen DNA sequence, and ligate it into an E. coli expression vector to construct a recombinant human type III collagen expression vector, which is then transformed into E. coli. S2. Hyp was added to induce the expression of recombinant human type III collagen, and the contents of proline and hydroxyproline in recombinant human type III collagen were detected by liquid chromatography. S3. By altering cell membrane permeability, the hydroxylation rate of recombinant human type III collagen was further increased. After purification, the content of proline and hydroxyproline was detected by liquid chromatography.
6. The method for increasing the hydroxylation rate of recombinant human type III collagen according to claim 5, characterized in that: The nucleotide sequence encoding the full-length human type III collagen without N-terminal peptide in step S1 is shown in SEQ ID No.
3.
7. The method for increasing the hydroxylation rate of recombinant human type III collagen according to claim 5, characterized in that: The concentration of Hyp in step S2 is 50~200 mmol / L.
8. The method for increasing the hydroxylation rate of recombinant human type III collagen according to claim 5, characterized in that: The method for changing cell membrane permeability in step S3 is to add inorganic salts or to treat with ultrasound.
9. The method for increasing the hydroxylation rate of recombinant human type III collagen according to claim 8, characterized in that: The concentration of the inorganic salt is 100~150 mmol / L.
10. The method for increasing the hydroxylation rate of recombinant human type III collagen according to claim 9, characterized in that: The inorganic salt is CaCl2 or MgSO4.