Recombinant human-derived collagen and a method for purifying the same

Abstract

This invention provides a recombinant human collagen and its purification method. The method involves expressing recombinant human collagen using microorganisms, then heating the supernatant of the lysate or extract at a temperature above 60°C for at least 15 minutes to achieve solid-liquid separation. Finally, column chromatography using a cation exchange column is performed. Compared to commonly used purification methods employing two chromatographic columns, this invention significantly reduces time and economic costs while further improving the purity and activity of the recombinant human collagen. Furthermore, the entire method can be performed under neutral conditions, avoiding protein denaturation or the introduction of new impurities due to acidic solvents.

Description

Technical Field

[0001] This invention belongs to the field of biopharmaceutical manufacturing, separation and purification technology. More specifically, it relates to a recombinant human collagen and its purification method. Background Technology

[0002] Collagen consists of three intertwined α chains forming a right-handed superhelical structure (triple helix). Each chain contains approximately 1000 amino acid residues, with glycine (Gly) comprising 30% and proline (Pro) and hydroxyproline (Hyp) comprising 25%. This high proportion of glycine and hydroxyproline stabilizes the triple helix structure through hydrogen bonds and van der Waals forces, giving collagen high mechanical strength, thermal stability, and controllable biodegradability. Collagen is widely used in biopharmaceutical and cosmetic manufacturing. For example, in biopharmaceutical manufacturing, collagen is often used as a sustained-release carrier to encapsulate drugs, enabling continuous drug release. Similarly, in cosmetic manufacturing, collagen is frequently used to create products that repair the skin barrier, whiten the skin, and provide antioxidant benefits.

[0003] Currently, there are several methods for purifying collagen: (1) Animal-derived extraction: Collagen is extracted from animal tissues using acid or enzymatic hydrolysis. This method is low-cost, but it is prone to leaving impurities (such as lipids and nucleic acids) and pathogens (such as mad cow disease virus), and the purity is insufficient (usually <80%). It is also inefficient (requiring multiple centrifugation, filtration, salting out, etc.). (2) Chemical synthesis: Although it can precisely control the structure, it is expensive and prone to introducing chemical residues, which limits its application in fields such as biopharmaceutical manufacturing and cosmetic manufacturing. (3) Chromatography: Collagen is separated from impurities by the difference in charge, which does not introduce other impurities. However, it requires the use of two chromatographic columns, which is time-consuming and costly, and inefficient.

[0004] Therefore, there is an urgent need to find a low-cost, high-purity method that does not introduce other impurities for the efficient extraction of collagen, which is of great importance to fields such as biopharmaceutical manufacturing and cosmetics manufacturing. Summary of the Invention

[0005] This invention addresses the shortcomings of existing technologies by providing a purification method for recombinant human collagen. By performing a heat treatment before column chromatography, the extraction purity of recombinant human collagen can be further improved using only one chromatographic column, significantly reducing time and economic costs, without introducing other impurities.

[0006] The primary objective of this invention is to provide a method for purifying recombinant human collagen.

[0007] A second objective of this invention is to provide a recombinant human collagen.

[0008] A third objective of this invention is to provide the application of the above-mentioned recombinant human collagen in the preparation of medical aesthetic products, cosmetics, or pharmaceuticals.

[0009] The fourth objective of this invention is to provide a product.

[0010] The above-mentioned objective of this invention is achieved through the following technical solution:

[0011] This invention provides a purification method for recombinant human collagen, specifically: after expressing recombinant human collagen by microorganisms, the supernatant of the lysis product or extract is heated at a temperature above 60 °C for more than 15 min to separate solid and liquid, and finally column chromatography is performed using a cation exchange column.

[0012] Compared to the commonly used purification methods that employ two chromatographic columns, this invention not only significantly reduces time and economic costs but also further improves the purity and activity of recombinant human collagen. Furthermore, the method of this invention can be carried out entirely under neutral conditions, avoiding protein denaturation or the introduction of new impurities caused by acidic solvents.

[0013] Preferably, the amino acid sequence of the recombinant human collagen is shown in SEQ ID NO:1. When using this recombinant human collagen, the pH and salt concentration during the lysis process do not cause protein denaturation and have no significant impact on the purification effect; therefore, it is not necessary to specifically adjust the pH or salt concentration during the lysis process.

[0014] Preferably, the microorganism is Escherichia coli or Pichia pastoris.

[0015] As a preferred embodiment, when the microorganism is Escherichia coli, the purification method of the recombinant human collagen is as follows: after expressing recombinant human collagen in Escherichia coli, the product is lysed, then heated at a temperature above 60 °C for more than 15 min for solid-liquid separation, and finally column chromatography using a cation exchange column is performed.

[0016] Preferably, before lysis, E. coli is induced to express recombinant human collagen, and then the cells are suspended in sodium phosphate buffer.

[0017] Optionally, the inducing agent used is isopropyl-β-D-thiogalactoside.

[0018] Optionally, the pH of the sodium phosphate buffer solution is 4 to 9, for example, 4, 5, 6, 7, 7.5, 8, or 9.

[0019] Optionally, the sodium phosphate buffer contains 100 to 500 mM NaCl, for example, 100, 200, 300, 400, or 500 mM NaCl.

[0020] Preferably, the pyrolysis is achieved by ultrasonic pyrolysis or pressure pyrolysis.

[0021] Optionally, the frequency of the ultrasound is 18–22 kHz, for example, 20 kHz.

[0022] Optionally, the power of the ultrasound is 25% to 35%, for example, 30%.

[0023] Optionally, the ultrasound mode is a pulse mode, such as 30 seconds on / 30 seconds off.

[0024] Optionally, the pressure used for pressurization is 70 to 90 MPa, for example, 80 MPa.

[0025] Optionally, the number of cycles of pressurized pyrolysis is 2 to 4, for example, 3.

[0026] As another preferred embodiment, when the microorganism is Pichia pastoris, the purification method of the recombinant human collagen is as follows: after expressing recombinant human collagen by Pichia pastoris, the supernatant of the product is extracted, and then heated at a temperature above 60°C for more than 15 minutes to separate the solid and liquid, and finally column chromatography is performed using a cation exchange column.

[0027] Preferably, the heating temperature is above 75°C.

[0028] Preferably, the heating is achieved via a water bath.

[0029] Preferably, the solid-liquid separation is performed by centrifugation.

[0030] Optionally, the centrifugation speed is 12,000 to 18,000 × g, for example, 15,000 × g.

[0031] Optionally, the centrifugation time is 25 to 35 minutes, for example, 30 minutes.

[0032] Preferably, before column chromatography, the system is diluted to a conductivity of 0.8–1.2 ms / cm, most preferably 1 ms / cm.

[0033] Alternatively, the dilution may be performed using water.

[0034] Preferably, the cation exchange column is SP Sepharose FF.

[0035] Preferably, the elution buffer used in the column chromatography is sodium phosphate buffer.

[0036] Optionally, the concentration of the sodium phosphate buffer is 18–22 mM, for example 20 mM.

[0037] Optionally, the pH of the sodium phosphate buffer solution is 7.

[0038] Optionally, the sodium phosphate buffer contains 100–500 mM NaCl, for example, 300 mM NaCl.

[0039] The recombinant human collagen of this invention, with the amino acid sequence shown in SEQ ID NO:1, not only achieves efficient soluble expression, avoiding complex and inefficient refolding operations, but also maintains high activity such as promoting cell adhesion. Furthermore, the purity of the recombinant human collagen does not change significantly when the heating temperature increases from 75 °C, and pH value and salt concentration do not significantly affect the purity of the recombinant human collagen of this invention, indicating that the recombinant human collagen of this invention has extremely high thermal stability, acid and alkali resistance, and salt resistance, and is widely applicable to the preparation of cosmetics or pharmaceuticals. Therefore, a recombinant human collagen with the amino acid sequence shown in SEQ ID NO:1, the application of the above-mentioned recombinant human collagen in the preparation of medical aesthetic products, cosmetics, or pharmaceuticals, and a product containing the above-mentioned recombinant human collagen should all be within the protection scope of this invention.

[0040] The present invention has the following beneficial effects:

[0041] Compared to the commonly used purification methods that employ two chromatographic columns, this invention not only significantly reduces time and economic costs but also further improves the purity and activity of recombinant human collagen. Furthermore, the method of this invention can be carried out entirely under neutral conditions, avoiding protein denaturation or the introduction of new impurities caused by acidic solvents. Attached Figure Description

[0042] Figure 1 These are electrophoresis images numbered 1 to 14.

[0043] Figure 2 These are electrophoresis images numbered 15 to 28.

[0044] Figure 3 These are electrophoresis images numbered 29 to 40.

[0045] Figure 4 These are electrophoresis images numbered 41 to 49.

[0046] Figure 5 These are electrophoresis images numbered 50-61.

[0047] Figure 6 This is the result of the average analysis of heating temperatures.

[0048] Figure 7 This is the result of the mean analysis of heating time.

[0049] Figure 8This is the mean analysis result for pH values.

[0050] Figure 9 The results are the average values ​​of the NaCl concentration.

[0051] Figure 10 The results are from the high-performance liquid chromatography (HPLC) of Scheme 1.

[0052] Figure 11 The results are from the high-performance liquid chromatography (HPLC) of Scheme 2.

[0053] Figure 12 The results are from the high-performance liquid chromatography (HPLC) of scheme 3.

[0054] Figure 13 The results are from the high-performance liquid chromatography (HPLC) of scheme 4.

[0055] Figure 14 The melting curve of wild-type recombinant human collagen.

[0056] Figure 15 The melting curve of the improved recombinant human collagen. Detailed Implementation

[0057] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0058] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0059] Example 1: Construction of Escherichia coli expressing recombinant human collagen

[0060] Based on the recombinant human collagen amino acid sequence shown in SEQ ID NO:1, the amino acid sequence of recombinant human collagen (synthesized by Qingke Biotechnology Co., Ltd.) was obtained by repeating the sequence three times after the start codon. A recombinant plasmid expressing recombinant human collagen was constructed using pET26b as the recombinant expression vector. The recombinant plasmid was then transformed into BL21 (DE3) competent cells via heat shock, inoculated onto LB agar plates containing 0.01 wt% kanamycin, and cultured statically at 37 °C for 20 h to obtain single colonies. Single-clone cells were selected, activated, and amplified to obtain seed culture. The seed culture was inoculated into TB medium at a volume ratio of 1:100 and cultured at 37 ℃ until the OD600 reached 0.4. Isopropyl-β-D-thiogalactoside was added to a final concentration of 1 mM, and protein expression was induced at 32 ℃ for 20 h. Then, the cells were centrifuged at 8000×g for 30 min, collected, and stored at -80 ℃.

[0061] Example 2: Preliminary purification of recombinant human collagen

[0062] Cells collected in Example 1 were suspended in sodium phosphate buffer with a specific pH and NaCl concentration, and then subjected to sonication lysis in a pulse mode with a frequency of 20 kHz, a power of 30%, and a 30-second on / 30-second off cycle. 0.2 mL of the lysis buffer was transferred to a PCR centrifuge tube and incubated in a PCR thermal cycler at a set temperature for a period of time. After incubation, the PCR centrifuge tube was removed and centrifuged at 11000×g for 10 min. 2 μL of the supernatant obtained from centrifugation was taken for gel electrophoresis, stained, and photographed. The purity of the recombinant human collagen was further analyzed using ImageLab software.

[0063] Using heating temperature, heating time, pH value, and NaCl concentration as the four factors in the orthogonal experiment, with 6 to 7 levels respectively, and the purity of recombinant human collagen as the evaluation index, the orthogonal experiment was conducted.

[0064] Electrophoresis images are shown in Figures 1-5, where... Figure 1 These are electrophoresis images numbered 1 to 14. Figure 2 These are electrophoresis images numbered 15-28. Figure 3 These are electrophoresis images numbered 29-40. Figure 4 These are electrophoresis images numbered 41 to 49. Figure 5 These are electrophoresis images numbered 50-61.

[0065] The results of the orthogonal experiment are shown in Table 1. Mean analysis and variance analysis were performed on the four factors in Table 1. The mean analysis results are as follows: Figures 6-9 As shown, the results of the analysis of variance are presented in Tables 2-5. Among them, Figure 6 The results are the average values ​​of the heating temperatures. Figure 7 The results are the average values ​​of the heating time. Figure 8 The results are the mean values ​​of the pH values. Figure 9 The results are the average values ​​of the NaCl concentration.

[0066] Table 1. Results of the orthogonal experiment

[0067]

[0068]

[0069] Table 2. Results of Analysis of Variance for Heating Temperature

[0070]

[0071] Table 3. Results of Analysis of Variance for Heating Time

[0072]

[0073] Table 4. Results of analysis of variance for pH values

[0074]

[0075] Table 5. Results of ANOVA for NaCl concentration

[0076]

[0077] Analyze Tables 2-5. Figures 6-9 It can be known that:

[0078] (1) Heating temperature is one of the biggest factors affecting the orthogonal experiment. When the temperature is 60 °C, the purity of recombinant human collagen is significantly improved. When the temperature is 75 °C, the purity of recombinant human collagen reaches its highest level. When the temperature is further increased, the purity of recombinant human collagen does not change significantly. This also proves that the recombinant human collagen of the present invention has extremely high thermal stability.

[0079] (2) Heating time is one of the biggest factors affecting this orthogonal experiment. When the time is 15 min, the purity of recombinant human collagen is significantly improved. When the time is further extended, the purity of recombinant human collagen does not change significantly (57% to 62%).

[0080] (3) Significant differences in pH and salt concentration during the pyrolysis process p All values ​​are greater than 0.05, indicating that pH value and salt concentration will not have a significant impact on the purity of recombinant human collagen. That is, the recombinant human collagen of the present invention has extremely high acid and alkali resistance and salt resistance.

[0081] Example 3 Purification of recombinant human collagen

[0082] I. Purification Methods

[0083] (1) Scheme 1: Purification using two chromatographic columns at room temperature (25 ℃)

[0084] Cells collected in Example 1 were suspended in sodium phosphate buffer with pH 7.5 and NaCl concentration of 0.1 M, and then subjected to high-pressure lysis in a high-pressure homogenizer at 80 MPa for 3 cycles. After centrifugation at 15000×g for 30 min, the supernatant was diluted with water to a conductivity of 1 ms / cm. Then, flow-through was performed using an anion exchange column (Q Sepharose FF) (some non-target proteins were adsorbed into the packing material during this process). The target protein flow-through was then eluted with 20 mM sodium phosphate buffer (pH=7) containing 300 mM sodium chloride and washed with a cation exchange column (SP Sepharose FF). The resulting eluent was the target protein.

[0085] (2) Scheme 2: Purification using a single chromatographic column at room temperature (25 ℃)

[0086] Cells collected in Example 1 were suspended in sodium phosphate buffer with pH 7.5 and NaCl concentration of 0.1 M, and then subjected to high-pressure lysis in a high-pressure homogenizer at 80 MPa for 3 cycles. After centrifugation at 15000×g for 30 min, the supernatant obtained by centrifugation was diluted with water to a conductivity of 1 ms / cm. Then, using 20 mM sodium phosphate buffer (pH=7) containing 300 mM sodium chloride as the elution buffer, the cells were adsorbed and washed with a cation exchange column (SP Sepharose FF). The resulting eluent was the target protein.

[0087] (3) Scheme 3: Purification under heating conditions

[0088] The cells collected in Example 1 were suspended in sodium phosphate buffer with a pH of 7.5 and a NaCl concentration of 0.1 M. Then, they were subjected to high-pressure lysis in a high-pressure homogenizer at 80 MPa for 3 cycles. The cell lysate was then heated at 75 °C for 30 min using a water bath method and centrifuged at 15000×g for 30 min. The resulting supernatant was the target protein.

[0089] (4) Scheme 4: Purification using a single chromatographic column under heating conditions

[0090] Cells collected in Example 1 were suspended in sodium phosphate buffer with a pH of 7.5 and a NaCl concentration of 0.1 M. They were then subjected to high-pressure lysis in a high-pressure homogenizer at 80 MPa for 3 cycles. The cell lysate was then heated at 75 °C for 30 min using a water bath method and centrifuged at 15000 × g for 30 min. The supernatant obtained by centrifugation was diluted with water to a conductivity of 1 ms / cm. The eluent was then used to adsorb and wash impurities using a cation exchange column (SP Sepharose FF) with 20 mM sodium phosphate buffer (pH=7) containing 300 mM sodium chloride. The resulting eluent was the target protein.

[0091] II. Purification Results

[0092] (1) Purity of recombinant human collagen

[0093] The target proteins obtained from schemes 1 to 4 were filtered through a 0.22 μm syringe filter. The total protein content in the solution sample was measured using biuret reagent. The solution sample was then diluted to 0.1 μg / mL with 50 mM sodium phosphate buffer (pH=7) containing 150 mM sodium chloride. 10 μL of the diluted solution sample was taken and the content of recombinant human collagen was determined by high performance liquid chromatography (mobile phase: 50 mM sodium phosphate buffer (pH=7) containing 150 mM sodium chloride). The purity of recombinant human collagen in the solution sample was then calculated.

[0094] The results are as follows Figures 10-13 As shown, where, Figure 10 The results are from the high-performance liquid chromatography (HPLC) of Scheme 1. Figure 11 The results are from the high-performance liquid chromatography (HPLC) of Scheme 2. Figure 12 The results are from the high-performance liquid chromatography (HPLC) of scheme 3. Figure 13 The results are shown in the high-performance liquid chromatography (HPLC) results for Scheme 4. It can be seen that the purities of Schemes 1 through 4 are 98.2%, 40.4%, 47.7%, and 99.6%, respectively. This indicates that Scheme 4, purified according to the method of this invention, yields the highest purity of recombinant human collagen, even slightly higher than Scheme 1, which uses two chromatographic columns for purification. In other words, the method of this invention can further improve the purity of recombinant human collagen while significantly reducing time and economic costs.

[0095] (2) Activity of recombinant human collagen

[0096] The target proteins obtained from schemes 1 and 4 were diluted with pure water to a concentration of 500 μg / mL. The diluent was added to a 96-well plate at a rate of 50 μL / well. After incubation at 37 °C for 2 h, the diluent was removed, and then trypsin-digested HS27 cells (103) were added. 4(100 cells / well) were used as the experimental group. The group using PBS instead of diluent was the negative control group, and the group using PBS instead of diluent and without HS27 cells was the blank control group.

[0097] The cells were then incubated at 37 ℃ in a CO2 incubator for 3 h. After washing twice with PBS to remove unadhered cells, 200 μL of DMEM medium and 10 μL of CCK-8 reagent were added. The cells were incubated at 37 ℃ in a 5% CO2 incubator for 1 h. The absorbance (A) of each well at 450 nm was then read using a microplate reader. Finally, the cell adhesion rate (%) was calculated as follows: (A...) 实验组 -A 空白对照组 ) / (A 阴性对照组 -A 空白对照组 The cell adhesion promotion rates of schemes 1 and 4 were calculated by multiplying the percentage by 100%.

[0098] The results showed that the cell adhesion promotion rates of Scheme 1 and Scheme 4 were 350% and 358%, respectively, indicating that the recombinant human collagen obtained by Scheme 4, which was purified according to the method of the present invention, has excellent cell adhesion promotion activity, and is even slightly better than Scheme 1, which was purified using two chromatographic columns. That is, the method of the present invention can further improve the cell adhesion promotion activity of recombinant human collagen while significantly reducing time and economic costs.

[0099] Example 4: Thermal stability of recombinant human collagen

[0100] Based on the amino acid sequence of wild-type recombinant human collagen as shown in SEQ ID NO:3, the amino acid sequence of wild-type recombinant human collagen as shown in SEQ ID NO:4 was obtained by repeating the sequence three times after the start codon (synthesized by Qingke Biotechnology Co., Ltd., human collagen type 17 a1 chain UniProtKB:Q9UMD9).

[0101] This embodiment compares the thermal stability of recombinant human collagen (amino acid sequence as shown in SEQ ID NO:2, hereinafter referred to as "improved recombinant human collagen") purified in Scheme 4 of Example 3 with that of wild-type recombinant human collagen (amino acid sequence as shown in SEQ ID NO:4). First, *E. coli* expressing wild-type recombinant human collagen was constructed according to the method in Example 1. Then, the wild-type recombinant human collagen was purified according to Scheme 4 of Example 3. The melting stability of the wild-type and improved recombinant human collagen was determined using a Protein Thermal Shift™ (PTS) kit. The results were then analyzed based on the melting curves (…). Figures 14-15 The melting temperature Tm was calculated, and then the thermal stability of the two was compared.

[0102] The results showed that the melting temperature Tm of wild-type recombinant human collagen was 80.6497 ℃, while the melting temperature Tm of the improved recombinant human collagen was higher than 100 ℃, indicating that the thermal stability of the recombinant human collagen with the amino acid sequence shown in SEQ ID NO:1 was significantly higher than that of wild-type recombinant human collagen.

[0103] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for purifying recombinant human collagen, characterized in that, After recombinant human collagen is expressed by microorganisms, the supernatant of the lysate or extract is heated at 60–100 °C for 15–120 min for solid-liquid separation, and finally column chromatography is performed using a cation exchange column; wherein, the amino acid sequence of the recombinant human collagen is shown in SEQ ID NO:

2.

2. The purification method according to claim 1, characterized in that, The heating temperature is 75–100 °C.

3. The purification method according to claim 1, characterized in that, Prior to column chromatography, the system was diluted to a conductivity of 0.8–1.2 ms / cm.

4. The purification method according to claim 1, characterized in that, The cation exchange column is SP Sepharose FF.

5. The purification method according to claim 1, characterized in that, The elution buffer used in the column chromatography was sodium phosphate buffer.

6. The purification method according to claim 1, characterized in that, The microorganisms mentioned are Escherichia coli or Pichia pastoris.

7. A recombinant human collagen, characterized in that, The amino acid sequence is shown in SEQ ID NO:

2.

8. The use of the recombinant human collagen according to claim 7 in the preparation of medical aesthetic products or cosmetics.

9. A medical aesthetic product or cosmetic, characterized in that, Contains the recombinant human collagen as described in claim 7.

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