A method for preparing recombinant humanized type III collagen with a triple helix structure

Abstract

This invention relates to bioengineering, specifically disclosing a method for preparing recombinant humanized type III collagen with a triple-helix structure, comprising the following steps: S1, constructing a ppICZαA recombinant expression plasmid containing a human type III collagen fragment, and transforming the recombinant expression plasmid into yeast; S2, screening high-copy transformant strains from the strain and performing shake-flask level induction expression tests; S3, conducting high-density fermentation in a fermenter to obtain a large amount of secretible target protein. This invention, by constructing a ppICZαA recombinant expression plasmid containing a human type III collagen fragment and electroporating it into a yeast expression system, screens for yeast strains capable of expressing recombinant humanized type III collagen fragments, and conducts high-density fermentation in a fermenter, followed by purification after fermentation, yields pure recombinant collagen with a triple-helix structure, which can be applied in skincare products, functional skincare products, medical devices, and biomedical materials.

Description

Technical Field

[0001] This invention relates to bioengineering, and more specifically to a method for preparing recombinant humanized type III collagen with a triple helix structure. Background Technology

[0002] Collagen is a major structural protein in the extracellular matrix of animals, possessing multiple biological functions. It is widely distributed in the connective tissues of the human body, making up one-third of human protein and three-quarters of the dry weight of skin. It plays a crucial role in connecting bones, skin, and joints. At least 28 types of collagen have been identified, mainly classified as fibrous collagen, reticular collagen, beaded filamentous collagen, anchoring fibrous collagen, membrane proteins, and multiplexin collagen. Different structures result in different functions of collagen. For example, type I collagen primarily supports skin contours, while type III collagen determines skin elasticity. The most common types are: Type I (dermis, bones, tendons, ligaments, etc., the most abundant, accounting for approximately 80%–90% of total collagen), Type II (cartilage, vitreous humor, etc.), Type III (skin, blood vessels, intestines, etc.), Type IV (basement membrane, etc.), and Type V (bone, dermis, cornea, placenta, etc.).

[0003] The basic unit of collagen is procollagen, which is assembled from multiple procollagen molecules to form a fibrous protein. Procollagen consists of three polypeptide chains, and the primary structure of the polypeptide chain has a repeating unit (Gly-XY)n, where G is glycine (Gly), which accounts for about 30% of the amino acid content of collagen, and X and Y are usually proline and hydroxyproline, and less commonly lysine and hydroxylysine. The three procollagen polypeptide chains can intertwine with each other through interchain hydrogen bonds to form a stable triple helix structure.

[0004] In recent years, collagen has been widely used in fields such as biological scaffold materials, cosmetics, food and medical devices. The development of collagen-based products mainly focuses on bone repair, skin wound repair dressings, tendon repair, drug delivery and beauty.

[0005] Currently, based on their origin, collagen is broadly classified into animal-derived collagen and recombinant collagen. Animal-derived collagen mainly comes from terrestrial and marine animals, possessing advantages such as mature processing and high activity, but also disadvantages such as high immunogenicity and viral susceptibility. Recombinant collagen refers to the protein obtained by cloning the human collagen gene into a selected expression vector, transforming it into expression cells, and finally purifying it. Expression systems mainly include prokaryotes (primarily Escherichia coli), yeast, plants, baculoviruses, and mammalian cell expression systems.

[0006] Currently, expression systems primarily utilize Escherichia coli and yeast. While microbial fermentation systems using E. coli and yeast are low-cost, have short cycles, are relatively easy to cultivate, and are more readily commercially viable, the pyrogens (such as endotoxins) produced by E. coli make it difficult to apply the expressed products clinically. Target proteins are often expressed in inclusion body form, even if intracellularly soluble, resulting in a large amount of host protein, making product purification difficult. For example, the collagen purified by Jiangxi Chongshan Biological Products Co., Ltd. in patent CN117510618 B only achieved a purity of 89%; and the collagen purified by Xi'an Giant Biogene Technology Co., Ltd. in patent CN119775391 A only achieved a purity of 95%, with neither patent mentioning endotoxin removal. Furthermore, the post-translational modification system of prokaryotic expression systems is imperfect, resulting in low biological activity of the expressed products. Yeast expression systems do not have the problem of endotoxin pyrogens from the strain itself, and have molecular chaperones and post-translational modifications that are present in eukaryotes. Therefore, they have advantages such as good safety, stable quality, no pyrogens, simple purification, low cost, and good reproducibility. However, there are difficulties in preparation, including the selection of gene fragments, the construction of triple helix structures, and the easy degradation of the target protein. Summary of the Invention

[0007] The purpose of this invention is to provide a method for preparing recombinant humanized type III collagen with a triple helix structure, which enables high-yield production of this collagen in yeast, has the easiest purification method, extremely low cost, good degradation resistance, and further improves its stability.

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing recombinant humanized type III collagen with a triple helix structure, comprising the following steps:

[0009] S1. Construct a recombinant expression plasmid containing a human type III collagen fragment ppICZαA, mainly including the construction of recombinant expression plasmid 6×His-DDDDK-2C×3, and after linearizing the recombinant expression plasmid, transform it into yeast X33. Then, use PCR to identify and screen recombinant strains containing the target gene.

[0010] S2. High-copy transformant strains were screened using bleomycin (Zeocin) at a concentration of 300 μg / mL and induced expression was performed at the shake-flask level. The strains with the highest protein expression levels were screened by SDS-PAGE.

[0011] S3. The strain with the highest expression level obtained in S2 is then subjected to high-density fermentation in a fermenter to obtain a large amount of secretible target protein (2C×3). Specifically, fermentation is carried out at an inoculum volume of 10% of the fermenter volume. The culture medium consists of: 26.7 mL / L 85% phosphate, 0.93 g / L calcium sulfate dihydrate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 4.13 g / L potassium hydroxide, 40 g / L glycerol, and 14 mL / L PTM1, wherein PTM1 is sterilized by membrane filtration.

[0012] The cultivation process also includes: fed culture medium: 650 g / L glycerol, 800 g / L methanol (12 mL PTM1 added per L methanol). The PTM1 formulation is: 6 g / L copper sulfate pentahydrate, 0.08 g / L sodium iodide, 3 g / L manganese sulfate monohydrate, 0.2 g / L sodium molybdate dihydrate, 0.02 g / L boric acid, 0.5 g / L cobalt chloride, 20 g / L zinc chloride, 65 g / L ferrous sulfate heptahydrate, and 0.2 g / L biotin. The PTM1 cultivation conditions are: 30℃, pH ≥ 5 controlled with ammonia, and aeration rate of 1 L / min (compressed air).

[0013] Feeding process control: After the initial carbon source is depleted, add 650 g / L glycerol until the OD600 reaches 95-105. Add 2 g / L methanol to initiate the switching phase, reducing the glycerol feeding rate to 0 in four stages. After the carbon source is depleted, add methanol in batches to initiate the induction phase. Induction ends after 36-48 hours. The cell percentage in the fermentation broth is approximately 20%. Collect the fermentation broth supernatant for SDS-PAGE analysis; the purity of the target protein should be at least ≥90%.

[0014] S4. Purify the large quantity of secretible target protein (2C×3) using methods including G25 molecular sieve filtration, ammonium sulfate precipitation, activated carbon adsorption, hollow fiber ultrafiltration concentration, or ion exchange chromatography to obtain recombinant humanized type III collagen. Specifically, this includes:

[0015] A. Desalting was performed using molecular sieve G25. The desalting buffer consisted of 20 mM Tris, 100 mM NaCl, and pH 8.0, resulting in purified protein.

[0016] B. The pigment was removed by ammonium sulfate precipitation combined with activated carbon adsorption to obtain purified protein;

[0017] C. After microfiltration with 0.45μm and 0.22μm hollow fibers, the protein was concentrated by ultrafiltration with 5kDa hollow fibers to obtain the purified protein.

[0018] D. Further purification using ion exchange chromatography packing material can further improve the purity of the target protein, with a protein purity ≥95%. Combined with ultrafiltration to control endotoxin <0.5 EU / mg, it can meet the requirements for raw materials for Class II medical devices.

[0019] E. Further optimize the conditions for controlling ion exchange, collect target proteins with higher purity (≥99%), and combine this with ultrafiltration to control endotoxin levels to <0.05 EU / mg, which can meet the requirements for raw materials for Class III medical devices.

[0020] In the above technical solution, the method for preparing recombinant humanized type III collagen with a triple helix structure provided by the present invention has the following beneficial effects:

[0021] 1. The recombinant humanized type III collagen fragment obtained through analysis and optimization screening is relatively stable in the fermentation broth, without exhibiting collagen degradation after fermentation. Several other collagen fragments selected simultaneously in our laboratory all showed significant protein degradation during fermentation. Therefore, this provides the greatest possible inherent guarantee for subsequent purification, preservation, and transportation operations.

[0022] 2. High-density fermentation is carried out in the fermenter, which enables high-yield, soluble expression of recombinant humanized type III collagen in yeast, with a yield of up to 7.5 g / L, which is a high expression level in the industry. It is feasible and practical to scale up to 15 tons of fermentation production.

[0023] 3. The purity of the recombinant humanized type III collagen obtained from fermentation broth is at least ≥90%. Only the simplest replacement of the preservation buffer or chromatography treatment is needed to achieve a purity of 95% or higher. Therefore, it has an extremely easy purification method with few purification steps, which directly reduces the purification cost to the greatest extent.

[0024] 4. The endotoxin detection method of this invention yields recombinant humanized type III collagen with controllable endotoxin content (<1000 EU / mg, 100 EU / mg, etc.) or even below 5 EU / mg, meeting the requirements for use as a cosmetic raw material. Further purification reduces the endotoxin content to below 0.5 EU / mg, meeting the requirements for use as a Class II medical device raw material. Further refinement reduces the endotoxin content to below 0.05 EU / mg, meeting the requirements for use as a Class III medical device raw material. In terms of endotoxin level control, this invention represents a top-tier level among all patents.

[0025] 5. The recombinant humanized type III collagen obtained by this invention, after purity testing, can produce a product with a purity >99% according to SDS-PAGE and HPLC analysis. This is higher than the protein purity >90% prepared by conventional methods, such as the collagen purified by CN119775391 A, which only reaches a purity of 89%, representing a significant improvement. For target proteins with relatively relaxed purity requirements of 90% and 95%, the preparation difficulty is lower, and the preparation target can be quickly achieved using mature processes.

[0026] 6. The recombinant humanized type III collagen obtained by this invention, after stability testing, can be stored in dry powder form at 40°C for more than 40 days under accelerated experimental conditions. It is generally believed that it can be stored at 4°C for at least one year.

[0027] 7. The recombinant humanized type III collagen obtained by the preparation method of the present invention has excellent water solubility and stability, high purity, and effectively avoids the problems of endotoxin pyrogens. It is free of animal-derived viruses, the preparation method is simple, and high-yield collagen can be obtained at low cost. It meets the requirements for high-purity collagen in cosmetic raw materials and medical device raw materials. Attached Figure Description

[0028] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0029] Figure 1 The image shows the PCR amplification band pattern of the recombinant plasmid ppICZα-6×His-DDDDK-2C×3.

[0030] Figure 2 The PCR band pattern during high copy selection after electroporation of ppICZα-6×His-DDDDK-2C×3 at X33;

[0031] Figure 3 Results of shake-flask expression identification of recombinant humanized type III collagen (SDS-APGE);

[0032] Figure 4 Results of fermentation expression of recombinant humanized type III collagen in a 10L fermenter (SDS-APGE);

[0033] Figure 5 Results of fermentation expression of recombinant humanized type III collagen in a 50L fermenter (SDS-APGE);

[0034] Figure 6Results of fermentation expression of recombinant humanized type III collagen in a 15-ton fermenter (SDS-APGE);

[0035] Figure 7 Results of G25 desalting for purification of recombinant humanized type III collagen (SDS-APGE);

[0036] Figure 8 Results of ammonium sulfate precipitation and activated carbon adsorption for the purification of recombinant humanized type III collagen (SDS-APGE);

[0037] Figure 9 Results of hollow fiber ultrafiltration concentration for purification of recombinant humanized type III collagen (SDS-APGE);

[0038] Figure 10 Total ion chromatogram of recombinant humanized type III collagen as identified by LC-MS / MS mass spectrometry.

[0039] Figure 11 This is a schematic diagram of the purity test results for recombinant humanized type III collagen (SDS-PAGE).

[0040] Figure 12 This is a schematic diagram of the purity detection results (HPLC) for recombinant humanized type III collagen.

[0041] Figure 13 The results of stability testing for recombinant humanized type III collagen;

[0042] Figure 14 The image shows a circular dichroism chromatogram of recombinant humanized type III collagen. Detailed Implementation

[0043] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. 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 of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0044] A method for preparing recombinant humanized type III collagen with a triple helix structure, comprising:

[0045] Example 1

[0046] 1. Sequence selection

[0047] This invention provides a recombinant humanized type III collagen and its preparation method. The amino acid sequence 2C×3 is selected from 36 amino acids repeated 3 times in the triple helix region of human type III collagen (NM_000090.4). It contains 3 GER and 3 GFP cell binding motifs and 3 RGD cell adhesion motifs. It has good water solubility, which is beneficial to promoting cell adhesion and improving cell repair function.

[0048] 2. Construction and identification of recombinant expression plasmids

[0049] A 6×His-DDDDK gene was added to the 5' end of the humanized type III collagen fragment 2C×3 gene. The Pichia pastoris codon was optimized, and EcoRI and NotI restriction sites were added to the 5' and 3' ends, respectively. The gene was cloned into the vector pPICZαA via 5'EcoRI and 3'NotI to construct the recombinant plasmid pPICZαA-6×His-DDDDK-2C×3. The gene was synthesized by Genewiz Biotechnology Co., Ltd.

[0050] The plasmid was extracted from E. coli skeletal culture containing the recombinant plasmid by PCR and the quality of the extracted plasmid was verified by nucleic acid electrophoresis (power supply of DYY-6D electrophoresis instrument, Beijing Liuyi Biotechnology).

[0051] Nucleic acid electrophoresis results as follows Figure 1 As shown, with routine spotting, the plasmid bands are very bright and uniform, indicating good quality and suitability for the following linearization experiments. Lane 1: M is DNA standard (DL2000 Plus DNA Marker, Vazyme), from top to bottom: 2000, 1500, 1000, 750, 500, 250, 100 bp; Lane 2: PCR verification sample of the extracted recombinant plasmid.

[0052] 3. Linearization of recombinant plasmids, followed by electroporation of X33 plasmids.

[0053] Take 20 μg of recombinant plasmid, linearize it by digestion with PmeI (NEB) enzyme, digest it at 37℃ for 3 h, and then purify it by agarose gel extraction.

[0054] Table 1. Configuration of Enzyme Digestion Linearization System

[0055]

[0056] Prepared Pichia pastoris X33 competent cells (laboratory strain) were slowly revived on ice. A sterile electroporation cuvette was prepared in advance, irradiated with UV light in a clean bench for at least 15 minutes, and then pre-cooled on ice. 10 μL of the linearized recovered plasmid was added to the Pichia pastoris X33 competent cells, gently mixed, and all competent cells were aspirated using a pipette and transferred to the pre-cooled electroporation cuvette. The cuvette was then inserted into the electroporator (electrode converter), and electroporation was performed at 1500V. After electroporation, 1 mL of LYPD medium was added to the cuvette, gently mixed with a pipette tip, and all liquid was aspirated and transferred to the original competent cell EP tubes. The cells were incubated at 30°C and 160 rpm for 2 hours. 200 μL of the revived bacterial culture was plated on YPD medium containing 300 μg / mL Zeocin (Thermo Fisher Scientific, requires storage at -20°C protected from light) and incubated at 30°C for at least 2 days.

[0057] 4. High-copy screening and identification

[0058] Uniform yeast clones were selected from revived YPD plates and transferred to YPD medium containing 100 μg / mL Zeocin resistance. The clones were incubated overnight at 30°C and 200 rpm. Eleven clones were selected, their genomes were extracted, and genotyping was performed by PCR, followed by verification by 1% agarose gel electrophoresis.

[0059] Electrophoresis results as follows Figure 2 As shown, clone 1 showed two bands in the electrophoresis result, which did not meet the target size. Clones 2 through 11 showed single PCR bands, especially clones 3, 6, and 11, whose electrophoretic bands were very bright. The band sizes were as expected. Lane 1: M is DNA standard, from top to bottom 2000, 1500, 1000, 750, 500, 250, 100 bp; Lane 2: clone 1, Lane 3: clone 2, Lane 4: clone 3, Lane 5: clone 4, Lane 6: clone 5, Lane 7: clone 6, Lane 8: clone 7, Lane 9: clone 8, Lane 10: clone 9, Lane 11: clone 10, Lane 12: clone 11.

[0060] Example 2

[0061] 100 μL of the resuscitation solution was transferred to 100 mL of BMGY medium and cultured on a shaker (YC-05 three-layer horizontal shaker, Suzhou Jiemei Electronics) at a small shake flask level (250 mL shake flask) for 24 h at 30 °C and 200 rpm. Then, methanol (Thermo Fisher Scientific, HPLC Grade) was added every 24 h to a final concentration of 1%. The culture was continued for 2 days, and protein expression was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Electrophoresis equipment: DYY-6D electrophoresis apparatus; power supply: Beijing Liuyi Biotechnology; stacking gel concentration: 5%; separating gel concentration: 12%.

[0062] Electrophoresis results as follows Figure 3 As shown, all 11 clones exhibited distinct and relatively uniform protein bands, >90%. Among them, 2C×3-9 (the 9th clone of 2C×3) showed the highest expression level according to SDS-PAGE analysis after shake-flask induction. Lane 1: 2C×3-1 (referring to the first clone of 2C×3), Lane 2: Protein Marker, Lane 3: 2C×3-2 (referring to the second clone of 2C×3), Lane 4: 2C×3-3 (referring to the third clone of 2C×3), Lane 5: 2C×3-4 (referring to the fourth clone of 2C×3), Lane 6: 2C×3-5 (referring to the fifth clone of 2C×3), Lane 7: 2C... Lane 8: 2C×3-6 (referring to the 6th clone of 2C×3), Lane 9: 2C×3-8 (referring to the 8th clone of 2C×3), Lane 10: 2C×3-9 (referring to the 9th clone of 2C×3), Lane 11: 2C×3-10 (the 10th clone of 2C×3), Lane 12: 2C×3-11 (referring to the 11th clone of 2C×3).

[0063] Example 3

[0064] The clone with the highest expression level selected in Example 2 was plated on YPD plates and cultured. Single colonies were picked and transferred to test tubes and cultured at 30°C and 200 rpm for 14-18 h to obtain primary seed culture. 600 μL of primary seed culture was transferred to 60 mL of YPD medium for small-scale amplification culture at 30°C and 200 rpm for 6-10 h to obtain secondary seed culture.

[0065] Secondary seed stock was inoculated into the fermenter (10L fermenter, MC-JGF-10L, Beijing Mancang Technology) at a 10% inoculation rate. BSM medium was used in the fermenter. Culture conditions were: 30℃, pH ≥ 5 controlled with ammonia, and aeration rate of 1 VVM (compressed air). After the initial carbon source was depleted, 650 g / L glycerol was added. A strategy linking dissolved oxygen and rotation speed was used to maintain 30%-50% dissolved oxygen. When the OD600 (OD was measured using a UV-Vis spectrophotometer, model: Purkinje TU-1810, Beijing Purkinje General Instrument Co., Ltd.) reached 95-105, 2 g / L methanol was added in one go. Simultaneously, the glycerol feeding rate was gradually reduced to 0 g / L in four stages. After the carbon source was depleted (residual sugar concentration ≤ 0.5 g / L), methanol was added in batches, maintaining the rotation speed at maximum, 6-12 g / L each time. Bacterial cell growth lasted 20-24 hours, and induction was performed for 36-48 hours or longer. Protein expression was detected by SDS-PAGE, and the separating gel concentration was 12%.

[0066] Electrophoresis results from a 10L fermenter are as follows: Figure 4 As shown, protein expression levels gradually increased over time, with protein purity in the fermenter at least >90% and no obvious impurities. Lane 1: Protein Marker; Lane 2: 2C×3-40h (for 40h expression level detection); Lane 3: 2C×3-48h (for 48h expression level detection); Lane 4: 2C×3-64h (for 64h expression level detection).

[0067] Fermentation was conducted in a 50L fermenter (0.05m³ fermenter, product number H22-015, Lianyungang Hechang Machinery Co., Ltd.; fermenter control system HC-BIO-8000, Lianyungang Hechang Bioengineering Equipment Co., Ltd.). BSM medium was used, and the culture conditions were: 30℃, pH ≥ 5 controlled with ammonia, initial aeration rate of 1 VVM (compressed air), and tank pressure of 0.05 MPa. As the fermentation time increased, the aeration rate and rotation speed were adjusted to maintain dissolved oxygen at 30%-50%. The feeding strategy was the same as that for the 10L fermenter. Protein expression was detected by SDS-PAGE, and the separating gel concentration was 12%.

[0068] Electrophoresis results from a 50L fermenter are as follows: Figure 5 As shown, the protein expression level gradually increased with time, reaching its peak between 112 and 120 hours. The protein expression purity at each time point was >90%, and even after fermentation for 132 hours, the protein did not degrade, demonstrating its stability. Lane 1: Protein Marker; Lane 2: Sample fermented for 64 hours; Lane 3: Sample fermented for 89 hours; Lane 4: Sample fermented for 96 hours; Lane 5: Sample fermented for 112 hours; Lane 6: Sample fermented for 120 hours; Lane 7: Sample fermented for 136 hours.

[0069] Fermentation scale-up production was carried out in a 15-ton fermenter using BSM medium. Culture conditions included: 30℃, pH ≥ 5 controlled with ammonia, and an initial aeration rate of 480 m³ / h. 3 The fermentation rate was approximately 1 h (~1 VVM), with a tank pressure of 0.05 MPa. As fermentation time increased, the aeration rate and rotation speed were adjusted to maintain dissolved oxygen at 30%-50%. Larger tanks may experience insufficient dissolved oxygen, requiring the aeration rate and rotation speed to be increased to the maximum allowable capacity. The feeding strategy was basically the same as for the 10L fermenter. Protein expression was detected by SDS-PAGE, with a separating gel concentration of 12%.

[0070] Electrophoresis results of 15-ton fermenter are as follows Figure 6 As shown, the protein expression level gradually increased over time, reaching its highest level at 112h. The protein expression purity at each time point was >90%. Lane 1: Protein Marker; Lane 2: Samples fermented for 64h; Lane 3: Samples fermented for 88h; Lane 4: Samples fermented for 112h.

[0071] The results from scale-up experiments at 10L, 50L, and 15 tons show that the supernatant of the fermentation broth is mainly composed of the target recombinant humanized collagen, with a purity significantly greater than 90%. This demonstrates the feasibility and practicality of scaling up the production of this protein. Even at a scale-up of 15 tons, the yield reaches 7.5 g / L, which is a high expression level within the industry.

[0072] Example 4

[0073] The fermentation products were purified as follows:

[0074] A. Desalting treatment: Take 100 mL of the supernatant from centrifuged culture medium and desalinate it using molecular sieve G25 (G25 packing material, Changzhou Tiandi Renhe Biotechnology Co., Ltd.); The protein purification system (Cytiva, formerly GE Healthcare) was used for desalting.

[0075] During desalting, samples were loaded at 10% CV (CV represents column volume). Due to the unique amino acid composition of collagen GXY, most of these proteins exhibit weak absorption at 280 nm; therefore, it is necessary to collect samples by combining the 280 nm and 215 nm UV absorption peaks. Desalting buffer: 20 mM PB, 100 mM NaCl, pH 8.0. SDS-PAGE was used to analyze samples before and after desalting; the separating gel concentration was 12%.

[0076] Electrophoresis results as follows Figure 7 As shown, the protein sample size remained consistent before and after desalting, and the sample purity was slightly improved after desalting. Lane 1: Protein Marker; Lane 2: Sample loading (2C×3) before desalting; Lane 3: Sample desalting (2C×3).

[0077] B. After fermentation, the broth was centrifuged at 4000 rpm for 5 min, then filtered through a 0.45 μm hollow fiber microfiltration system (hollow fiber membrane element, PES material, model BMV2-18-0.45, Shandong Bona Biotechnology). 35% saturated ammonium sulfate (ammonium sulfate powder, MACKLIN, analytical grade, 99% purity) was added and stirred for 1 h, then allowed to stand overnight. The fermentation broth after overnight ammonium sulfate precipitation was centrifuged at 12000 rpm for 10 min, and the precipitate was reconstituted with water. The reconstituted protein was adsorbed with 2.5‰ activated carbon (MACKLIN, Activated Charcoal, 200 mesh powder) for 1 h to remove pigments, then centrifuged at 12000 rpm for 10 min. The supernatant was filtered sequentially through filter paper, 0.8 μm, 0.45 μm, and 0.22 μm filter membranes to remove residual activated carbon. Process samples were analyzed by SDS-PAGE; the separating gel concentration was 12%.

[0078] Electrophoresis results as follows Figure 8 As shown: Ammonium sulfate precipitation can precipitate and enrich most proteins, with an estimated yield >90%; after reconstitution of the ammonium sulfate-treated sample and subsequent activated carbon treatment, there is minimal protein loss, with an estimated yield >90%. Lane 1: Protein Marker; Lane 2: Control before ammonium sulfate treatment; Lane 3: Ammonium sulfate precipitation supernatant (AS supernatant); Lane 4: Precipitate after centrifugation of ammonium sulfate precipitation (AS precipitation); Lane 5: Activated carbon adsorption supernatant.

[0079] C. After fermentation, the broth was centrifuged at 4000 rpm for 5 min, and then sequentially passed through 0.45 μm and 0.22 μm hollow fiber microfiltration (hollow fiber membrane element, PES material, model BMV2-18-0.22, Shandong Bona Biotechnology). The transmembrane pressure difference was controlled at 0.1-0.2 MPa, the temperature at 15-25℃, and the turbidity of the microfiltrate was controlled at 5-10 NTU. After the ultrafiltration membrane fouling rate was reduced by approximately 30%, ultrafiltration concentration was performed using a 5 kDa hollow fiber membrane element (hollow fiber membrane element, PES material, model BFV-5 kDa, Shandong Bona Biotechnology), controlling the transmembrane pressure at 0.05-0.15 MPa, the temperature at 20-25℃, and the turbidity of the microfiltrate at <5 NTU. Process samples were analyzed by SDS-PAGE, with a separating gel concentration of 12%.

[0080] Electrophoresis results as follows Figure 9As shown, after 0.45μm and 0.22μm hollow fiber microfiltration, the purified samples remained single, and the target protein recovery rate was ≥90%. After 5kDa hollow fiber filtration, almost all samples remained in the concentration solution and did not permeate the membrane, with a yield >95%. Lane 1: Protein Marker, Lane 2: 0.45μm hollow fiber microfiltrate, Lane 3: 0.22μm hollow fiber microfiltrate, Lane 4: 5kDa hollow fiber ultrafiltration concentrate, Lane 5: 5kDa hollow fiber ultrafiltration permeate.

[0081] The purification process of fermentation broth, using hollow fiber membranes to achieve linear scale-up of microfiltration and ultrafiltration by increasing membrane area, and using cation exchange columns to scale up according to column bed volume ratios, are all conventional scale-up methods in the field, enabling large-scale production. However, it is essential to extend the service life of equipment consumables through maintenance and upkeep, thereby comprehensively reducing the production cost per unit output. For example:

[0082] Based on our company's actual production experience with recombinant humanized collagen series products: When operating various hollow fiber membranes such as 0.45μm, 0.22μm, and 5kDa, pay attention to controlling the transmembrane pressure. After each use, handle them in accordance with the maintenance manual. For example, depending on the type of hydrolyzed protein or saponified fat, treat with 0.5M NaOH for 30-120 minutes. For organic compounds and cell debris, treat with 300-500ppm NaClO available chlorine for 30-60 minutes. They can be reused 100-200 times without any problems.

[0083] When using ion exchange packing, be careful not to exceed the packing's withstand pressure of 0.3 MPa for extended periods. After use, treat the packing with NaOH solution, NaCl solution, or low-concentration organic reagents as needed, depending on the degree of contamination. For large-scale fermentation broth processing, treatment is required after every 3-5 sample loadings to extend its service life. It can be used more than 120 times without major issues.

[0084] Example 5

[0085] Samples were sent to Beijing Baitai Packer Biotechnology Co., Ltd. for testing (Project No.: BTP-HSX-20241226-01). After SDS-PAGE, reductive alkylation, SP3 enrichment (Trypsin digestion), recovery of the digest and lyophilization, and C18 (Stage-Tip) desalting pretreatment, the samples were analyzed using LC-MS / MS (Vanquish Neo / Orbitrap FusionLimos, Thermo Fisher Scientific). The C18 column was 150 μm id × 170 mm, packing: Reprosil-Pur 120C18-AQ 1.9 μm. Mobile phase A: 0.1% FA, Mobile phase B: 0.1% FA, 80% ACN. Raw mass spectrometry data were acquired, and the target protein database was searched using MaxQuant (2.4.9.0). The raw mass spectrometry files were analyzed, and the identification results were obtained by database matching using the software.

[0086] The high-quality peptide sequences of the actual sample were compared with the theoretical sequence of the target protein, recombinant humanized type III collagen. The mass spectrometry identification results are shown in Table 2. The target protein has 108 amino acids, and the identified target protein was divided into 6 peptides with 100% peptide coverage. In most cases, mass spectrometry identification involves only a single Trypsin digestion, and the peptide coverage is usually 60-80%. Because this amino acid peptide itself only has 108 amino acids, and it is a triple repetition of 36 amino acids, the mass spectrometry can easily cover it completely, fully confirming that the purified product is the target recombinant humanized type III collagen. Figure 10 The total ion chromatogram shows that the peptides are well separated with no obvious interference peaks.

[0087] Table 2 Mass spectrometry identification results

[0088]

[0089] Example 6

[0090] The 2C×3 protein obtained in Example 4, or the pure lyophilized powder of different application grades prepared by further ion exchange chromatography, was made into a 1 mg / mL solution, or diluted in an appropriate ratio for detection. The dilution water was the endotoxin detection-specific water provided with the kit.

[0091] The test reagents include: a gel-gel method horseshoe crab endotoxin test reagent, containing horseshoe crab reagents with different sensitivity levels (0.5 EU / ml, 0.25 EU / mL, and 0.03 EU / mL); a positive control: a bacterial endotoxin working standard (potency 10 EU / vial); and a negative control: water specifically for endotoxin testing, manufactured by Zhanjiang Andus Biotechnology Co., Ltd.

[0092] The centrifuge tubes and pipette tips used in the detection process need to be pyrogen-free treated. Refer to the "Pharmacopoeia of the People's Republic of China": 2020 Edition, Volume III, 1143 Bacterial Endotoxin Test for detection. The reaction temperature is 37 ± 1 °C, and the reaction time is 60 ± 2 minutes. Gently remove the reaction tube from the thermostat and slowly invert it 180°. If a gel forms in the tube and the gel does not deform and does not slip off the tube wall, it is positive and recorded as (+); if no gel forms or the formed gel is not firm, deformed, and slips off the tube wall, it is negative and recorded as (-).

[0093] The results show that the endotoxin detection at all levels is qualified:

[0094] Table 3 Endotoxin Detection Results

[0095]

[0096] Specifically, the endotoxin content is controllable (<1000 EU / mg, 100 EU / mg levels) or even lower than 5 EU / mg, which can meet the requirements as a raw material for cosmetics;

[0097] After further fine treatment, the endotoxin content is lower than 0.05 EU / mg, which can meet the requirements as a raw material for Class III medical devices. Entrust Shanghai Fuda Testing Technology Group Co., Ltd. for detection, report number FT-20250910053, the bacterial endotoxin test result of recombinant humanized type III collagen is <

[0098] 0.03 EU / mg.

[0099] Example 7

[0100] SDS-PAGE Detection of Protein Purity

[0101] The freeze-dried powder of 2C×3 protein (batch 007240801-02) obtained in Example 4 was made into a 1 mg / mL solution, with a sample loading volume of 10 μL, and detected by SDS-PAGE. The concentration of the separating gel was 12%.

[0102] The electrophoresis results are as Figure 11 shown. Without gray-scale analysis, directly observing the sample lane shows obvious and relatively single protein bands, and the purity is at least >95%. Lane 1: Protein Marker, Lane 2: freeze-dried powder of 2C×3 protein (batch 007240801-02), and the target protein shows obvious single protein bands.

[0103] HPLC Detection of Protein Purity

[0104] Agilent 1260 high performance liquid chromatography was used with the following parameters:

[0105] Chromatographic column: Galaxy SECS2000, specifications: 5μm, 300×7.8mm, model:

[0106] FMH-3145-KONU (Guangzhou Philomen)

[0107] Mobile phase: 150 mmol / L sodium phosphate buffer

[0108] Preparation method: Weigh 3.145g of NaH2PO4 and 17.572g of Na2HPO4, dissolve them in pure water, mix well, adjust the pH to 7.4 with sodium hydroxide or phosphoric acid, make up to 1L, and filter through a 0.22μm PES membrane.

[0109] Column temperature: 25℃

[0110] Flow rate: 1 mL / min

[0111] Detection wavelength: 205nm

[0112] Injection volume: 10 μL

[0113] Gradient degree: isocratic elution for 20 min

[0114] The 2C×3 lyophilized protein powder obtained in Example 3 was analyzed by HPLC, and the results are shown in the figure below. Figure 12 As shown, using the area normalization method, peaks with a peak area ratio of <0.5% are considered impurity peaks. It can be seen that the peak area ratio of the main peak is 99.56%, indicating that the target protein has extremely high purity.

[0115] Example 8

[0116] The 2C×3 protein lyophilized powder obtained in Example 4 was sealed in a sterile centrifuge tube and placed in a constant temperature and humidity incubator (temperature 40℃, relative humidity RH 60%±5%) for accelerated degradation experiment. The control sample was placed at 4℃. Samples were taken periodically to detect changes in purity and molecular weight. SDS-PAGE analysis was performed, and the separating gel concentration was 12%.

[0117] The protein content of each sample was analyzed and statistically analyzed. The sample at 4℃ and 0d was used as a control (100%), and the percentage of other accelerated samples was compared with that control. GraphPadPrism 8.0 software was used for plotting and analysis. Each sample was tested in 3 replicates, and the RSD was ≤5%.

[0118] The results are as follows Figure 13 The blue curve represents the control at 4℃, and the red curve represents the treated sample at 40℃. The protein is very stable. It can be seen that the lyophilized powder maintained at least 95% stability after 40 days at 40℃, with no significant difference from the 4℃ control (a decrease of less than 10% is considered significant degradation).

[0119] Based on the Arrhenius equation (which describes the effect of temperature on reaction rate or material aging) and practical experience, the freeze-dried powder is expected to be stored for at least 6 months at room temperature and for at least one year under refrigeration conditions of 2-8℃.

[0120] Example 9

[0121] The 2C×3 protein lyophilized powder obtained in Example 4 was processed and then subjected to circular dichroism (CD) spectroscopy. The CD spectrum is the average value of three scans. After the data was analyzed and calculated in the software, a circular dichroism chromatogram of recombinant humanized type III collagen was obtained.

[0122] CD results are as follows Figure 14 As shown, recombinant humanized type III collagen 2C×3 has a negative peak at a wavelength near 195 nm and a positive peak at a wavelength near 221 nm, indicating that the protein has a triple helix structure under these conditions. The basis for judgment is YY / T1888-2023, 5.5.2.2 Circular dichroism (CD) spectrum.

[0123] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0124] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

[0125] Example 10

[0126] The product's anti-wrinkle, soothing, and cell migration-promoting effects were tested by CCIC Testing & Inspection (Tianjin) Co., Ltd., confirming its actual efficacy. Specifically:

[0127] Anti-wrinkle efficacy: Test acceptance number TJGF001720242180-3, the recombinant humanized type III collagen in the test sample at a concentration of 1.25% to 5% can significantly upregulate the content of Collagen I protein (p<0.05), indicating that the test sample has the ability to promote the synthesis of type I collagen and has anti-wrinkle efficacy.

[0128] Soothing Efficacy: Test case number TJGF001720242180-1 showed that, compared with the negative control, the recombinant humanized type III collagen in the test sample at concentrations of 1.25%–5% significantly reduced the content of the TNF-α inflammatory factor (P<0.05). This indicates that the test substance has an inhibitory effect on TNF-α and thus a soothing effect.

[0129] Promoting Cell Migration: Test case number TJGF001720242180-4, using recombinant humanized type III collagen at concentrations of 1.25%–5%, analysis showed that the test group had a significantly reduced cell scratch area compared to the negative control group, and the relative migration rate was statistically different (p<0.05). This indicates that the sample promotes cell migration and has repairing effects.

Claims

1. A method for preparing recombinant humanized type III collagen with a triple helix structure, characterized in that, Includes the following steps: S1. Construct a recombinant expression plasmid containing a human type III collagen fragment ppICZαA, and transform the recombinant expression plasmid into yeast; S2. High-copy transformant strains were screened from the strains and induced expression tests were performed in shake flasks to screen for strains with the highest protein expression levels. S3. The strain with the highest expression level obtained in S2 above is fermented at high density in a fermenter to obtain a large amount of secretible target protein of 2C×3 (36 amino acids repeated 3 times in recombinant humanized type III collagen). S4. The target protein that can be secreted and expressed by the 2C×3 is purified to obtain recombinant humanized type III collagen.

2. The method for preparing recombinant humanized type III collagen with a triple helix structure according to claim 1, characterized in that, The amino acid sequence of human type III collagen in step S1 includes, but is not limited to, amino acid sequences that are identical to or have a homology (NCBI Protein BLAST comparison) of >90% with the amino acid sequence shown in SEQ ID NO.

1.

3. The method for preparing recombinant humanized type III collagen with a triple helix structure according to claim 2, characterized in that, The SEQ ID NO.1 is a 2C×3 fragment of human type III collagen after analysis and screening. It is a 36-amino acid triple repeat fragment containing 3 GER and 3 GFP cell binding motifs and 3 RGD cell adhesion motifs.

4. The method for preparing recombinant humanized type III collagen with a triple helix structure according to claim 1, characterized in that, The nucleotide sequence corresponding to the fragment containing human type III collagen in step S1 includes, but is not limited to, sequences that encode the same protein as the nucleotide sequence shown in SEQ ID NO.2 or sequences with homology (NCBINucleotide BLAST alignment) > 90%, or sequences that are different from the nucleotide sequence of SEQ ID NO.2 due to genetic code degeneracy and optimized using different software, or other truncated fragments or small molecule fragments contained in SEQ ID NO.

2.

5. The method for preparing recombinant humanized type III collagen with a triple helix structure according to claim 1, characterized in that, The main structure of the recombinant expression plasmid in step S1 is 6×His-DDDDK-2C×3, wherein 6×His is a histidine tag commonly used in genetic engineering, and DDDDK is the restriction site of enterokinase EK. According to application requirements, the 6×His tag can be removed by enterokinase EK digestion to obtain tag-free recombinant protein.

6. The method for preparing recombinant humanized type III collagen with a triple helix structure according to claim 1, characterized in that, The yeast in step S1 is Pichia pastoris, specifically wild-type Pichia pastoris X33.

7. The method for preparing recombinant humanized type III collagen with a triple helix structure according to claim 1, characterized in that, The purification in step S4 includes a combination of at least two of the following methods: G25 molecular sieve desalting, 35% saturated ammonium sulfate precipitation, activated carbon adsorption, 0.45μm / 0.22μm hollow fiber microfiltration, 5kDa hollow fiber ultrafiltration concentration, and ion exchange chromatography. In this purification method combination, the basic purification steps include G25 molecular sieve desalting + 35% saturated ammonium sulfate precipitation + 0.45μm / 0.22μm hollow fiber microfiltration, and the refining steps include 5kDa hollow fiber ultrafiltration concentration + ion exchange chromatography. The final product is recombinant humanized type III collagen that can be stably stored, with accelerated stability at 40℃ ≥ 40 days.

8. The method for preparing recombinant humanized type III collagen with a triple helix structure according to claim 1, characterized in that, The prepared recombinant humanized type III collagen was tested for purity using HPLC area normalization method, with a purity ≥90%, or ≥95% and / or ≥99%, and endotoxin ET (tested according to method 1143 of Part III of the 2020 edition of the Pharmacopoeia of the People's Republic of China, with ET <5 EU / mg, or ET <0.5 EU / mg, or ET <0.05 EU / mg) and can be used for different applications.

9. A recombinant humanized type III collagen with a triple helix structure prepared by the method described in claims 1-8, characterized in that, Purity > 90% and endotoxin < 5 EU / mg, used as cosmetic raw materials; or purity > 95% and endotoxin < 0.5 EU / mg, used as raw materials for Class II medical devices; or purity > 99% and endotoxin < 0.05 EU / mg, used as raw materials for Class III medical devices or biomedical materials.

10. A recombinant humanized type III collagen with a triple helix structure prepared by the method described in claims 1-8, characterized in that, This recombinant humanized type III collagen has undergone efficacy tests for anti-wrinkle, soothing, and cell migration promotion, demonstrating its practical effectiveness.

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