A method for collagen purification based on fluorescent tracer and non-chromatographic chromatography

Abstract

The application discloses a collagen purification method based on fluorescent tracing and non-chromatography technology, and is based on analysis of a type III collagen functional domain, a small molecule active polypeptide is obtained by chemical solid-phase synthesis, and the small molecule active polypeptide is precisely screened in and out of the body, and an active site with cell migration, proliferation and wound repair in the type III collagen is determined. By adopting an eGFP-SUMO and ELPs double-tag fusion strategy, the application realizes heterologous expression of the polypeptide 5-ELPs, and completes purification without chromatography technology. Compared with a traditional nickel column purification process, the application is not limited by a high-cost chromatography system and filler, and can obviously shorten the purification time and cost. The application relates to the technical field of biological medicines. The introduction of the eGFP protein realizes visualization of recombinant protein expression and purification, facilitates tracing and monitoring of protein expression and purification in a subsequent production process, and makes the whole cycle production of the polypeptide 5-ELPs fermentation and purification visualized.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography techniques. Background Technology

[0002] Recombinant type III collagen, as a novel biomaterial, has shown great potential to replace animal-derived collagen in wound repair due to its excellent biocompatibility, efficacy, and low immunogenicity. With the development of proteomics and bioinformatics technologies, screening and identifying active fragments with specific biological functions from natural proteins has become an important direction in biomedical research. Type III collagen, after enzymatic hydrolysis, can produce a variety of peptides with potential biological activities, which may have important applications in tissue regeneration and disease treatment. Recombinant technology can be used to directionally enrich these functional active domains. However, the efficient separation and purification of recombinant proteins has always been a key challenge in the biopharmaceutical field. Traditional protein purification methods mainly rely on chromatographic separation techniques, such as ion exchange and affinity chromatography. While these methods can obtain high-purity target proteins, they have limitations such as high cost, complex operation, and difficulty in scaling up. ELPs are a class of artificially designed polypeptide sequences derived from elastin, exhibiting temperature-responsive phase transition properties. When the ambient temperature is higher than their phase transition temperature, ELPs undergo hydrophobic association, forming insoluble aggregates; when the temperature decreases, they can redissolve. Furthermore, ELPs may also improve the stability and bioactivity of target proteins. This reversible phase transition behavior provides a new approach for non-chromatographic purification of proteins.

[0003] To overcome the shortcomings of existing technologies, this invention provides the following technical solution: constructing a fusion protein expression system containing the bioactive functional domains of type III collagen and a SUMO-eGFP dual tag to achieve efficient and soluble expression of the target protein; fusing elastin (ELPs) with phase transition properties to develop a chromatography-free purification process, reducing separation costs; and using eGFP fluorescent labeling to visualize the entire fermentation and purification cycle, facilitating dynamic tracking and real-time monitoring throughout the process. This strategy not only reduces the production cost of type III collagen and its derivatives but also provides a more economical and environmentally friendly protein purification platform for the biopharmaceutical field, offering new insights into overcoming current technical bottlenecks in protein purification. Summary of the Invention

[0004] (a) Technical problems to be solved

[0005] To address the shortcomings of existing technologies, this invention provides a collagen purification method based on fluorescence tracing and non-chromatographic chromatography techniques. By fusing expression visualization markers with temperature-responsive purification tags, it achieves non-chromatographic purification of the target protein and its full-cycle visualization.

[0006] (II) Technical Solution

[0007] To achieve the above objectives, the present invention provides the following technical solution: a method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography techniques, specifically comprising the following steps:

[0008] S1. The sequence of type III collagen was analyzed using bioinformatics methods, five polypeptide fragments with potential biological activity were selected, and then candidate polypeptides were prepared by in vitro chemical synthesis.

[0009] S2. The biological activity of the selected polypeptide fragments with potential biological activity is evaluated through cell proliferation, cell migration, cell adhesion experiments and animal experiments to determine the polypeptide fragments for constructing the fusion protein.

[0010] S3. Using seamless cloning technology, eGFP, SUMO, peptide 5-ELPs and vector pET3c were amplified and linked to obtain the prokaryotic expression vector pET3c-GS-peptide 5-ELPs.

[0011] S4. Induction of expression of fusion protein GS-peptide 5-ELPs: The recombinant plasmid pET3c-GS-peptide 5-ELPs was transformed into the expression strain E.coli BL21(DE3) pLysS. After IPTG induction, the plasmid was broken and the supernatant containing GS-peptide 5-ELPs was collected for SDS-PAGE and WB verification.

[0012] S5. Obtain the supernatant solution containing GS-peptide 5-ELPs. Based on the reversible phase transition characteristics of the ELPs tag in GS-peptide 5-ELPs, the protein is purified by temperature-regulated ITC method, and the purification effect is analyzed by SDS-PAGE electrophoresis.

[0013] S6. Perform cell proliferation, cell migration and stability experiments on the protein obtained by phase transition purification.

[0014] Preferably, in step S1, the amino acid sequence of the human type III collagen A1 chain is obtained using the uniport database, and the sequence is analyzed using bioinformatics methods.

[0015] Preferably, the cell migration in step S2 specifically involves: co-culturing NIH / 3T3 cells with a polypeptide concentration of 2.5 μg / mL for 48 h and observing the migration of NIH / 3T3 cells under an inverted microscope after 48 h.

[0016] Preferably, in step S2, in order to evaluate the effect of the five polypeptide fragments on HaCaT cell adhesion, the polypeptides at a concentration of 1 μg / mL were co-cultured with HaCaT cells for 48 h, and the results were detected using the CCK-8 assay.

[0017] Preferably, in step S4, the recombinant plasmid is transformed into E. coli BL21(DE3) pLysS competent cells to obtain the expression strain E. coli BL21(DE3) pLysS / pET3c-GS-peptide 5-ELPs. Positive single clones are picked from the transformed plates and transferred to 50 mL of LB medium containing ampicillin. The culture is incubated overnight at 37°C with shaking at 220 rpm. The culture is then inoculated at a ratio of 1% into 100 mL of fresh medium and cultured further. The OD600 value is monitored periodically. When it reaches 0.8, IPTG is added to induce the expression of the target protein.

[0018] Preferably, after induction, the culture is transferred to a centrifuge tube, centrifuged at 8000 rpm for 5 minutes to collect the cells, and premixed hammer lysis buffer is added at a mass-to-volume ratio of 1:10 according to the wet weight of the cells. After resuspending thoroughly, the cells are centrifuged and the supernatant is collected. At the same time, the precipitate is resuspended with an equal volume of 10% SDS solution. 120 μL of the supernatant and the precipitate resuspended sample are taken separately, and 30 μL of 5× protein electrophoresis loading buffer is added to each. After mixing, the samples are denatured in a water bath at 100°C for 10 minutes. After the samples are cooled to room temperature, 10 μL is taken for SDS-PAGE analysis.

[0019] Preferably, the phase transition purification method for GS-peptide 5-ELPs in step S5 specifically includes the following steps:

[0020] T1. Sample preparation: Collect the induced bacterial cells, resuspend them in 20mM PB Buffer, homogenize them under low temperature conditions, centrifuge at 12000 rpm for 20 min, and collect the supernatant for ITC purification.

[0021] Enzymatic digestion of T2 and GS-peptides 5-ELPs;

[0022] First phase transition purification of T3 and peptide 5-ELPs: Add NaCl to the enzyme-digested sample to a final concentration of 2M, incubate at 50℃ for at least 20 min to ensure complete salt dissolution, centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant, retain the protein precipitate, resuspend the precipitate with 0.5 times the original volume of 20 mM PB Buffer, incubate on ice for 20 min, centrifuge at 4℃ and 12,000 rpm for 10 min, and collect the supernatant containing the target protein;

[0023] Further phase transition purification of T4 and peptide 5-ELPs: Add NaCl to the collected supernatant to a final concentration of 2M, incubate at 65°C for at least 20 min to ensure complete salt dissolution, centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant, retain the protein precipitate, resuspend the precipitate with 0.5 times the original volume of 20 mM PB Buffer, incubate on ice for 20 min, centrifuge at 4°C and 12,000 rpm for 10 min, collect the supernatant containing the target protein, and then perform a second and third round of phase transition purification.

[0024] Preferably, the enzymatic digestion of GS-peptide 5-ELPs in step T2 specifically involves mixing the supernatant of the fusion protein GS-peptide 5-ELPs with Ulp1p enzyme and digesting it at 37°C for 2 hours.

[0025] (III) Beneficial Effects

[0026] This invention provides a method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography techniques. Compared with existing technologies, it has the following advantages:

[0027] (1) The collagen purification method based on fluorescence tracing and non-chromatographic chromatography technology, based on the analysis of the functional domains of type III collagen, uses chemical solid-phase synthesis to obtain small molecule active peptides, and accurately screens the small molecule active peptides in vivo and in vitro to identify active sites in type III collagen that promote cell migration, proliferation and wound repair.

[0028] (2) The collagen purification method based on fluorescence tracing and non-chromatographic chromatography technology achieves heterologous expression of peptide 5-ELPs by adopting the eGFP-SUMO and ELPs dual-tag fusion strategy and completes the process without chromatographic chromatography. The introduction of eGFP protein enables visualization of recombinant protein expression and purification, which facilitates the tracking and monitoring of protein expression and purification in subsequent production processes. The introduction of eGFP makes the entire production cycle of peptide 5-ELPs fermentation and purification visualized.

[0029] (3) This collagen purification method based on fluorescence tracing and non-chromatographic chromatography technology purifies peptide 5-ELPs by fusing small molecule active peptides with ELPs and using the ITC purification method with ELP tags. Compared with nickel column affinity chromatography, it is faster and simpler, thus demonstrating its potential as a novel purification tool. After the introduction of ELPs, it exhibits good resistance to enzymatic digestion and stability. Due to the introduction of ELP tags, peptide-ELPs have good stability, which facilitates their better application. Attached Figure Description

[0030] Figure 1This is a schematic diagram showing the positions of the five polypeptide fragments in the type III collagen sequence in Example 1 of the present invention;

[0031] Figure 2 The figure shows the experimental results of the effects of five polypeptide fragments on cell proliferation in Example 1 of this invention;

[0032] Figure 3 The figure shows the experimental results of the effects of five polypeptide fragments on cell migration in Example 1 of this invention;

[0033] Figure 4 This is a diagram showing the experimental results of the five polypeptide fragments' effects on cell adhesion in Example 1 of the present invention;

[0034] Figure 5 This is a graph showing the experimental results of the effect of five polypeptide fragments on the wound healing process in Example 1 of the present invention;

[0035] Figure 6 This is a schematic diagram of the construction process of the recombinant plasmid pET3c-GS-peptide 5-ELPs of the present invention;

[0036] Figure 7 This is an agarose gel electrophoresis image of the amplified products of the target gene eGFP, SUMO, polypeptide 5-ELPs and pET3c in Example 2 of the present invention.

[0037] Figure 8 This is a double enzyme digestion verification diagram of the pET3c-GS-peptide 5-ELPs recombinant plasmid in Example 2 of the present invention;

[0038] Figure 9 These are electrophoresis images, immunoblotting images, and visualization results of the fermentation expression process of the polypeptide 5-GS-ELPs / pET3c / BL21(DE3)pLysS strain in Example 3 of this invention.

[0039] Figure 10 This is a schematic diagram of the phase transition purification process and a protein electrophoresis diagram in Example 4 of the present invention;

[0040] Figure 11 This is a diagram showing the experimental results of the effect of polypeptide 5-ELPs on cell proliferation in Example 5 of the present invention;

[0041] Figure 12 This is a diagram showing the experimental results of the effect of polypeptide 5-ELPs on cell migration in Example 5 of the present invention;

[0042] Figure 13 The figure shows the results of the stability test of polypeptide 5-ELPs in Example 5 of this invention. Detailed Implementation

[0043] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] Please see Figure 1-13 The present invention provides five technical solutions: a collagen purification method based on fluorescence tracing and non-chromatographic chromatography, specifically including the following embodiments:

[0045] Example 1: Screening of active fragments and cell viability testing

[0046] (1) Sequence analysis of type III collagen

[0047] The amino acid sequence of human type III collagen A1 chain (COL3A1) was obtained using the Uniport database. Bioinformatics methods were used to analyze the sequence, identifying key functional domains with potential biological activity. Five polypeptide fragments were selected, and their positions within the full-length COL3A1 chain are as follows: Figure 1 As shown in Table 1, the amino acid sequence information and nucleotide sequence are as follows.

[0048] Table 1 Amino acid sequence information

[0049]

[0050] (2) Cell proliferation activity of five polypeptide fragments

[0051] Five polypeptide fragments were synthesized using a chemical solid-phase synthesis method, and their effects on the proliferation of L929 cells were evaluated. Each of the five polypeptide fragments was co-cultured with L929 cells for 48 h, and the results were analyzed using the CCK-8 assay. The results are as follows: Figure 2 The results showed that all five polypeptide fragments had certain cell proliferation-promoting activities, but polypeptides 1-4 did not significantly increase the cell proliferation rate of the control group at any concentration. In contrast, polypeptide 5 promoted the proliferation of L929 cells well at all concentrations, with the most significant proliferation-promoting effect at a concentration of 0.04 μg / mL.

[0052] (3) Cell migration activity of the five polypeptide fragments

[0053] To evaluate the effects of five peptide fragments on the migration ability of NIH / 3T3 cells, NIH / 3T3 cells were co-cultured with each peptide at a concentration of 2.5 μg / mL for 48 h, and the migration of NIH / 3T3 cells was observed under an inverted microscope. The results are as follows: Figure 3As shown in the figure. The results showed that at a concentration of 2.5 μg / mL, compared with the Control group, the cell migration rates of peptides 1, 2, and 4 were lower, while the cell migration rate of peptide 3 was significantly increased, and peptide 5 also showed a significant migration-promoting effect.

[0054] (3) Cell adhesion activity of five polypeptide fragments

[0055] To evaluate the effects of five peptide fragments on HaCaT cell adhesion, HaCaT cells were co-cultured with each peptide at a concentration of 1 μg / mL for 48 h, and the results were analyzed using the CCK-8 assay. The results are as follows: Figure 4 As shown in the figure. The results showed that, compared with the control, peptides 3 and 5 had certain cell adhesion effects, especially peptide 5, which showed better cell adhesion ability.

[0056] (4) Animal experiments

[0057] To further evaluate the effects of the five peptides on the wound healing process, the complete healing time of each group was statistically analyzed. The experimental results are as follows: Figure 5 As shown in the figure, the healing time of the peptide 5 group was significantly shorter than that of the other groups. This indicates that peptide 5 can significantly accelerate the wound healing process and shorten the time required for healing.

[0058] Example 2: Vector construction of recombinant plasmid pET3c-GS-peptide 5-ELPs

[0059] Using seamless cloning technology, the pET3c vector was amplified with the target genes eGFP, SUMO, and peptide 5-ELPs (AE in Figure 7) and ligated to construct the recombinant plasmid pET3c-GS-peptide5-ELPs. Figure 6 To verify the correct construction of the recombinant plasmid pET3c-GS-peptide 5-ELPs, a double enzyme digestion strategy was employed for identification. Two restriction endonucleases, BamHI (NEB, R3136V) and NdeI (NEB, R0111V), were selected for double digestion. The experimental results are as follows: Figure 8 As shown, after double enzyme digestion, two specific bands were clearly visible on the agarose gel. The smaller band, approximately 1.8 kbp, corresponds to the GS-peptide 5-ELPs insert fragment; the larger band, approximately 4.6 kbp, is consistent with the theoretical size of the pET3c vector backbone. The migration distances of these two bands were as expected, and the bands were clear and specific, with no non-specific bands or degradation observed. The band sizes in the electrophoresis pattern were highly consistent with the theoretical values ​​predicted by gene sequence analysis, confirming the successful construction of the recombinant plasmid.

[0060] Example 3: Induction of recombinant expression strain E. coli BL21(DE3) pLysS / pET3c-GS-peptide 5-ELPs

[0061] The recombinant plasmid was transformed into *E. coli* BL21(DE3) pLysS competent cells (Beyotime, D1015) to obtain the expression strain *E. coli* BL21(DE3) pLysS / pET3c-GS-peptide 5-ELPs. Positive single clones were picked from the transformed plates and transferred to 50 mL of LB medium containing ampicillin (100 μg / mL), and cultured overnight at 37 ℃ with shaking at 220 rpm. The culture was then inoculated at a rate of 1% into 100 mL of fresh medium (containing the same concentration of antibiotic) and cultured further. The OD600 value was monitored periodically; when it reached 0.8, IPTG (final concentration 1 mM) (GBCBIO, 1758-1700) was added to induce target protein expression.

[0062] After induction, the culture was transferred to centrifuge tubes and centrifuged at 8000 rpm for 5 minutes to collect the bacterial cells. Based on the wet weight of the bacterial cells, premixed hammer lysis buffer was added at a mass-to-volume ratio of 1:10. After thorough resuspending, centrifugation was performed, and the supernatant was collected. Simultaneously, the precipitate was resuspended using an equal volume of 10% SDS solution. 120 μL of both the supernatant and the resuspended precipitate were taken, and 30 μL of 5× protein electrophoresis loading buffer was added to each. After mixing, the samples were denatured at 100 °C for 10 minutes. After cooling to room temperature, 10 μL of the samples were analyzed by SDS-PAGE.

[0063] SDS-PAGE results are as follows Figure 9 As shown in Figure 9A, lane 1 is the uninduced sample, and lane 2 is the IPTG-induced sample. Figure 9B shows the immunoblotting results, with lane 1 being the IPTG-induced sample and lane 2 being the uninduced sample. Electrophoresis and immunoblotting results indicate that the molecular weight of the target protein GS-peptide 5-ELPs is approximately 70 kDa.

[0064] Visualization results of the fermentation process, such as Figure 9 As shown in (CE), single colonies of this expression strain appear light green under natural light. Figure 9 (C), while fluorescent green single colonies can be observed more clearly under ultraviolet irradiation (C). Figure 9 The color of the fermentation cells gradually turned bright green as the fermentation tank induction time increased (D); Figure 9 The expression level of the target protein gradually increased with induction time (E), while the expression level of the target protein also gradually increased with induction time. Figure 9 The F), indicating that fusion expression with GFP enabled real-time visualization and monitoring of the peptide 5 fermentation process.

[0065] Example 4: Phase transition purification of GS-peptide 5-ELPs

[0066] (1) Sample preparation:

[0067] Collect the induced bacterial cells, resuspend them in 20 mM PB Buffer, and homogenize them at low temperature. Centrifuge at 12000 rpm for 20 min, and collect the supernatant for ITC purification.

[0068] (2) Enzymatic digestion of GS-peptide 5-ELPs:

[0069] The supernatant of the fusion protein GS-peptide 5-ELPs was mixed with Ulp1p enzyme and digested at 37 °C for 2 h.

[0070] (3) Phase transition purification of peptide 5-ELPs

[0071] Add NaCl to the enzyme-digested sample to a final concentration of 2 M, and incubate at 50°C for at least 20 min to ensure complete salt dissolution. Centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant, and retain the protein precipitate. Resuspend the precipitate in 0.5 times its original volume of 20 mMPB buffer, incubate on ice for 20 min, centrifuge at 4°C and 12,000 rpm for 10 min, and collect the supernatant containing the target protein.

[0072] (4) Phase transition purification of peptide 5-ELPs

[0073] Add NaCl to the collected supernatant to a final concentration of 2 M, and incubate at 65°C for at least 20 min to ensure complete salt dissolution. Centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant, and retain the protein precipitate. Resuspend the precipitate in 0.5 times its original volume of 20 mM PB Buffer, incubate on ice for 20 min, centrifuge at 4°C and 12,000 rpm for 10 min, and collect the supernatant containing the target protein. Then perform a second and third round of phase transition purification.

[0074] Experimental results show that Figure 10 Image (A) shows that the supernatant after enzyme digestion becomes lighter in color after one phase transition purification, and becomes almost colorless and transparent after a second phase transition purification, indicating that the eGFP protein has been removed. This demonstrates that the method achieves visualization of the peptide 5 purification process. Further verification was performed by protein electrophoresis, and the results are as follows... Figure 10 As shown in (B), after repeating the above cycle 3 times, the purity of the target protein reached over 95%.

[0075] Example 5: Activity Verification of Peptide 5-ELPs

[0076] (1) Cell proliferation

[0077] To evaluate the effect of peptide 5-ELPs on the proliferation of NIH / 3T3 cells, NIH / 3T3 cells were co-cultured with peptide 5-ELPs at concentrations ranging from 0.78 to 100 μg / mL for 24 h, and the results were analyzed using the CCK-8 assay. The results are as follows: Figure 11 The results showed that when NIH / 3T3 cells were co-cultured with 25 μg / mL peptide 5-ELPs for 24 h, peptide 5-ELPs promoted cell proliferation. When the concentration of peptide 5-ELPs increased to 50 μg / mL, it significantly promoted cell proliferation, and when the concentration increased to 100 μg / mL, it still significantly promoted cell proliferation. Other concentration groups did not show significantly higher cell proliferation rates than the control group. This experiment demonstrates that peptide 5-ELPs retain the cell proliferation-promoting ability of peptide 5.

[0078] (2) Cell migration

[0079] To evaluate the effect of peptide 5-ELPs on the migration ability of NIH / 3T3 cells, NIH / 3T3 cells were co-cultured with peptide 5-ELPs at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL for 24 h. The migration of NIH / 3T3 cells was observed under an inverted microscope, and a scratch assay was performed. The results are as follows: Figure 12 The results showed that after co-culturing with peptide 5-ELPs at concentrations of 25 μg / mL, 50 μg / mL, and 100 μg / mL for 24 h, the cell migration rate of all three experimental groups was significantly increased compared with the Control group.

[0080] (3) Stability test

[0081] Analysis of bands by non-reducing SDS-PAGE gel electrophoresis (e.g.) Figure 13 (A and B) It can be observed that the bands of samples stored at 25 °C did not weaken over time. Samples stored for 0-40 days showed a single, clear main band on the gel, representing intact peptide 5-ELPs, and no degradation products were detected, indicating that long-term storage at room temperature does not lead to significant degradation of peptide 5-ELPs.

[0082] (4) Resistance to type III collagen hydrolysis

[0083] To simulate the in vitro anti-collagenase degradation process of peptide 5-ELPs, type III collagenase was selected to test its anti-collagenase degradation ability. Peptide 5-ELPs were added to a wound microenvironment simulation solution (PBS + 1 mM CaCl2) containing 2 μg / mL type III collagenase to a final concentration of 0.5 mg / mL. The reaction was carried out at 37 ℃ with shaking (200 rpm), and samples were collected at 0, 4, 8, 12, and 24 hours for SDS-PAGE analysis of protein degradation results. The results are as follows: Figure 13 As shown in Figure C, after 12 h of reaction, the peptide 5-ELPs band gradually degraded, with the size of the degraded band around 10-15 kDa, similar to the molecular weight of the ELPs. After 24 h of reaction, the peptide 5-ELPs band was completely degraded.

[0084] This invention Figure 7 In the diagram, lane 1: target gene eGFP; lane 2: target gene SUMO; lane 3: target gene polypeptide 5-ELPs; lanes 4 / 5: plasmid vector pET3c.

[0085] This invention Figure 8 Lane 1: The target gene GS-peptide 5-ELPs and pET3c vector backbone after double enzyme digestion of the recombinant plasmid.

[0086] This invention Figure 9 (A)1: Before induction; 2: After induction; Figure 9 (B) 1: after induction; 2: before induction; Figure 9 Visualization of single colonies of (CD) expression strains. Figure 9 Changes in cell color during induction at 0, 3, 6, and 9 h in an (E) fermenter. Figure 9 (F) SDS-PAGE was used to detect the expression of GS-peptide 5-ELPs during fermentation. M: protein marker; lanes 1-7: induction time 0 h, 2 h, 4 h, 6 h, 8 h, 10 h, 12 h.

[0087] This invention Figure 10 In the diagram, lane 1: bacterial culture sample after lysis; lane 2: bacterial culture sample after lysis and enzyme digestion; lane 3: sample after one purification after enzyme digestion; lane 4: sample after two purifications after enzyme digestion; lane 5: sample after three purifications after enzyme digestion.

[0088] This invention Figure 13 A: Electrophoresis image of protein stored at 25 ℃ for 0 days; M: Protein marker; Lane 1: Sample of protein stored at 25 ℃ for 0 days; Lane 2: Control sample stored at 4 ℃. Figure 13B: Electrophoresis image of protein stored at 25 ℃ for 40 days; M: Protein marker; Lane 1: Sample of protein stored at 25 ℃ for 40 days; Lane 2: Control sample stored at 4 ℃. Figure 13 C: Electrophoresis image of peptide 5-ELPs after enzymatic digestion with type III collagenase; M: Protein Marker; Lane 1: Peptide 5-ELPs sample before enzymatic digestion; Lanes 2-5: Samples of peptide 5-ELPs after enzymatic digestion with type III collagenase at 4, 8, 12, and 24 hours.

[0089] Furthermore, any content not described in detail in this specification is existing technology known to those skilled in the art.

[0090] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0091] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography techniques, characterized in that: Specifically, the following steps are included: S1. The sequence of type III collagen was analyzed using bioinformatics methods, five polypeptide fragments with potential biological activity were selected, and then candidate polypeptides were prepared by in vitro chemical synthesis. S2. The biological activity of the selected polypeptide fragments with potential biological activity is evaluated through cell proliferation, cell migration, cell adhesion experiments and animal experiments to determine the polypeptide fragments for constructing the fusion protein, and the optimal polypeptide fragment, namely polypeptide 5, is selected. The amino acid sequence of polypeptide 5 is shown in SEQ ID NO 5. S3. Using seamless cloning technology, eGFP, SUMO, peptide 5-ELPs and vector pET3c were amplified and linked to obtain the prokaryotic expression vector pET3c-GS-peptide 5-ELPs. S4. Induction of expression of fusion protein GS-peptide 5-ELPs: The recombinant plasmid pET3c-GS-peptide 5-ELPs was transformed into the expression strain E.coli BL21(DE3) pLysS. After IPTG induction, the plasmid was broken and the supernatant containing GS-peptide 5-ELPs was collected for SDS-PAGE and WB verification. S5. Obtain the supernatant solution containing GS-peptide 5-ELPs. Based on the reversible phase transition characteristics of the ELPs tag in GS-peptide 5-ELPs, the protein is purified by temperature-regulated ITC method, and the purification effect is analyzed by SDS-PAGE electrophoresis. S6. Perform cell proliferation, cell migration and stability experiments on the protein obtained by phase transition purification.

2. The method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography as described in claim 1, characterized in that, The phase transition purification method for GS-peptide 5-ELPs in step S5 specifically includes the following steps: T1. Sample preparation: Collect the induced bacterial cells, resuspend them in 20mM PB Buffer, homogenize them under low temperature conditions, centrifuge at 12000 rpm for 20 min, and collect the supernatant for ITC purification. Enzymatic digestion of T2 and GS-peptides 5-ELPs; First phase transition purification of T3 and peptide 5-ELPs: Add NaCl to the enzyme-digested sample to a final concentration of 2M, incubate at 50℃ for at least 20 min to ensure complete salt dissolution, centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant, retain the protein precipitate, resuspend the precipitate with 0.5 times the original volume of 20 mM PB Buffer, incubate on ice for 20 min, centrifuge at 4℃ and 12,000 rpm for 10 min, and collect the supernatant containing the target protein; Further phase transition purification of T4 and peptide 5-ELPs: Add NaCl to the collected supernatant to a final concentration of 2M, incubate at 65℃ for at least 20 min to ensure complete salt dissolution, centrifuge at 12,000 rpm for 10 min at room temperature, discard the supernatant, retain the protein precipitate, resuspend the precipitate with 0.5 times the original volume of 20 mM PB Buffer, incubate on ice for 20 min, centrifuge at 4℃ and 12,000 rpm for 10 min, collect the supernatant containing the target protein, and then perform a second and third round of phase transition purification. In step T2, the enzymatic digestion of GS-peptide 5-ELPs specifically involves mixing the supernatant of the fusion protein GS-peptide 5-ELPs with Ulp1p enzyme and digesting it at 37°C for 2 hours.

3. The method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography as described in claim 1, characterized in that: In step S1, the amino acid sequence of the human type III collagen A1 chain is obtained using the uniport database, and the sequence is analyzed using bioinformatics methods.

4. The method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography as described in claim 1, characterized in that: In step S2, cell migration specifically involves: co-culturing NIH / 3T3 cells with a peptide at a concentration of 2.5 μg / mL for 48 h and observing the migration of NIH / 3T3 cells under an inverted microscope after 48 h.

5. The method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography as described in claim 1, characterized in that: In step S2, to evaluate the effect of the five polypeptide fragments on HaCaT cell adhesion, HaCaT cells were co-cultured with 1 μg / mL of the polypeptide for 48 h, and the results were detected using the CCK-8 assay.

6. The method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography as described in claim 1, characterized in that: In step S4, the recombinant plasmid is transformed into E. coli BL21(DE3) pLysS competent cells to obtain the expression strain E. coli BL21(DE3) pLysS / pET3c-GS-polypeptide 5-ELPs; positive single clones are picked from the transformed plate and transferred to 50 mL of LB medium containing ampicillin, and cultured overnight at 37°C and 220 rpm with shaking. The culture is then inoculated into 100 mL of fresh medium at a ratio of 1% and cultured for a longer period. The OD600 value is monitored periodically, and when it reaches 0.8, IPTG is added to induce the expression of the target protein.

7. The method for purifying collagen based on fluorescence tracing and non-chromatographic chromatography according to claim 2, characterized in that: After induction, the culture was transferred to centrifuge tubes and centrifuged at 8000 rpm for 5 minutes to collect the cells. Based on the wet weight of the cells, premixed hammer lysis buffer was added at a mass-to-volume ratio of 1:

10. After thorough resuspending, the cells were centrifuged again and the supernatant was collected. At the same time, the precipitate was resuspended using an equal volume of 10% SDS solution. 120 μL of the supernatant and the precipitate resuspended were taken separately, and 30 μL of 5× protein electrophoresis loading buffer was added to each. After mixing, the samples were denatured in a water bath at 100°C for 10 minutes. After the samples were cooled to room temperature, 10 μL was taken for SDS-PAGE analysis.

Images