Recombinant humanized collagen and methods of making the same

Abstract

This invention provides recombinant humanized collagen, comprising a recombinant humanized type I collagen α1 chain fragment or recombinant humanized type III collagen, wherein the amino acid fragment of the recombinant humanized type I collagen α1 chain fragment is shown in SEQ ID NO: 1; or, the amino acid fragment of the recombinant humanized type III collagen is shown in SEQ ID NO: 2. A method for preparing the recombinant humanized collagen is also disclosed. The preparation process of the method provided by this invention is optimized, particularly by employing a feed strategy during the glycerol-to-methanol transition period to improve protein yield. This invention, through the screening of truncated fragments of recombinant humanized collagen and the optimization of the preparation process, obtains a high-yield, high-purity, and low-endotoxin recombinant humanized collagen product.

Description

Technical Field

[0001] This invention relates to the field of bioengineering, and more specifically, to a recombinant humanized collagen and its preparation method. Background Technology

[0002] Collagen is the most abundant protein in animals, accounting for approximately 25-35% of human protein and equivalent to 6% of body weight. It is widely distributed throughout various tissues and organs, such as bones, cartilage, ligaments, skin, cornea, various membranes, and fascia, and is particularly abundant in human skin and connective tissue. It is a polymer that constitutes various extracellular matrix components, playing a role in binding tissues in animal cells. It is a major component of supporting and connective tissues, performing multiple functions including support, protection, and connection. Collagen is formed by the assembly of multiple protoplasmic collagen molecules. Protoplasmic collagen is its basic unit, composed of three polypeptide chains. These three protoplasmic collagen polypeptide chains can intertwine with each other through interchain hydrogen bonds to form a stable triple helix structure. To date, 27 types of collagen have been discovered, and approximately 29 types of collagen have been encoded. Different types of collagen play different roles in connective tissue. Type I collagen is the most abundant, accounting for about 80-90% of the total collagen. It is mainly distributed in bones, tendons, skin (adults), and blood vessel walls. It has a relatively hard texture and plays a supporting role in the skin. It is widely used in medical aesthetic injections and cosmetics. Type III collagen is found in higher concentrations in blood vessels and can be used as an important raw material for tissue repair, improving dry skin, enlarged pores, and skin aging.

[0003] Type I collagen is the most abundant collagen in the human body. Its fibers are relatively coarse and tightly packed, primarily determining the toughness and strength of skin and mucous membranes. Its core function is to provide structural support and mechanical strength to tissues, lifting skin cells to smooth wrinkles and reduce fine lines. It also forms the "steel mesh" of bones, making them both strong and resilient. Furthermore, it can transmit mechanical and chemical signals into cells through transmembrane receptors such as integrins and glycoproteins, acting as a signaling molecule. Simultaneously, it plays a crucial role in tissue repair and wound healing.

[0004] Type III collagen is a type of collagen found primarily in the dermis of the skin, responsible for providing elasticity and smoothness. With age, the synthesis of type III collagen gradually decreases, leading to loss of skin elasticity and wrinkles. Therefore, supplementing with type III collagen is an important anti-aging measure. Recombinant humanized collagen is a fragment of the amino acid sequence encoded by a specific type of human collagen gene, prepared using DNA recombination technology, or a combination of functional fragments containing human collagen. Recombinant type III humanized collagen is one such type; structurally identical to human type III collagen, this characteristic makes it easily absorbed and utilized by the body, making it a high-quality raw material. Type III humanized collagen shares amino acid sequence homology with natural human type III collagen, exhibiting good biocompatibility. Once it enters the skin, it triggers a tissue response, stimulating the production of collagen. By forming a collagen fiber network, it supports cells and tissues, thereby repairing and physically filling wrinkles and sagging areas. This replenishes lost collagen, increases skin elasticity and firmness, improves skin texture, helps reduce collagen degradation, reduces cell apoptosis, alleviates inflammation, enhances the skin barrier, and repairs damaged skin.

[0005] The most common expression system for recombinant humanized type III collagen is the *E. coli* expression system. It uses *E. coli* cells as the host cell, expressing the target protein through *E. coli* cells containing a foreign gene expression vector. Its advantages include ease of operation, low cost, short preparation cycle, and high protein expression levels. However, the *E. coli* expression system lacks hydroxylases that stabilize the triple helix structure of collagen, posing a challenge for large-scale production of recombinant human collagen. Although some researchers have addressed this issue by adding the P4H gene and corresponding collagen genes, compared to plant and mammalian cell expression systems, it still lacks a complete triple helix structure and good thermal stability.

[0006] Traditional collagen extraction processes primarily rely on animal tissues as starting materials, but this method inevitably faces challenges such as viral contamination risks, immune rejection reactions, and significant batch-to-batch quality variations. In contrast, genetic engineering technology has opened up new avenues for the efficient and safe production of collagen. Currently, recombinant collagen has been successfully expressed in various expression systems, including yeast, E. coli, mammalian cells, insect cells, and tobacco. Collagen produced through microbial fermentation offers advantages such as reduced costs, increased production efficiency, and ease of large-scale production.

[0007] However, prokaryotic expression products such as E. coli are prone to forming inclusion bodies, making it difficult to remove endotoxins, resulting in low protein secretion efficiency and a lack of post-translational modification mechanisms found in eukaryotes. This leads to unstable product structures and ultimately poor yield and quality. On the other hand, the expression of collagen using mammalian, insect cells, and transgenic plants is more often used in laboratory research due to the demanding culture conditions, long growth cycles, and high costs.

[0008] Pichia pastoris, as a eukaryotic cell, is highly similar to CHO cells and is widely used in the production of proteins for vaccines and pharmaceuticals. It has advantages such as relatively easy gene manipulation, a clear genetic background, and a well-defined expression regulation mechanism. Furthermore, it possesses post-translational modification mechanisms, such as glycosylation, hydroxylation, and acetylation, which are not present in prokaryotic systems, enabling it to maintain the physiological activity of the product and making it an excellent expression system for humanized collagen. However, the Pichia pastoris expression system has high requirements for culture conditions. Fluctuations in culture conditions can lead to instability in collagen expression, thus affecting the final yield and quality. For example, unsuitable temperature may affect the growth rate and metabolic processes of Pichia pastoris, thereby affecting the amount of collagen expressed; fluctuations in pH may alter the physiological state of Pichia pastoris cells, such as enzyme activity, which is detrimental to collagen synthesis and secretion.

[0009] Research on the recombinant expression of type I humanized collagen fragments using the Pichia pastoris system in China has been increasing year by year. Jiangxi Chongshan Biological Products Co., Ltd. disclosed a method for synthesizing type I humanized collagen, its preparation, and its applications in Chinese Patent CN117510618B, published on July 5, 2024, and application number 202311463544.7, filed on November 6, 2023. The amino acid sequence of this collagen is a nine-fold repeat of the amino acid 674-736 peptide segment of the type I collagen α1 chain protein, containing nine GFPGER structures. This domain can bind to cytokine receptors. Increasing the number of this domains in a single protein molecule through tandem expression can enhance the affinity of collagen for cells and promote cell adhesion. The purified synthetic humanized type I collagen is a monomer with an apparent molecular weight of 56 kDa, close to its theoretical molecular weight, but the purity of the purified collagen only reaches 89%. Xi'an Giant Biogene Technology Co., Ltd. disclosed recombinant human type I collagen and its uses in Chinese patent CN119775391A, published on April 8, 2025, application number 202411978420.7, filed on December 30, 2024. The recombinant human type I collagen has good cell proliferation, cell migration, and cell adhesion activities (all superior to full-length recombinant human type I collagen or commercially available human collagen) and is easy to produce. Therefore, it is feasible to express the full-length α1 chain single-chain fragment of collagen. However, this protein carries a His tag. Although the designed purification label is beneficial for the purification, detection, and identification of the recombinant protein, if the purification label is not removed, there is a potential risk of allergic reactions in the application. Adding an enzymatic digestion process to remove the purification label would complicate the production process and greatly increase the production cost.

[0010] In existing technologies, Escherichia coli expressing type III collagen lacks hydroxylase (requiring additional P4H gene transfer), plant cell expression levels are <0.5 g / L, and mammalian cell costs are high (culture cost is 5 times that of Pichia pastoris); although Pichia pastoris is a potential host, publicly available literature, such as the article "Research Progress in the Development and Application of Collagen" published in the Chinese Journal of Biotechnology (Mar. 25, 2023, 39(3): 942-960), Table 2 (page 948), shows the expression system for generating recombinant collagen, indicating that its fermentation conditions are sensitive and the product is easily degraded. P. pastoris The highest yield of type III collagen was 1.47 g / L. Therefore, there is an urgent need to develop new technologies for the expression, construction, and stabilization of recombinant humanized collagen. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of existing methods for preparing recombinant humanized collagen, while improving the production capacity and purity of recombinant humanized collagen, and obtaining novel humanized type I collagen α1 chain fragments and recombinant humanized type III collagen fragments with good biological properties. This invention employs a high-density fermentation method during the fermentation process, optimizing the fermentation process in a fermenter, including optimizing the culture medium, culture conditions, and glycerol supplementation strategy, to achieve high-density fermentation of the highly expressed recombinant strain.

[0012] To achieve the above objectives, the present invention provides recombinant humanized collagen, comprising a recombinant humanized type I collagen α1 chain fragment or a recombinant humanized type III collagen, wherein the amino acid fragment of the recombinant humanized type I collagen α1 chain fragment is shown in SEQ ID NO: 1; or, the amino acid fragment of the recombinant humanized type III collagen is shown in SEQ ID NO: 2.

[0013] The recombinant humanized type I collagen α1 chain fragment with the amino acid sequence shown in SEQ ID NO: 1 is fragment T11 of the humanized type I collagen α1 chain that has been analyzed, screened, and experimentally verified in this invention. The recombinant humanized type I collagen α1 chain fragment with the amino acid sequence shown in SEQ ID NO: 1 is rich in cell-binding motifs such as 2 cell adhesion sites RGD, 4 KGD, 7 GER, and 1 GEK, as well as a highly hydrophilic region.

[0014] The recombinant humanized type III collagen with the amino acid sequence shown in SEQ ID NO: 2 is the humanized type III collagen fragment T5 obtained through analysis and screening in this invention. The recombinant humanized type III collagen with the amino acid sequence shown in SEQ ID NO: 2 contains four cell adhesion sites (KGD).

[0015] Preferably, the amino acid nucleotide encoding the recombinant humanized type I collagen α1 chain fragment is shown in SEQ ID NO: 3; or, the amino acid nucleotide encoding the recombinant humanized type III collagen is shown in SEQ ID NO: 4.

[0016] The present invention also provides a method for preparing recombinant humanized collagen according to any one of the above claims, comprising the following steps:

[0017] Step 1: Construct engineered bacteria using the encoding nucleotides of recombinant humanized collagen to obtain highly expressed recombinant strains:

[0018] Step 2: The high-expression recombinant strain obtained by screening was inoculated into BSM medium. The culture conditions were: temperature 30℃, pH ≥ 5 controlled with ammonia water, and aeration rate of 1 VVM.

[0019] Step 3: After the initial carbon source is depleted, feed is added using glycerol at a concentration of 650 g / L until the bacterial culture OD600 reaches 95 to 105;

[0020] Step 4: Feeding strategy during the transition period from glycerol to methanol: Add methanol to enter the transition period, with the final methanol concentration in the fermentation broth at 2 g / L. At the same time, continue feeding glycerol and control the feeding rate of glycerol so that the feeding rate of glycerol is reduced from 8 g / L to 0.

[0021] Step 5: After the carbon source is exhausted, methanol is added to enter the induction period. The final methanol concentration in the fermentation broth is 8 g / L, and the expression of recombinant humanized collagen fragments is carried out. Whenever the dissolved oxygen in the fermentation broth reaches 50%, the methanol addition step is repeated to make the methanol concentration reach 8 g / L.

[0022] Step 6: Purify the recombinant humanized collagen fragment expressed in Step 5.

[0023] Preferably, in step 2, a dissolved oxygen and rotation speed linkage strategy is adopted. This strategy involves controlling the dissolved oxygen in the fermentation broth to maintain it between 30% and 50% by adjusting the rotation speed. That is, whenever dissolved oxygen fluctuations exceed the 30%-50% range, the rotation speed is adjusted to restore the dissolved oxygen to the 30%-50% range. More preferably, the rotation speed is reduced whenever dissolved oxygen exceeds 50%; or, the rotation speed is increased whenever dissolved oxygen falls below 30%, maintaining dissolved oxygen at 30%-50%.

[0024] Preferably, in step 2, casein is added to the BSM culture medium. Preferably, the final concentration of casein is 0-40 g / L. Most preferably, the casein concentration is 30 g / L. This invention significantly reduces the probability of protease attacking the target collagen by adding casein, thus blocking the degradation of the recombinant humanized collagen at its source. More preferably, in the process of preparing the recombinant humanized type III collagen, in step 2, 30 g / L of casein is added to the BSM culture medium. More preferably, in the process of preparing humanized type I collagen, in step 2, casein is not added to the BSM culture medium, because the α1 chain fragment of the recombinant humanized type I collagen with the amino acid sequence shown in SEQ ID NO: 1 is relatively stable and not easily degraded.

[0025] In any of the above-mentioned preferred embodiments, in step 3, glycerol is continuously added at a rate of 8 g / L / h. Here, 8 g / L means that when glycerol with a concentration of 650 g / L is added, the increase in glycerol in the fermentation broth is 8 g per liter per hour. Preferably, in step 3, glycerol with a concentration of 650 g / L is continuously added at a rate of 8 g / L / h.

[0026] In any of the above-mentioned preferred embodiments, in step 4, the method of controlling the glycerol feeding rate is to reduce the glycerol feeding rate from 8 g / L / h to 0 g / L / h within 2 hours.

[0027] In any of the above-mentioned preferred embodiments, in step 4, the method of controlling the glycerol feeding rate is to reduce the glycerol feeding rate in 3-5 stages over 2 hours, so that the glycerol feeding rate drops from 8 g / L / h to 0 g / L / h. Preferably, the glycerol feeding rate is reduced by 2 g / L every 0.5 hours from 8 g / L / h until the glycerol feeding rate drops to 0 g / L.

[0028] Preferably, in steps 4 and 5, a feeding strategy is adopted during the transition period from glycerol to methanol. Preferably, in step 4, after OD600 reaches 95-105, 2 g / L of methanol is added at once, while the glycerol feeding rate is gradually reduced to 0 g / L / h in four stages: 8, 6, 4, 2, and 0 g / L / h, with each rate maintained for 30 minutes. Preferably, in step 5, when all carbon sources are depleted (dissolved oxygen DO is again >50% and no longer decreasing), the amount of methanol is increased to enter the formal induction period. Preferably, methanol is added in batches, with 8 g / L of anhydrous methanol added each time DO >50%, while the rotation speed is maintained at maximum (preferably 400, 500, 600, 700, 800, 900 rpm and a range thereof). The mixture is discharged into the tank when OD600 is approximately between 175-250 (preferably 200).

[0029] Once the OD600 reaches 95-105, a single addition of 2 g / L methanol is used to rapidly increase the amount of alcohol oxidase in the cells, thereby enabling them to begin utilizing methanol. Gradually decreasing the glycerol feeding rate can maintain the supply of carbon source to the yeast cells for a period of time, preventing a significant decrease in metabolic level or cell death. As the glycerol feeding rate slows down, the inhibition of alcohol oxidase and its promoter PAOX1 in Pichia pastoris by glycerol is gradually relieved, preparing for the subsequent large-scale methanol-induced expression.

[0030] Compared to the common method of constant methanol replenishment (maintaining a final concentration of 2 g / L for a long period) in existing technologies, the advantage of batch methanol replenishment in this invention is that there is sufficient methanol to allow the target protein to be expressed in large quantities in a short period of time. During this period, the methanol is periodically consumed and will not accumulate continuously, which can shorten the total fermentation time to a certain extent without affecting the protein yield. On the other hand, after adding methanol during the fermentation process, there is no need for special and frequent monitoring of methanol concentration; only the dissolved oxygen (DO) needs to be observed. Methanol is replenished once when DO > 50%, eliminating the need for professional methanol concentration detection equipment and saving the workload of fermentation personnel.

[0031] In any of the above-mentioned preferred embodiments, the bacterial growth time is 20 hours, that is, the total duration of steps 2 and 3 is 20 hours.

[0032] Preferably, the switching period lasts for 2 hours, i.e., step 4 lasts for 2 hours.

[0033] Preferably, the induction time for any of the above is 96 hours, i.e., the duration of step 5 is 96 hours.

[0034] In any of the above-mentioned preferred embodiments, in step 1, the encoding nucleotides of recombinant humanized collagen are ligated to an expression vector to construct a recombinant expression plasmid; the recombinant expression plasmid is transformed into yeast; and a high-expression recombinant strain is obtained through screening and identification. Preferably, the expression vector is the pPICZαA plasmid. The pPICZαA plasmid is a commercially available vector and can be purchased.

[0035] In any of the above-mentioned preferred embodiments, in step 1, the yeast is Pichia pastoris.

[0036] Preferably, the yeast is Pichia pastoris X33. Pichia pastoris X33 is a commonly used strain in the prior art and can be purchased commercially.

[0037] In any of the above-mentioned preferred embodiments, in step 6, the purification method is preferably a combination of at least one or more purification processes including centrifugation, microfiltration, ultrafiltration concentration, cation exchange chromatography, desalting, endotoxin removal, filtration sterilization, low-temperature vacuum freeze drying, and cobalt-60 irradiation, with each process step proceeding in order of impurity removal priority.

[0038] In any of the above preferred embodiments, in step 6, the recombinant humanized type I collagen fragment is purified by a combination of purification processes including centrifugation, microfiltration, ultrafiltration concentration, cation exchange chromatography, desalting, endotoxin removal, filtration sterilization, low-temperature vacuum freeze drying, and cobalt-60 irradiation.

[0039] Preferably, in step 6, the purification step of the fermentation broth of the recombinant humanized type III collagen fragment includes membrane ultrafiltration purification and endotoxin removal. Preferably, the membrane ultrafiltration purification method includes: first, using a 100kDa membrane ultrafiltration membrane to remove macromolecules from the obtained fermentation broth of the recombinant humanized type III collagen fragment; then, using a 30kDa ultrafiltration membrane to further remove small molecules; and finally, replacing the broth with pure water to achieve purification through a replacement buffer.

[0040] In any of the above-mentioned preferred embodiments, the fermentation system of the preparation method of recombinant humanized collagen provided by the present invention is 2L to 1000L; preferably 50L to 1000L, and more preferably 2, 5, 10, 50, 100, 300, 500, 700, 1000L and the range thereof.

[0041] Preferably, for fermentation systems of different volumes from 50L to 1000L, the stirring speed and aeration rate are adjusted to maintain the dissolved oxygen in the fermentation broth at 30%-50%. By maintaining dissolved oxygen, the yield of the target protein expressed in different fermentation systems is stabilized at around 10 g / L.

[0042] In any of the above preferred embodiments, an initial rotation speed of 100-300 rpm and / or an initial aeration rate of 1 VVM (VVM, Volume per Volume per Minute, aeration ratio, m³ / (m³·h)) are preferably used in the fermentation system. As fermentation proceeds, the rotation speed and / or aeration rate are adjusted upward or downward to maintain the dissolved oxygen in the fermentation broth at 30%-50%.

[0043] In a preferred embodiment of the present invention, a process for preparing a high-purity recombinant humanized type I collagen α1 chain fragment includes the following steps:

[0044] Step 1: Construction of recombinant expression vector: The gene encoding the recombinant humanized type I collagen α1 chain fragment T11 shown in SEQ ID NO: 3 was ligated into the pPICZαA vector to obtain the recombinant expression vector pPICZαA-T11. The nucleotide sequence shown in SEQ ID NO: 3 is a nucleotide sequence optimized by Pichia pastoris codons and fused with the α-factor signal peptide gene on the pPICZαA vector;

[0045] Step 2: Screening of engineered bacteria: The pPICZαA-T11 plasmid obtained in Step 1 was transformed into Pichia pastoris strain X33. After methanol-induced expression, engineered bacteria that could express high molecular weight of the target protein fragment and relatively few other miscellaneous proteins were screened.

[0046] Step 3: Protein expression: The engineered strains screened in Step 2 were cultured in a 50 L fermenter in inorganic salt medium (BSM medium) for high-density fermentation to obtain a supernatant containing recombinant humanized type I collagen α1 chain fragments.

[0047] Step 4: Purification: The fermentation broth obtained in Step 3 is purified by a combination of processes including centrifugation, microfiltration, ultrafiltration concentration, cation exchange chromatography, desalting, endotoxin removal, filtration sterilization, low-temperature vacuum freeze drying, and cobalt-60 irradiation. The purification process is achieved through the superposition of these processes.

[0048] The preferred embodiment of the above is that the specific method for constructing the recombinant expression vector is as follows: after high-fidelity PCR amplification of SEQ ID NO:3, it is seamlessly cloned and ligated downstream of the α-factor signal peptide Kex2 protease recognition site of the pPICZαA vector (i.e. immediately after the Ker2 protease recognition site Lys-Arg). After verification by colony PCR and sequencing, the vector is enriched to obtain the recombinant expression vector pPICZαA-T11.

[0049] Preferably, the screening method for the engineered strain is as follows: the recombinant expression vector pPICZαA-T11 is linearized by enzyme digestion and transformed into Pichia pastoris competent cells X33. Positive transformants are selected, and high-copy recombinants are screened through an antibiotic resistance gradient to obtain the Pichia pastoris genetically engineered strain X33-pPICZαA-T11 that produces the recombinant humanized type I collagen α1 chain fragment T11. The X33-pPICZαA-T11 engineered strain is inoculated into BMGY medium for shake-flask experiments, methanol is used to induce expression, and the supernatant is collected for SDS-PAGE electrophoresis detection to screen for engineered strains with the same molecular weight as the theoretical molecular weight and relatively low expression of other miscellaneous proteins.

[0050] Preferably, the specific method for protein expression is as follows: The screened engineered strain is inoculated into BSM medium under the following culture conditions: 30 ℃, pH ≥ 5 controlled with ammonia, and aeration rate of 1 L / min (compressed air). After the initial carbon source is depleted, glycerol at a concentration of 650 g / L is added until the OD600 of the fermentation broth reaches 95-105. Methanol is added to initiate the switching phase, with a final methanol concentration of 2 g / L in the fermentation broth. The glycerol feeding rate is controlled to decrease to 0 g / L / h in four stages. After the carbon source is depleted, methanol is added in batches to initiate the induction phase for protein expression.

[0051] Preferably, the purification method is as follows: preferably, the fermentation supernatant is taken and microfiltered using a 0.45 μm hollow fiber membrane to remove large particulate impurities; preferably, a concentrated desalting solution is obtained by ultrafiltration using a 30 kDa hollow fiber membrane to remove a large amount of inorganic salts; preferably, the desalting solution is subjected to cation (SP) exchange chromatography, and the eluent is collected in segments; preferably, the eluent obtained by chromatography is concentrated and desalted by 2 kDa spiral wound membrane ultrafiltration; preferably, endotoxins are removed using a 50 kDa hollow fiber membrane; preferably, sterilization is achieved by filtration using a 0.22 μm sterile membrane sterilized by autoclaving; preferably, lyophilization is performed using a low-temperature vacuum freeze dryer; finally, preferably, the solution is purified by a 6 kGy dose of cobalt-60 (… 60Irradiation with Co) γ-rays yielded high-quality recombinant humanized type I collagen α1 chain fragment freeze-dried fibers that simultaneously met the following requirements: purity ≥99% (HPLC), endotoxin <0.05 EU / mg (Limulus amebocyte lysate (LAL) reagent detection), protein content ≥95% (Kjeldahl nitrogen determination), and passed microbiological testing. Protein identification by LC-MS / MS and raw files acquired by mass spectrometry were analyzed using MaxQuant (2.4.9.0) database search software, and the peptide coverage (amino acid sequence coverage) reached as high as 92.5%.

[0052] In another preferred embodiment of the present invention, a process for preparing recombinant humanized type III collagen fragments is provided, using a similar method to prepare the recombinant humanized type III collagen fragments.

[0053] This invention provides a recombinant humanized type III collagen and its preparation method, the main steps of which are as follows:

[0054] Preferably, the method for preparing the recombinant humanized type III collagen includes: constructing a pPICZαA recombinant expression plasmid containing a human type III collagen fragment and transforming it into yeast; screening high-copy transformant strains from the strain and performing shake-flask level induction expression tests; performing high-density fermentation in a fermenter; purifying the fermentation product from the fermenter to obtain recombinant humanized type III collagen, and performing specific enzyme digestion tests, MS identification, and endotoxin tests.

[0055] In any of the above-mentioned preferred embodiments, the method for constructing the engineered bacteria is as follows: ligating the nucleic acid encoding the recombinant humanized type III collagen fragment T5 shown in SEQ ID NO: 4 into the pPICZαA plasmid to construct a recombinant expression plasmid; and transforming the recombinant expression plasmid into yeast.

[0056] In any of the above preferred embodiments, the nucleotide fragment shown in SEQ ID NO:4 is seamlessly cloned and ligated downstream of the α-factor signal peptide Kex2 protease recognition site of the pPICZαA vector (i.e. immediately after the Ker2 protease recognition site Lys-Arg), enriched after verification by colony PCR and sequencing, to obtain the recombinant expression vector pPICZαA-T11.

[0057] Preferably, the screening method for high-expression recombinant strains is as follows: high-copy transformants are screened from YPD plates containing 300 μg / mL Zeocin (bleomycin); the high-copy transformants are identified by PCR; after correct identification, shake-flask induction expression test is performed to obtain high-expression recombinant strains.

[0058] Preferably, the purification step is membrane ultrafiltration purification and endotoxin removal to obtain recombinant humanized type III collagen fragments that can be stably stored.

[0059] In any of the above preferred embodiments, the preparation method further includes identifying the purified product by enzymatic digestion with a specific type III collagenase, wherein the purified product is digested with a collagenase that can specifically recognize the Pro-X-Gly-Pro sequence (where X is a neutral amino acid), while the control non-collagen protein is not cleaved.

[0060] Preferably, the recombinant humanized type III collagen prepared by the method described in any of the above methods has been proven by efficacy testing to have actual effects on skin tightening and tissue repair.

[0061] This invention provides a novel recombinant humanized type I collagen and a new process for fermenting and producing recombinant humanized type I collagen, overcoming the technical problems in the prior art. Specifically, the full-length α1 chain of recombinant humanized type I collagen contains 1014 amino acids, and its molecular weight reaches 90.4 kDa after single-chain expression. Even without the participation of proline hydroxylase P4H, the collagen itself will form a certain triple helix structure, although it is looser than natural collagen. Nevertheless, the molecular weight will reach over 270 kDa. Whether expressed as a single chain or forming a certain triple helix structure, it will place a great burden on the host cell and may even make expression difficult. Basic research shows that the expression level of the full-length α1 chain of recombinant humanized type I collagen is low, and it is easily degraded during fermentation and purification.

[0062] The high-purity recombinant humanized type I collagen α1 chain fragment provided by this invention retains the core function of the type I collagen α1 chain and is 100% homologous to the corresponding sequence of the human type I natural protein, thus avoiding immune rejection. After shake-flask expression and fermentation in a fermenter, the purity of the target protein fragment reached a very high level, with no degradation observed, demonstrating its good stability. This lays a solid foundation for subsequent purification and transfer to other products, facilitating the maintenance of extremely high protein purity and avoiding protein degradation caused by prolonged processing. The recombinant humanized type I collagen provided by this invention, fermented in a 50 L fermenter, yielded approximately 10 g / L of the target protein in the supernatant after centrifugation, which is a high expression level in the industry, proving that the fragment is easily expressed and secreted into the culture medium. It is well known that traditional E. coli systems for expressing exogenous proteins mostly involve intracellular expression, especially when expressing proteins from higher organisms, which easily leads to the formation of inclusion bodies. However, this invention uses Pichia pastoris for secretory expression, eliminating the need for cell disruption, cell wall removal, centrifugation, and even the very cumbersome denaturation and renaturation purification steps.

[0063] The recombinant humanized type I collagen α1 chain fragment shown in SEQ ID NO: 1 of this invention can retain cell adhesion while having high solubility (solubility of at least 50 g / L and above).

[0064] The recombinant humanized type I collagen freeze-dried fibers prepared by the process of this invention simultaneously meet the following requirements: purity ≥99%; protein content 95%-103.5% (calculated using Kjeldahl nitrogen determination, based on an average coefficient of 6.25 for converting nitrogen content to protein content); endotoxin <0.05 EU / mg (Limulus amebocyte lysate (LAL) reagent detection). These fibers can be used as raw materials in the production of products for various applications, including cosmetics, Class II medical devices, and even Class III medical devices, thus injecting new momentum into the diversified development of the collagen market. Compared with existing technologies, the recombinant humanized type I collagen provided by this invention includes, but is not limited to, the following beneficial effects:

[0065] 1. This invention optimizes Pichia pastoris based on its preferred codons, eliminating uncommon codons and hairpin structures, making it more suitable for stable expression in Pichia pastoris.

[0066] 2. The recombinant plasmid designed and constructed in this invention has the target gene inserted downstream of the recognition site of the α factor secretion signal peptide Kex2 protease. It has no tag and no redundant amino acids, which can greatly avoid allergies. The resulting recombinant collagen α1 chain fragment is 100% identical to the corresponding part of the amino acid sequence of type I human collagen.

[0067] 3. The recombinant humanized type I collagen α1 chain fragment provided by the present invention has important functional sites of human type I collagen, contains two RGD motifs that can improve cell adhesion rate, and has good biocompatibility.

[0068] 4. This invention utilizes Pichia pastoris to secrete and express recombinant type I humanized collagen α1 chain fragments. Compared with animal-derived type I collagen, it has the advantages of no risk of viral transmission and low immunogenicity. It can also avoid the disadvantages of E. coli expression systems, such as difficulty in purification and easy generation of pyrogens.

[0069] 5. The recombinant humanized type I collagen α1 chain fragment obtained by the present invention through analysis, optimization and screening is very stable in the fermentation broth and does not exhibit collagen degradation problems during fermentation.

[0070] 6. The high-density fermentation in the fermenter of this invention enables high-yield, soluble expression of recombinant humanized type I collagen α1 chain fragments in yeast. Through optimized fermentation processes, this invention achieves a purity of at least ≥90% for the recombinant humanized type I collagen in the fermentation broth. Its yield reaches 10 g / L, which is a high expression level in the industry, exceeding the typical yield of 3 to 5 g / L for existing Pichia pastoris expression of type I collagen.

[0071] 7. This invention, through the combined use of multiple purification processes, obtains recombinant humanized type I collagen α1 chain fragment freeze-dried fibers that simultaneously meet several core indicators of high-quality collagen, such as purity ≥99%, which is higher than the purity of proteins prepared by conventional single processes or simple superposition in existing technologies. Through the ingenious combination of chromatography and multiple filtration methods, this invention produces products with endotoxin <0.05 EU / mg, protein content ≥95%, and no microbial contamination, greatly reducing immunogenicity and pyrogenic reactions. It can be used as a raw material in the production of products in different application scenarios such as cosmetics, Class II medical devices, and even Class III medical devices.

[0072] This invention analyzes the T5 fragment of human type III collagen to obtain the amino acid fragment of recombinant humanized type III collagen, as shown in SEQ ID NO: 2. The purified recombinant humanized type III collagen, identified by LC-MS / MS, and the raw file acquired by mass spectrometry, was retrieved using the MaxQuant (1.6.2.10) database, showing a peptide coverage of up to 89.38%. In the amino acid fragment shown in SEQ ID NO: 2, amino acid sequences from position 64 to 584 are all covered, including all four KGD active sites; and specific type III collagenase digestion confirms that the purified product is the target recombinant humanized type III collagen fragment. Compared with existing technologies, the humanized type III collagen and its preparation method provided by this invention offer, but are not limited to, the following beneficial effects:

[0073] 1. The recombinant humanized type III collagen fragment obtained through analysis and optimization screening in this invention exhibits slight degradation in the fermentation broth. However, by supplementing casein during fermentation, no degradation issues were observed in the collagen after fermentation. Therefore, this provides maximum inherent assurance for subsequent purification, preservation, and transportation operations.

[0074] 2. The high-density fermentation on the fermenter of this invention enables high-yield, soluble expression of recombinant humanized type III collagen in yeast, with a yield of up to 5.4 g / L, which is a high expression level in the industry.

[0075] 3. The recombinant human type III collagen obtained by this invention can be fermented in yeast at low cost and in high yield, and its purification process is simple and efficient. The optimized fermentation process of this invention yields recombinant humanized type III collagen in the fermentation broth with a purity of at least ≥90%. Only the simplest replacement of the preservation buffer is needed to achieve a protein purity of 97%, thus providing a simple and efficient purification method that minimizes purification costs. The entire process employs a staged ultrafiltration purification process, resulting in a short experimental cycle, high yield, and no need for chromatography steps, thus reducing the overall purification cost.

[0076] 4. The recombinant humanized type III collagen obtained by the preparation method of the present invention maintains its adhesive properties while possessing excellent water solubility and stability. It also effectively avoids the endotoxin pyrogen problem (endotoxin < 0.5 EU / mg), is free of animal-derived viruses, and has high product purity (97.47%). The recombinant humanized type III collagen has undergone firming and repair efficacy tests by a third-party company, demonstrating practical efficacy and meeting the common requirements for medical device and cosmetic raw materials.

[0077] 5. This invention selected truncated fragments from the N-terminus (T5), C-terminus (T6), and middle (T7) of human type III collagen. Under the same conditions, the T5 fragment, containing only four KGD cell adhesion sites (i.e., the recombinant humanized type III collagen shown in SEQ ID NO: 2 of this invention), showed significantly higher protein expression than the T6 and T7 fragments, which contain six KGD sites (T6 also contains one additional RGD site). Moreover, third-party testing proved that T5 still has firming and repairing effects (the test results showed significant differences). Therefore, this invention is not simply an arbitrary extraction of a protein sequence; the selected fragments have high protein expression levels, and through optimization of fermentation and purification processes, the obtained protein has high purity, low endotoxin content, and practical efficacy. Attached Figure Description

[0078] Figure 1 This is an SDS-PAGE electrophoresis image of 10 clones of the recombinant humanized type I collagen α1 chain fragment T11 expressed in shake flasks in preferred embodiment 2 of the present invention.

[0079] Figure 2 This is an SDS-PAGE electrophoresis image of eight different recombinant humanized type I collagen truncated fragments expressed in a 2L fermenter in the preferred embodiment 3 of the present invention.

[0080] Figure 3 This is an SDS-PAGE electrophoresis image of the recombinant humanized type I collagen α1 chain fragment T11 fermented in a 50L fermenter in the preferred embodiment 3 of the present invention.

[0081] Figure 4 This is an SDS-PAGE electrophoresis image of three batches of recombinant humanized type I collagen α1 chain fragment T11 from a 50 L fermenter in preferred embodiment 3 of the present invention.

[0082] Figure 5 The image shown is an SDS-PAGE electrophoresis result of the purification of the recombinant humanized type I collagen α1 chain fragment T11 in the preferred embodiment 4 of the present invention.

[0083] Figure 6 The purity test result (HPLC) of the recombinant humanized type I collagen α1 chain fragment T11 in preferred embodiment 4 of the present invention.

[0084] Figure 7 The total ion chromatogram is the LC-MS / MS mass spectrometry identification result of the recombinant humanized type I collagen α1 chain fragment T11 in the preferred embodiment of the present invention.

[0085] Figure 8 This is an LC-MS / MS diagram showing the peptide coverage of the recombinant humanized type I collagen α1 chain fragment T11 in preferred embodiment 5 of the present invention.

[0086] Figure 9 This is the PCR band pattern during high-copy transformant screening after electroporation of pPICZα-T5 to X33 in the preferred embodiment of the present invention, which is a preferred embodiment of the present invention.

[0087] Figure 10 This is an SDS-PAGE image of the recombinant humanized type III collagen T5 expression and identification in shake flask in preferred embodiment 7 of the present invention.

[0088] Figure 11 This is an SDS-PAGE image of recombinant humanized type III collagen T5, T6 and T7 expressed in a fermenter in preferred embodiment 7 of the present invention.

[0089] Figure 12 This is an HPLC chromatogram for the purity identification of recombinant humanized type III collagen T5 in preferred embodiment 8 of the present invention.

[0090] Figure 13 The total ion chromatogram of recombinant humanized type III collagen T5 in preferred embodiment 8 of the present invention, as identified by MS.

[0091] Figure 14 This is a peptide coverage diagram of recombinant humanized type III collagen T5 in preferred embodiment 8 of the present invention, as identified by MS.

[0092] Figure 15 This is an SDS-PAGE identification map of recombinant humanized type III collagen hydrolyzed by collagenase in preferred embodiment 9 of the present invention. Detailed Implementation

[0093] The present invention will be further described in detail below with reference to embodiments and accompanying drawings. The present invention discloses a high-purity recombinant humanized collagen fragment and its preparation process. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the same result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of the present invention to realize and apply the technology of the present invention. The reagents, consumables, and instruments used in implementing the present invention, except where specifically mentioned, are all common knowledge and general knowledge in the art, and the present invention does not impose any special limitations.

[0094] The BSM culture medium in this invention consists of: 26.7 mL / L 85% phosphate, 0.93 g / L calcium sulfate, 18.2 g / L potassium sulfate, 14.9 g / L magnesium sulfate heptahydrate, 4.13 g / L potassium hydroxide, 40 g / L glycerol, and 12 mL / L PTM1. PTM1 is a trace element mixture comprising: 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, 0.916 g / L cobalt chloride hexahydrate, 20 g / L zinc chloride, 65 g / L ferrous sulfate heptahydrate, 0.2 g / L biotin, and 5 mL / L sulfuric acid, diluted to 1 L with water.

[0095] Low-salt LB medium: 10 g peptone, 5 g yeast extract, 5 g NaCl, distilled water to a final volume of 1 L, autoclave at 121℃ for 20 min.

[0096] YPD medium: 20 g glucose, 10 g yeast extract, 20 g peptone. Add distilled water to a final volume of 1 L and autoclave at 115°C for 30 min.

[0097] BMGY medium: 20 g peptone, 10 g yeast extract, 100 mL 1 M potassium phosphate buffer (pH 6.0), 10 g glycerol. Autoclave at 121°C for 20 min. After cooling, add 100 mL 10×YNB and 2 mL 500×Biotin, which have been sterilized by filtration through a 0.22 μm membrane.

[0098] Example 1 Construction of recombinant humanized type I collagen expression plasmid

[0099] 1. Sequence selection

[0100] Example 1 provides high-purity recombinant humanized type I collagen truncated fragments and their preparation process. A total of 8 truncated fragments were selected, of which 6 were from human type I collagen α1 chain (NM_000088), and their amino acid sequences are shown as SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 9, SEQ ID NO: 10 and SEQ ID NO: 11, respectively. The other 2 were from human type I collagen α2 chain (NM_000089), and their amino acid sequences are shown as SEQ ID NO: 7 and SEQ ID NO: 8, respectively.

[0101] The truncated fragments of recombinant humanized type I collagen have a significant impact on protein expression and purification depending on the truncation position. Among the many truncated fragments studied in this invention, Example 1 uses type I collagen T1, T2, T3, T4, T8, T9, and T10 as comparative examples, and compares them with the preferred recombinant humanized type I collagen T11 of this application.

[0102] The eight truncated protein fragments selected covered the full length of the triple helix regions of the α1 and α2 chains of human type I collagen. Each fragment was rich in different numbers of cell adhesion recognition sites (RGD) and cell binding motifs (GER and GEK), as shown in Table 1. Cell adhesion recognition sites theoretically provide a basis for humanized type I collagen to maintain its cell adhesion, migration, proliferation, and differentiation properties. Cell binding motifs, due to their adjacent arrangement of positive and negative charges, enhance protein hydrophilicity and solubility while improving cell adhesion, thus facilitating soluble protein expression and secretion.

[0103] Table 1: Basic Information Analysis of the 8 Selected Shortened Protein Fragments

[0104]

[0105] 2. Construction and identification of recombinant expression plasmids

[0106] Online optimization of Pichia pastoris preferred codons (GenSmart™, Version Beta 1.0) was performed, balancing factors such as GC content, codon usage frequency, and RNase splicing sites, and eliminating [unspecified codons]. Pme I, Sac I, Sal The full-length sequences of the α1 chain (Col1A1) (as shown in SEQ ID NO: 14) and α2 chain (Col1A2) (as shown in SEQ ID NO: 15) of human type I collagen were synthesized by Genewiz Biotechnology Co., Ltd. with restriction endonuclease sites such as I.

[0107] Based on gene information, T1, T2, T8, T9, T10, and T11 used the full-length sequence of the α1 chain of synthesized human type I collagen as a template, while T3 and T4 used the full-length sequence of the α2 chain of synthesized human type I collagen as a template. Primers were designed and the target gene was amplified using a high-fidelity enzyme (2×phanta Flash Master Mix, Vazyme). Simultaneously, the pPICZα plasmid vector was linearized. Eco After purification by agarose gel extraction (RI-HF, NEB) of the target gene and linearized vector (Agarose Gel Extraction Kit, Enhanced Version, Tiangen Biotech), homologous recombination was performed and transformed into E. coli clone Match-T1 (Biomed). Clones were screened using low-salt LB plates containing 25 μg / mL Zeocin (Thermo Fisher Scientific). Plasmids were extracted and sequenced to obtain recombinant expression plasmids containing the correct T1, T2, T8, T9, T10, T11 and T3, T4 genes.

[0108] Example 2 Preparation of recombinant humanized type I collagen recombinant engineered bacteria

[0109] 1. Linearization of recombinant plasmids, followed by electroporation of Pichia pastoris X33.

[0110] (1) Using restriction endonucleases Pme I (NEB) linearized the recombinant plasmids pPICZα-T1 / T2 / T3 / T4 / T8 / T9 / T10 / T11 constructed in Example 1 above and then recovered them.

[0111] (2) Take 1-10 μg of the linearized plasmid from (1) above and add it to 80 μL of Pichia pastoris X33 competent cells (Pichia pastoris X33 can be purchased commercially), mix well and transfer to an electroporation cup for electroporation (cell electrode converter, Eporator).

[0112] (3) Immediately after the electroporation is completed, add 1 mL of pre-cooled YPD medium to the conversion cup for activation culture at 30 ℃ for 2-3 h.

[0113] (4) Spread 200 μL of bacterial culture onto a Zeocin-resistant (300 μg / mL) YPD plate and incubate at 30 °C for 2–5 days until a single colony appears.

[0114] (5) Select uniform yeast clones and transfer them to YPD medium containing 100 μg / mL of Zeocin-resistant medium. Incubate overnight at 30°C and 200 rpm. After genome extraction (NaOH lysis method) and verification, add 20% glycerol to preserve the culture.

[0115] 2. Shaking bottle expression

[0116] 10 μL of T11 glycerol bacteria was transferred to 5 mL of YPD medium for resuscitation. 100 μL of the resuscitation solution was then transferred to 100 mL of BMGY medium and cultured on a shaker (YC-05 three-layer horizontal shaker, Suzhou Jiemei Electronics) at 30 ℃, 200 rpm for 24 h. The yeast culture was collected aseptically and centrifuged using a high-speed refrigerated centrifuge (Multifuge×1R, Thermo Fisher Scientific, 4000 rpm, 7 min). The precipitate was collected, the supernatant was discarded, and the precipitate was resuspended in an equal volume of BMMY medium. The culture was then incubated at 30 ℃, 200 rpm, with methanol (Thermo Fisher Scientific, HPLC grade) added every 24 h to a final concentration of 1%. After 2 days of culture, the protein expression of 10 selected clones was detected by SDS-PAGE (DYY-6D electrophoresis apparatus power supply, Beijing Liuyi Biotechnology). The results are as follows: Figure 1 As shown, after shaking flask expression of 10 clones selected from T11, clone 6 of T11 (T11-6) showed the highest protein expression level, and the glycerol bacterium corresponding to this clone can be identified as the engineered bacteria for further fermentation in the fermenter.

[0117] Figure 1 Lane 1: The first clone of T11 (T11-1), Lane 2: Marker, Lane 3: The second clone of T11 (T11-2), Lane 4: The third clone of T11 (T11-3), Lane 5: The fourth clone of T11 (T11-4), Lane 6: The fifth clone of T11 (T11-5), Lane 7: The sixth clone with the highest expression level of T11 (T11-6), Lane 8: The seventh clone of T11 (T11-7), Lane 9: The eighth clone of T11 (T11-8), Lane 10: The ninth clone of T11 (T11-9), Lane 11: The tenth clone of T11 (T11-10).

[0118] The operation is the same for T1 / T2 / T3 / T4 / T8 / T9 / T10, each selecting the engineered bacteria with the highest expression level in the shake flask for fermentation in the fermenter.

[0119] Example 3 Expression of recombinant humanized type I collagen

[0120] 1. Comparison of fermentation tank levels for T1 / T2 / T3 / T4 / T8 / T9 / T10 / T11

[0121] The engineered bacteria with the highest expression levels of T1 / T2 / T3 / T4 / T8 / T9 / T10 / T11 selected in Example 2 were plated on YPD plates. Single colonies were picked and transferred to test tubes and cultured at 30 ℃ and 200 rpm for 7-9 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 ℃ and 200 rpm for 16-20 h to obtain secondary seed culture.

[0122] Each secondary seed was inoculated into a 2L fermenter (MC-JGF-2L, Beijing Mancang Technology) at a 10% inoculation rate. The culture medium used in the fermenter was BSM inorganic salt medium, and the components of BSM were as follows:

[0123] 85% phosphoric acid 26.7 mL / L, calcium sulfate 0.93 g / L, potassium sulfate 18.2 g / L, magnesium sulfate heptahydrate 14.9 g / L, potassium hydroxide 4.13 g / L, glycerol 40 g / L, PTM1 12 mL / L.

[0124] PTM1 is a mixture of trace elements, including: copper sulfate pentahydrate 6 g / L, sodium iodide 0.08 g / L, manganese sulfate monohydrate 3 g / L, sodium molybdate dihydrate 0.2 g / L, boric acid 0.02 g / L, cobalt chloride hexahydrate 0.916 g / L, zinc chloride 20 g / L, ferrous sulfate heptahydrate 65 g / L, biotin 0.2 g / L, sulfuric acid 5 mL / L, and diluted with water to 1 L. No pH adjustment is required. Slight heating is necessary for dissolution, but the temperature should not be too high. Heating can be performed according to the temperatures described in existing technology. After dissolution, the solution should be filtered out and stored at room temperature.

[0125] Culture conditions: 30 ℃, pH ≥ 5 controlled with ammonia water, aeration rate of 1 L·min -1 (Compressed air), tank pressure controlled at 0.05±0.01MPa. During the initial culture stage, adjust the rotation speed (preferably not exceeding 900rpm) and aeration rate to maintain 20%-30% dissolved oxygen. When the initial carbon source is depleted (i.e., dissolved oxygen rebounds, DO>50%), add 650 g / L of glycerol at a glycerol feeding rate of 8g / L / h. Use a strategy that links dissolved oxygen and rotation speed to maintain 30%-50% dissolved oxygen and monitor changes in OD600 (UV-Vis spectrophotometer, model: Purkinje TU-1810, Beijing Purkinje General Instrument Co., Ltd.).

[0126] Feeding strategy: A feeding strategy is adopted during the transition period from glycerol to methanol. That is, after the OD600 reaches 95-105, 2 g / L of methanol is added at once. At the same time, the glycerol feeding rate is gradually reduced to 0 g / L / h in 4 stages, namely 8, 6, 4, and 2 g / L / h, and each feeding rate is maintained for 30 minutes. When all carbon sources are exhausted (dissolved oxygen DO is again >50% and no longer decreases), methanol is added in batches. Each time DO is >50%, 8 g / L of anhydrous methanol is added. At this time, the rotation speed is maintained at the maximum (preferably 400-900 rpm). When the OD600 is about 200, the product is discharged from the tank. Once the OD600 reaches 95-105, a single addition of 2 g / L methanol is given to rapidly increase the amount of alcohol oxidase in the cells, thereby enabling them to begin utilizing methanol. Gradually decreasing the glycerol feeding rate can maintain the supply of carbon source to the yeast cells for a period of time, preventing a significant decrease in metabolic level or cell death. As the glycerol feeding rate slows down, the inhibition of alcohol oxidase and its promoter PAOX1 in the oxidase cell by glycerol is gradually relieved, preparing for the subsequent large-scale methanol-induced expression.

[0127] Eight different recombinant humanized type I collagen truncated fragments (T1 / T2 / T3 / T4 / T8 / T9 / T10 / T11) were fermented at high density in a 2L fermenter. Expression was induced with methanol. The supernatant of the fermentation broth was subjected to SDS-PAGE (12% separating gel). The electrophoresis results are shown below. Figure 2 As shown: the expression band of T1 (SEQ ID NO:5) was significantly weak; the expression level of T2 (SEQ ID NO:6) was very high, but protein degradation bands were observed immediately adjacent to and below its main band; T3 (SEQ ID NO:7) showed multiple bands, suggesting that the protein was not expressed or expressed at a weak level, and severe degradation occurred simultaneously with expression; T4 (SEQ ID NO:8) showed low protein expression and significant protein degradation; T8 (SEQ ID NO:9) and T9 (SEQ ID NO:10) showed relatively significant expression at 49 h, but significant degradation occurred at 66 h with increasing expression time; T10 (SEQ ID NO:11) and T11 (SEQ ID NO:1) showed significant and singular expression, but the expression level of T10 was significantly lower at the same fermentation time, approximately less than 1 / 3 of that of T11; among all 8 truncated fragments, T11 (SEQ ID NO:1) showed the weakest expression. NO:1 showed the highest expression level, with a protein content of 10 g / L, which is a high expression level in the industry. Moreover, the protein band was relatively simple, and the protein purity in the fermentation broth supernatant was at least >95% (SDS-PAGE), making it suitable as the preferred fragment for the biosynthesis of truncated fragments of recombinant humanized type I collagen.

[0128] Lane 1: T1 fermentation expression for 66 h, Lane 2: T2 fermentation expression for 66 h, Lane 3: T3 fermentation expression for 66 h, Lane 4: T4 fermentation expression for 66 h, Lane 5: Protein Marker, Lane 6: Protein Marker, Lane 7: T8 fermentation expression for 49 h, Lane 8: T8 fermentation expression for 66 h, Lane 9: T9 fermentation expression for 49 h, Lane 10: T9 fermentation expression for 66 h, Lane 11: T10 fermentation expression for 49 h, Lane 12: T10 fermentation expression for 66 h, Lane 13: T11 fermentation expression for 49 h, Lane 14: T11 fermentation expression for 66 h.

[0129] From the protein expression results, T11 (SEQ ID NO:1), which showed the highest and most stable protein expression, contained two cell adhesion sites (RGD), seven GER, and one GEK cell binding motif. However, its number was not the highest among all eight truncated proteins. This indicates that truncated fragments derived from type I collagen, whether from the α1 or α2 chain, cannot simply be selected arbitrarily to construct an engineered bacterium that can express the target protein or express it well. Instead, extensive comparative screening is required to select truncated protein fragments with high expression levels, stable fermentation without degradation, and a single target protein band that facilitates downstream purification before proceeding to the next step of industrial scale-up.

[0130] 2. Scale up and replicate the best-expressing T11 in a 50L fermenter.

[0131] The clone with the highest expression level selected in Example 2 was plated on a YPD plate. A single colony was picked and transferred to a test tube and cultured at 30 ℃ and 200 rpm for 7-9 h to obtain the primary seed culture. 600 μL of the primary seed culture was transferred to 60 mL of YPD medium for small-scale amplification by shaking at 30 ℃ and 200 rpm for 16-20 h to obtain the secondary seed culture.

[0132] Secondary seeds were inoculated into the fermenter at a rate of 10% (0.05m). 3 Fermenter, product number H22-015, Lianyungang Hechang Machinery Co., Ltd.; fermenter control system HC-BIO-8000, Lianyungang Hechang Bioengineering Equipment Co., Ltd. The fermenter also uses BSM culture medium. Culture conditions: 30 ℃, pH ≥ 5 controlled with ammonia, aeration rate of 1 L / min. -1(Compressed air), tank pressure controlled at 0.05±0.01MPa, maintaining dissolved oxygen at 20%-30% during the initial production stage. After the initial carbon source is depleted, 650 g / L glycerol is added at a feed rate of 8 g / L / h. When OD600 reaches 95-105, 2 g / L methanol is added in a single batch, while the glycerol feed rate is gradually reduced to 0 g / L / h in four stages: 8, 6, 4, and 2 g / L / h, each stage maintained for 30 minutes. After the carbon source is depleted (dissolved oxygen DO > 50% and no longer decreasing), methanol is added in batches, with 8 g / L anhydrous methanol added each time DO > 50%. Cell growth is 20 h, and induction is 96 h. SDS-PAGE analysis of protein expression at 48 h, 64 h, and 96 h is shown below. Figure 3 Lane 1: 48h, Lane 2: 80h, Lane 3: 96h.

[0133] Three batch replication experiments were conducted in a 50 L fermenter, maintaining consistent process and application parameters. Electrophoresis results showed that the target product molecular weight was consistent across all three batches, with no obvious impurities. Figure 4 As shown, lane 1: 50 L fermentation of batch 1; lane 2: 50 L fermentation of batch 2; lane 3: 50 L fermentation of batch 3; lane 4: Protein Marker. The target yields of recombinant humanized collagen in the fermentation broth supernatant were 10.2 g / L, 9.7 g / L, and 10.3 g / L, respectively, with an RSD (relative standard deviation) of 3%, meeting the stability requirements (<5%) for bio-fermentation experiments; demonstrating that the fermentation process of this invention is stable and controllable at a 50 L scale, with good reproducibility. Figure 1 , 2 Based on the area ratio of the target band to the contaminant protein band in the electrophoresis patterns of T11 fermentation broth in sections 3 and 4, it can be deduced that the concentration of the target protein in T11 fermentation broth exceeds 90%.

[0134] Example 4 Purification of recombinant humanized type I collagen

[0135] (1) The fermentation broth was centrifuged (high-speed refrigerated centrifuge, Multifuge×1R, Thermo Fisher Scientific) to remove the bacterial cells and obtain the supernatant. The supernatant was then filtered using a 0.45 μm hollow fiber membrane (hollow fiber membrane element, BMV2-18-0.45, 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. The subsequent ultrafiltration membrane fouling rate was reduced by approximately 30% or more. The microfiltration target protein recovery rate was ≥90%.

[0136] (2) The microfiltrate was concentrated and replaced with a 30 kDa hollow fiber membrane ultrafiltration solution (hollow fiber membrane element, BFV-18-30 kDa, Shandong Bona Biotechnology), and then replaced with a 50 mM sodium acetate (Tianjin Kaitong Chemical, analytical grade) buffer solution at pH 4.5 to obtain the chromatographic loading solution. The transmembrane pressure difference was controlled at 0.05-0.15 MPa, the temperature at 20-25 ℃, and the turbidity of the microfiltrate was controlled at <5 NTU.

[0137] (3) SP chromatography was performed (SP Beads 6FF packing material, Changzhou Tiandi Renhe Biotechnology Co., Ltd.; purification equipment used MTH3LPUMP 3L / MIN chromatography pump system, MTH04560M chromatography column), and the linear flow rate was maintained at about 50 cm / h during elution:

[0138] a. Equilibration: Equilibrate with 50 mM sodium acetate buffer, pH 4.5, for 3-5 CV.

[0139] b. Sample loading: The above ultrafiltration replacement solution is loaded with a dynamic loading of 30-40 g / L of packing material. This loading is within the reasonable range recognized in the industry. It avoids the low processing efficiency and high cost caused by low loading (such as <20 g / L), while high loading (such as >50 g / L) may cause risks such as co-adsorption of impurities and sample flow-through.

[0140] c. Rinsing: 50 mM sodium acetate buffer, pH 4.5, 3-5 CV;

[0141] d. Washing: 50 mM sodium acetate buffer, 50 mM sodium chloride, pH 4.5, 2 CV;

[0142] e. Elution: 50 mM sodium acetate buffer, 200 mM sodium chloride, pH 4.5, elute for 3 CV to obtain the target protein fraction. SDS-PAGE results of purified fractions are shown below. Figure 5 The diagram shows the following lanes used: 1: Marker, 2: Sample loading, 3: Flow-through, 4: Elution 1, 5: Elution 2, and 6: Elution merging.

[0143] (4) The collected eluent was desalted using a 2 kDa spiral wound membrane, then ultrafiltered with a 50 kDa hollow fiber membrane (BFH-18-50KDa, Shandong Bona Biotechnology) to remove endotoxins. It was then sterilized by filtration using a 0.22 μm sterile membrane sterilized under high pressure, and freeze-dried using a low-temperature vacuum freeze dryer (FDV-1200, Shanghai EYELA, cold trap temperature -50℃, vacuum <50Pa). Finally, it was treated with 6 kGy of cobalt-60 (… 60High-purity recombinant humanized type I collagen was obtained by irradiating with Co (γ-rays) for 11 hours (Tianjin Huaming High-Tech Irradiation Co., Ltd.). Figure 6 HPLC analysis showed that the purity of the obtained recombinant humanized type I collagen T11 lyophilized powder was ≥99%. Endotoxin levels of the T11 lyophilized powder were determined by the Limulus Amebocyte Lysate (LAL) gel electrophoresis, and the result was <0.05 EU / mg.

[0144] Thus, after a series of purification processes including centrifugation, microfiltration, ultrafiltration concentration, cation exchange chromatography, desalting, endotoxin removal, filtration sterilization, low-temperature vacuum freeze-drying, and cobalt-60 irradiation (6 kGy), each process step proceeds in order of impurity removal priority. The results of each step must meet the standards described in the above methods to meet the feed requirements of the next step. This yields recombinant humanized type I collagen α1 chain fragment freeze-dried fiber that simultaneously meets the following requirements: high purity (≥99%, HPLC), endotoxin <0.05 EU / mg, and protein content ≥95% (referencing the Pharmacopoeia of the People's Republic of China: 2020 Edition, Part III, 0731 Protein Content Determination Method, Method I Kjeldahl Nitrogen Determination, tested by Tianjin Houpu Technology Testing Co., Ltd.).

[0145] The 50 L fermentation and purification processes of this invention have good scale-up potential. The fermenter, designed with geometric similarity (height-to-diameter ratio unchanged at 2:1), can be scaled up to a 1000 L tank. At that point, by adjusting the stirring speed and aeration rate, the dissolved oxygen and mass transfer requirements of high-density Pichia pastoris fermentation can be met, ensuring a stable target protein yield of approximately 10 g / L. The subsequent purification process of the fermentation broth achieves linear scale-up of microfiltration and ultrafiltration by increasing the membrane area. The cation exchange column can be scaled up proportionally to the column bed volume; these are all conventional scale-up methods in the field, enabling large-scale production.

[0146] Preferably, during the fermentation process, the present invention adjusts the stirring speed and aeration rate to meet the dissolved oxygen and mass transfer requirements of Pichia pastoris in a 50L to 1000L fermentation system; specifically, the preferred solution is to maintain the dissolved oxygen of the fermentation broth at 30%-50% by adjusting the stirring speed and aeration rate.

[0147] In this invention, meeting the mass transfer requirement mainly means ensuring the efficiency of oxygen transfer. Specifically, through comprehensive engineering design of the stirring paddle material, blade length, shape, appearance, and shear force requirements, oxygen can be quickly and fully transferred to the fermentation system. Existing technologies based on the above comprehensive engineering design are not within the scope of protection of this invention. Any modification scheme that can adjust the fermenter speed and ultimately ensure 30%-50% dissolved oxygen mass transfer requirement is applicable to this invention.

[0148] Further preferably, an initial rotation speed of 100-300 rpm and / or an initial aeration rate of 1 VVM (VVM, Volume per Volume per Minute, aeration ratio, m³ / (m³·h)) are used in the fermentation system. As fermentation progresses, the rotation speed and / or aeration rate are adjusted upward or downward to maintain the dissolved oxygen in the fermentation broth at 30%-50% to meet its dissolved oxygen mass transfer requirements.

[0149] Example 4 also validated the expression in a 1000L fermentation system. By adjusting the rotation speed to 100-300 rpm and maintaining the aeration rate at 1 VVM (VVM, Volume per Volume per Minute, aeration ratio, m³ / (m³·h)) on a 1000L fermenter, dissolved oxygen was maintained at 30%-50%, ensuring that the yield of the target protein was stable at around 10 g / L.

[0150] Example 5: Identification of recombinant humanized type I collagen proteome

[0151] The recombinant humanized type I collagen sample with the amino acid sequence shown in SEQ ID NO: 1 was sent to Beijing Biotech Biotechnology Co., Ltd. for analysis. After SDS-PAGE, reductive alkylation, SP3 enrichment (Trypsin digestion), recovery of the digest and lyophilization of the digest, and C18 (Stage-Tip) desalting pretreatment, the sample was analyzed by LC-MS / MS (Ensy-nLC1200 / QExactive, Thermo Fisher Scientific). The C18 column was 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-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.

[0152] The high-quality peptide sequences of the actual sample were compared with the theoretical sequence of the target protein, recombinant type I humanized collagen. The mass spectrometry identification results are shown in Table 2. The target protein consists of 603 amino acids and was identified as 58 peptides, with a peptide coverage of 92.5% and a molecular weight of 53.393 kDa, consistent with the theoretical value. Total ion chromatogram (…) Figure 7The results showed good peptide separation with no obvious interference peaks. The peptide coverage diagram (Figure 8, red indicates the area covered by the matching amino acids) showed that most of the amino acid sequence from SEQ ID NO:1 to 603 was covered. The 45 uncovered amino acids were mainly concentrated at positions 1-3 at the N-terminus, positions 163-174 in the middle, and positions 574-603 at the C-terminus, all of which are non-functional regions. The main core functional sites (RGD, KGD, GER, GEK, etc.) were completely covered, which fully confirmed that the purified product was the target recombinant humanized type I collagen α1 chain fragment.

[0153] Table 2 Mass spectrometry identification results

[0154]

[0155] Example 6 Construction of recombinant humanized type III collagen recombinant expression plasmid

[0156] 1. Sequence selection

[0157] Example 6 provides a recombinant humanized type III collagen and its preparation method. The amino acid sequence T5 is selected from the 584 amino acids (amino acids 168 to 751) near the N-terminus of human type III collagen (NM_000090). This fragment is rich in four cell adhesion sites (KGD), and the amino acid sequence is shown in SEQ ID NO:2. Cell adhesion sites theoretically provide a basis for humanized type III collagen to maintain its cell adhesion, migration, proliferation, and differentiation characteristics.

[0158] The truncated fragments of recombinant humanized type III collagen have a significant impact on protein expression and purification depending on the truncation position. Among the many truncated fragments studied in this invention, Example 6 uses type III collagen T6 and type III collagen T7 as comparative examples to compare with the preferred recombinant humanized type III collagen T5 of this application.

[0159] T6 is selected from 574 amino acids (amino acids 623 to 1196) near the C-terminus of type III collagen (NM_000090). This fragment is rich in 6 cell adhesion sites KGD and 1 RGD, and the amino acid sequence is shown in SEQ ID NO:12.

[0160] T7 is selected from 587 amino acids (amino acids 447 to 1033) at the mid-terminus of type III collagen (NM_000090). This fragment is rich in 6 cell adhesion sites (KGD), and the amino acid sequence is shown in SEQ ID NO:13.

[0161] 2. Construction of recombinant expression plasmids

[0162] Codon optimization was performed on the GenSmart™ online codon optimization website (Version Beta 1.0) for Pichia pastoris expression hosts. Generally, a codon adaptation index (CAI, theoretical basis: PM Sharp, WH Li, DOI:10.1093 / nar / 15.3.1281) ≥0.80 is considered in the industry as the standard for predicting efficient expression of recombinant proteins. The CAI of T5, T6 and T7 reached 0.86, 0.81 and 0.83, respectively.

[0163] Each of the optimized humanized type III collagen fragments, T5, T6, and T7 genes, was augmented with additives at the 5' and 3' ends, respectively. EcoR I and Not The I restriction site was synthesized by Suzhou Genewise Biotechnology Co., Ltd. The gene sequence of T5 is shown in SEQ ID NO: 4, the gene sequence of T6 is shown in SEQ ID NO: 16, and the gene sequence of T7 is shown in SEQ ID NO: 17.

[0164] After transformation, the plasmid vector containing the gene was extracted and subjected to restriction endonuclease. EcoR I and Not I (NEB) was subjected to double enzyme digestion, and purified using agarose gel extraction kit (enhanced version, Tiangen Biotech). Similarly, the pPICZα plasmid vector was double-digested and purified using the same method.

[0165] Humanized type III collagen fragments T5, T6, and T7, along with the pPICZα plasmid vector fragment, were ligated using T4 DNA ligase and transformed into E. coli clone Match-T1 (Biomed). Clones were screened using low-salt LB plates containing 25 μg / mL Zeocin (Thermo Fisher Scientific), and the results were verified by sequencing to obtain recombinant expression plasmids containing the correct T5, T6, and T7 genes.

[0166] 3. Linearization of recombinant plasmids, followed by electroporation of X33 plasmids.

[0167] Take 20 μg of recombinant plasmid and use Pme Linearized by I enzyme digestion, digested at 37 °C for 2 h, and purified by agarose gel extraction.

[0168] Table 3 below shows the configuration table for the enzyme digestion linearization system.

[0169] Table 3: Configuration of Enzyme Digestion Linearization System

[0170]

[0171] Pichia Pastoris X33 competent cells were prepared using sorbitol. The prepared competent cells were slowly revived on ice. A sterile electroporation cuvette was prepared in advance and irradiated with UV light in a clean bench for at least 15 min, then pre-cooled on ice. 10 μL of linearized recovered plasmid was added to the Pichia Pastoris X33 competent cells and gently mixed. All competent cells were aspirated using a pipette and transferred to a pre-cooled electroporation cuvette. The cuvette was inserted into an electroporator (electrode converter) and electroporated at 1500V. After electroporation, 1 mL of YPD solution was added to the electroporation cuvette, and the mixture was gently agitated with a pipette tip. All liquid in the cuvette was aspirated and transferred to the original competent cell EP tubes. The cells were incubated at 30℃ and 160 rpm for 2 h. 200 μL of the revived bacterial culture was then plated onto YPD medium containing a final concentration of 300 μg / mL Zeocin-resistant cells and incubated at 30℃ for at least 2 days.

[0172] 4. High-copy screening and identification of Zeocin plate (taking T5 as an example; the operation is the same for T6 and T7)

[0173] Uniform yeast clones were selected from resuscitation plates and transferred to YPD medium containing 100 μg / mL Zeocin resistance. The cultures were incubated overnight at 30°C and 200 rpm. Eleven clones were selected, and their genomes were extracted (using the NaOH lysis method). Genotyping was performed by PCR, and the results were verified by 1% agarose gel electrophoresis.

[0174] Electrophoresis results as follows Figure 9 As shown, 11 randomly selected T5 clones were identified by PCR, and the target bands were all single and the band size was as expected, proving that all 11 clones had homologous recombination of the target gene into the genome and could be used for the next step of expression identification. Among them, the identification band of clone No. 4 was the brightest. Figure 9 In the diagram, lane 1:M represents DNA standards (from top to bottom: 2000, 1500, 1000, 750, 500, 250, 100bp); 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; and lane 12: clone 11.

[0175] Example 7 Expression of recombinant humanized type III collagen

[0176] 1. Shaking bottle expression (taking T5 as an example, the operation is the same for T6 and T7)

[0177] 100 μL of resuscitation solution was transferred to 100 mL of BMGY medium and cultured in small shake flasks (250 mL shake flasks) on a shaker (YC-05 three-layer horizontal shaker, Suzhou Jiemei Electronics) at 30 ℃, 200 rpm for 24 h. Yeast culture was collected under aseptic conditions, centrifuged at 4000 rpm for 7 min, the precipitate was collected, the supernatant was discarded, and the precipitate was resuspended in an equal volume of BMMY medium. The culture was then incubated at 30 ℃, 200 rpm, with 1% methanol (Thermo Fisher Scientific, HPLC grade) added every 24 h for 2 days. Protein expression was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Electrophoresis equipment: DYY-6D electrophoresis apparatus; power supply: Beijing Liuyi Biotechnology; stacking gel concentration: 5%; separating gel concentration: 12%.

[0178] Electrophoresis results as follows Figure 10 As shown, all 11 clones of T5 expressed a clear, single protein band with a purity >80%. Clone 6 showed a relatively high expression level and can be used for subsequent fermentation production. Two very weak bands were also observed below the target band, which may be due to degradation of the target band. This issue needs to be avoided during subsequent fermentation in the fermenter.

[0179] Figure 10 In the sequence, lane 1: the first clone of T5, lane 2: Protein Marker, lane 3: the second clone of T5, lane 4: the third clone of T5, lane 5: the fourth clone of T5, lane 6: the fifth clone of T5, lane 7: the sixth clone of T5, lane 8: the seventh clone of T5, lane 9: the eighth clone of T5, lane 10: the ninth clone of T5, lane 11: the tenth clone of T5, and lane 12: the eleventh clone of T5.

[0180] 2. Comparison of expression levels of T5, T6, and T7 proteins in fermenters

[0181] The clone No. 6 selected from the shake flask was plated on a YPD plate. The next day, a single colony was picked and transferred to a test tube and cultured at 30 ℃ and 200 rpm for 7-9 h to obtain the primary seed culture. 600 μL of the primary seed culture was transferred to 60 mL of YPD medium for small-scale shaking and expansion culture at 30 ℃ and 200 rpm for 16-20 h to obtain the secondary seed culture.

[0182] Secondary seed culture was inoculated into the fermenter (10L fermenter, MC-JGF-10L, Beijing Mancang Technology) at a 10% inoculation rate. The culture medium formulation in the fermenter was: yeast extract 10 g / L, peptone 20 g / L, dipotassium hydrogen phosphate 2.3 g / L, potassium dihydrogen phosphate 11.8 g / L, glycerol 10 g / L, 0.02% biotin 2 mL, and YNB 13.4 g / L (biotin and YNB were sterilized by passing through a 0.22 μm membrane). Based on the predicted results from the shake-flask stage, the protein did indeed degrade during fermentation in the fermenter. Therefore, an additional 30 g / L of casein (Casein, Maclean's, BR, C17372241) was added to the fermentation medium. Experiments were conducted at casein concentration gradients of 0, 10, 20, 30, and 40 g / L. At 30 g / L, the degradation of the protein was effectively inhibited without affecting the growth of Pichia pastoris. Because casein is a mixture rich in casein, its amino acid sequence contains a large number of hydrophobic peptide segments and peptide bond structures similar to collagen. When a high concentration of casein is added, it will act as a "bait protein" to compete with collagen for the active site of protease, greatly reducing the probability of protease attacking the target collagen and blocking degradation from the source.

[0183] Culture conditions: 30℃, pH ≥ 5 controlled with ammonia water, aeration rate of 1 VVM (compressed air). When the initial carbon source was depleted (i.e., dissolved oxygen rebound, DO > 50%), 650 g / L glycerol was added. A strategy of linking dissolved oxygen and rotation speed was adopted to maintain 30%-50% dissolved oxygen, and the change of OD600 was monitored (UV-Vis spectrophotometer, model: Purkinje TU-1810, Beijing Purkinje General Instrument Co., Ltd.).

[0184] Replenishment strategy:

[0185] The Pichia pastoris alcohol oxidase promoter PAOX1 is induced by methanol and inhibited by glucose or glycerol because methanol metabolism depends on alcohol oxidase (AOX) within peroxisomes. The alcohol oxidase required for methanol metabolism is sorted into peroxisomes, forming a compartmentalized structure. When glucose or glycerol is used as the carbon source, there are only one or a few small peroxisomes in the cell. However, when methanol is used as the carbon source, peroxisomes occupy almost 80% of the entire cell volume, and AOX increases to 35%-40% of the total cellular protein. Therefore, in high-density Pichia pastoris fermentation, the feeding strategy is crucial and directly affects protein expression.

[0186] The method of directly replenishing methanol after the initial glycerol in the system is simple and easy to implement, but this feeding strategy has potential problems. First, the Pichia pastoris cells do not yet have enough alcohol oxidase (which is produced in large quantities when methanol is present) to metabolize methanol. If methanol, as the only carbon source, cannot play its role in time, the yeast will reduce its metabolic level to maintain survival, resulting in decreased activity. Second, the long-term accumulation of methanol may lead to poisoning or even death of some cells, causing intracellular proteases to be secreted into the culture medium, potentially leading to the degradation of target proteins. Therefore, this invention explored a methanol switching method and selected a glycerol and methanol switching period strategy. That is, after the cells have grown for 20 hours and the OD600 reaches 95-105, 2 g / L of methanol is added at once, while the glycerol feeding rate is controlled at 8 g / L / h, and the feeding rate is reduced by 2 g / L / h every 0.5 hours, and reduced to 0 g / L / h in four stages within 2 hours.

[0187] The advantage of the switching-phase feeding strategy of this invention is that the addition of a small amount of methanol first rapidly increases the amount of alcohol oxidase in the cells, thereby enabling the cells to begin utilizing methanol. At the same time, the gradient reduction of the glycerol feeding rate can maintain the supply of carbon source to yeast cells for a period of time without causing a significant decrease in metabolic level or cell death. As the glycerol feeding rate slows down, the inhibition of alcohol oxidase and its promoter PAOX1 in the oxidase cell by glycerol is gradually reduced, preparing for the subsequent large-scale methanol-induced expression.

[0188] Once all carbon sources are depleted (dissolved oxygen rises significantly, DO > 50%), methanol is added to initiate the formal induction phase. Methanol is added in batches; each time methanol is depleted (i.e., when DO rebounds to > 50%), methanol is added again. The amount of methanol added each time is fixed, consistently 8g per liter of the initial fermentation volume. The fermentation speed is maintained at maximum (preferably 400-900 rpm). The fermentation tank is closed when the OD600 is approximately between 175-250. Compared to the common method of constant methanol addition (maintaining a final concentration of 2g / L for a long period), the advantage of batch methanol addition is that there is sufficient methanol for the target protein to be expressed in large quantities within a short time. During this period, the methanol is periodically consumed and does not accumulate continuously, which can shorten the total fermentation time to a certain extent without affecting protein yield. Furthermore, after adding methanol during fermentation, there is no need for dedicated and frequent monitoring of methanol concentration; only DO needs to be observed. Methanol is added once when DO > 50%, eliminating the need for specialized methanol concentration monitoring equipment and saving workload for fermentation personnel.

[0189] Three different recombinant humanized type III collagen truncated fragments (T5, T6, and T7) were simultaneously fermented at high density in a 10L fermenter, induced to express with methanol, and subjected to SDS-PAGE (12% separating gel). The electrophoresis results are shown below. Figure 11As shown, the protein expression levels in the two replicates of T5 were significantly higher than those in the two replicates of T6 and T7. Figure 11 In the diagram, lane 1: Protein Marker; lane 2: Repeat of T5, experiment 1 (T5-1); lane 3: Repeat of T5, experiment 2 (T5-2); lane 4: Repeat of T6, experiment 1 (T6-1); lane 5: Repeat of T6, experiment 2 (T6-2); lane 6: Repeat of T7, experiment 1 (T7-1); lane 7: Repeat of T7, experiment 2 (T7-2). Figure 11 The ratio of the area of ​​the target protein to the area of ​​the contaminant protein in the protein spectrum of the T5 fermentation broth can be used to estimate that the purity of the target protein in the T5 fermentation broth is over 90%.

[0190] Comparative analysis revealed that T5 is the 584 amino acids (amino acids 168 to 751) near the N-terminus of human type III collagen, a fragment rich in only 4 KGD cell adhesion sites; T6 is the 574 amino acids (amino acids 623 to 1196) near the C-terminus, a fragment rich in 6 KGD cell adhesion sites plus 1 RGD; and T7 is the 587 amino acids (amino acids 447 to 1033) at the mid-terminus, a fragment also rich in 6 KGD cell adhesion sites. From the protein expression results, under the same conditions, the T5 fragment containing only 4 cell adhesion sites (KGDs) showed significantly higher protein expression than the T6 and T7 fragments containing 6 KGDs (T6 also contains one RGD). T5, T6, and T7 cover the entire amino acid sequence of human type III collagen, indicating that it is not appropriate to simply select a truncated fragment from human type III collagen to construct engineered bacteria for expression. Instead, it is necessary to compare and screen to select the truncated fragment with high expression levels. In this invention, the T5 fragment near the N-terminus was selected for subsequent fermentation, and the protein yield reached 5.4 g / L, which is a high expression level in the industry.

[0191] Example 8 Purification and Detection of Recombinant Humanized Type III Collagen

[0192] 1. Protein purification

[0193] Table 4 shows the yield of the process steps in Example 8.

[0194] Centrifugation: Take 100 mL and centrifuge at 5000 rpm for 10 min using a high-speed refrigerated centrifuge (model Multifuge×1R, manufacturer Thermo Fisher Scientific). Discard the bacterial cells and collect 91 mL of the supernatant.

[0195] Microfiltration: The centrifuged supernatant was microfiltered using a 0.45μm hollow fiber membrane (PES material, Kebote). The transmembrane pressure difference (TMP) was controlled at 0.5-1.0 bar, and the temperature was controlled at 20-25℃. The permeate was collected. After the initial volume was concentrated 5 times, the filter was washed twice with pure water, with 20mL of pure water added each time. The permeate was collected again. 108ml of microfiltrate was obtained by mixing.

[0196] Ultrafiltration to remove impurities: The microfiltrate was subjected to ultrafiltration using a 100K ultrafiltration membrane (PES material, Kebotai) to remove impurities such as proteins. The transmembrane pressure difference (TMP) was controlled at 0.5-1.0 bar, and the temperature was controlled at 20-25℃. The permeate was collected; after being concentrated 5 times by volume, it was washed with PB buffer, and the permeate was collected again. After mixing, 135 mL of 100K ultrafiltrate was obtained.

[0197] Ultrafiltration solution replacement: The above 100K ultrafiltrate was ultrafiltered using a 30 kDa hollow cellulose membrane (PES material, Kebote) to remove small molecule proteins, desalinate, and concentrate. The transmembrane pressure difference (TMP) was controlled at 0.05-0.1 MPa, and the temperature was controlled at 20-25℃. After concentrating 3 times, PB was added for washing. After washing 225 mL, pure water was added for further washing until the conductivity was <0.5 ms / cm. 276 mL of the 30K concentrate was collected.

[0198] Table 4: Yield Table for Process Steps

[0199]

[0200] Note: To control endotoxin levels, each step of the microfiltration, ultrafiltration, and hollow fiber membrane process requires washing with 0.5M NaOH before and after use. The experimental water used is deionized water (prepared by a Thermo Fisher Scientific Smart2PurePro CV / UF16LPH pure water system).

[0201] 2. Endotoxin removal

[0202] Purcise TM A Q-membrane capsule adsorber (specification: CX0010EAQ16MM1P, PES membrane material, Cobot Corporation, nominal membrane volume 10mL) is used for further removal of endotoxins. The Q membrane contains anion exchange media and is modified with special functional groups in the cross-linked polymer coating to achieve the separation and purification of negatively charged components. Since endotoxins carry a significant negative charge at pH > 2, the Q membrane adopts a radial flow channel form, controlling the pressure to not exceed 3.0 bar and the flow rate to 10-250mL / min. The filtered components are collected for endotoxin detection, and the specific method is described in Example 4. The test is performed according to the Bacterial Endotoxin Test Method in Part III of the Pharmacopoeia of the People's Republic of China, 2020 edition, with a result <0.5EU / mg.

[0203] Compared with traditional column chromatography packing materials in existing technologies, membrane chromatography media have stronger chemical compatibility and ultra-low non-specific adsorption, higher process flow rates to shorten process time, higher dynamic binding capacity to reduce process costs, and linear scale-up of endotoxin removal can be achieved by increasing membrane area, number of membrane layers, and nominal membrane volume.

[0204] 3. Protein detection (Agilent 1260 high performance liquid chromatography)

[0205] Protein purity was determined using the SEC-HPLC method, as follows:

[0206] (1) Chromatographic column: Galaxy SEC S2000, specifications: 5μm, 300×7.8mm, model: FMH-3145-KONU (Guangzhou Feilomen)

[0207] (2) Mobile phase: 150 mmol / L sodium phosphate buffer

[0208] Preparation method: Weigh 3.145g of NaH2PO4 and 17.572g of Na2HPO4, dissolve them in 800ml of 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.

[0209] (3) Column temperature: 25℃

[0210] (4) Flow rate: 1 mL / min

[0211] (5) Detection wavelength: 205nm

[0212] (6) Injection volume: 10 μL

[0213] (7) Gradient degree: isocratic elution for 20 min

[0214] The T5 protein obtained from the above protein purification steps was analyzed by HPLC, and the results are as follows: Figure 12 As shown, using the area normalization method, the purity of target protein T5 (RT=5.815) was 97.47%, impurity 1 (RT=5.535) was 1.18%, and impurity 2 (RT=11.277) was 1.35%.

[0215] 4. Mass spectrometry identification

[0216] The samples were commissioned to Beijing Baitaipaike Biotechnology Co., Ltd. for testing.

[0217] SDS-PAGE, strip decolorization, reductive alkylation, in-gel digestion (Trypsin enzyme), peptide extraction, and C18 pretreatment were performed before analysis using a Vanqusih Neo / Orbitrap Exploris 480 liquid chromatography-mass spectrometry (LC-MS) system (Thermo Fisher Scientific). C18 pre-column: 75 μm × 2 cm, NanoViper C18 3 μm, 100A; analytical column: 75 μm × 25 cm, NanoViper C18 2 μm, 100A. Mobile phase A: 0.1% FA; mobile phase B: 0.1% FA, 80% ACN; flow rate: 300 nL / min; analysis time for each fraction: 66 min; mass spectrometry full scan range: 300–1800 m / z.

[0218] Raw mass spectrometry data were acquired, and the target protein databases were searched using PEAKSStudio. The raw mass spectrometry files were analyzed, and the identification results were obtained by matching the data to the relevant databases.

[0219] The high-quality peptide sequences of the actual samples were compared with the theoretical sequence of the target protein, recombinant type III humanized collagen. Figure 13 The total ion current chromatogram shows good peptide separation with no obvious interference peaks. The mass spectrometry identification results are summarized in Table 5. The target protein consists of 584 amino acids, and the identified target protein was divided into 66 peptides, with a peptide coverage rate of 89.38%. Figure 14 The red portion shows that positions 64 to 584 of the amino acid sequence SEQ ID NO: 2 are covered, including all four KGD active sites, confirming that the purified product is the target recombinant humanized type III collagen fragment.

[0220] Table 5: Mass Spectrometry Identification Results

[0221]

[0222] Example 9: Recombinant Human Type III Collagen Enzyme Hydrolysis Experiment

[0223] Collagenase is a peptide bond endopeptidase that specifically recognizes the Pro-X-Gly-Pro sequence (which appears frequently in collagen but rarely in other proteins) and cleaves the peptide bond between the neutral amino acid (X) and glycine (Gly) in the sequence to achieve the enzymatic hydrolysis of collagen.

[0224] This invention hydrolyzes T5 with a specific type III collagenase, while simultaneously using an equal amount of non-collagenous SOD protein as a control, which is also hydrolyzed with type III collagenase. The results are as follows. Figure 15As shown, compared with the control without collagenase (lane 2, with clear target bands), after hydrolysis with type III collagenase, the protein of T5 disappeared, indicating that type III collagenase had completely hydrolyzed T5 (lane 3), and only the bands of type III collagenase itself remained on the lane (compared with lane 5); after adding type III collagenase to the SOD protein (lane 5), compared with the control without type III collagenase (lane 4), the protein content only decreased slightly, and this slight decrease might be caused by the low specificity of the crude type III collagenase. The experiment proved that collagenase could specifically hydrolyze the T5 protein.

[0225] Figure 15 In it, lane 1: Protein Marker (from top to bottom: 240 kDa, 140 kDa, 115 kDa, 80 kDa, 58 kDa, 50 kDa, 31 kDa, 25 kDa, 15 kDa, 6.5 kDa), lane 2: T5 (without collagenase), lane 3: T5 + collagenase, lane 4: SOD (without collagenase), lane 5: SOD + collagenase, lane 6: control of collagenase itself.

[0226] Example 10 Detection of endotoxin in recombinant human-derived type III collagen

[0227] The T5 protein obtained in Example 8 was made into a solution of 1 mg / mL or used for detection after appropriate dilution. The dilution water was the special water for endotoxin detection provided in the kit.

[0228] The detection reagents included: Limulus reagent for endotoxin detection by gel method, Limulus reagent with a sensitivity level of 0.5 EU / mL; positive control: that is, the bacterial endotoxin working standard (titer 10 EU / vial); negative control: special water for endotoxin detection, manufacturer: Zhanjiang Andes Biological Co., Ltd.

[0229] The centrifuge tubes and pipette tips used in the detection process needed to be decontaminated. Refer to the "Pharmacopoeia of the People's Republic of China": 2020 Edition, Volume III, 1143 Bacterial Endotoxin Test for detection. The reaction temperature was 37 ± 1 °C, and the reaction time was 60 ± 2 minutes. The reaction tubes were gently taken out from the thermostat and slowly inverted 180°. If a gel formed in the tube and the gel did not deform and did not slip off the tube wall, it was positive, recorded as (+); if no gel formed or the formed gel was not firm, deformed, and slipped off the tube wall, it was negative, recorded as (-).

[0230] Table 6 results showed that the endotoxin detections at all levels were qualified:

[0231] Table 6 Endotoxin Detection Record Table

[0232]

[0233] Specifically, the endotoxin content of the recombinant humanized type III collagen obtained by this invention can be controlled to be below 5 EU / mg, which can meet the requirements for use as a cosmetic raw material;

[0234] Further Q-membrane treatment resulted in an endotoxin content of less than 0.5 EU / mg, meeting the requirements for use as a raw material for Class II medical devices. Testing was commissioned to Wolverhampton Testing Group Co., Ltd., and the bacterial endotoxin test result for recombinant humanized type III collagen was <0.5 EU / mg.

[0235] Example 11: Test of the firming and repairing effects of recombinant humanized type III collagen

[0236] The product's firming and repairing effects were tested by CCIC Testing & Inspection (Tianjin) Co., Ltd., confirming its actual efficacy. Specifically:

[0237] Firming effect: When the recombinant humanized type III collagen in the test samples was at a concentration of 1.25% to 5%, the ROS inhibition rate of each group was positive compared with the negative control, and the fluorescence intensity value was significantly different (P<0.05), indicating that the test substance has the effect of inhibiting ROS and has a firming effect.

[0238] Repair efficacy: When the recombinant humanized type III collagen in the test sample was at a concentration of 1.25% to 5%, it could promote a dose-dependent increase in the expression of human keratinocyte filaggrin (FLG), which was significantly different from the negative control (P<0.05), indicating that the test sample had a repair efficacy.

[0239] Example 12 Biosafety testing of recombinant humanized type I collagen

[0240] Recombinant humanized type I collagen T11 is used as a filler material in medical aesthetics, preferably for filling wrinkles, nasolabial folds, etc. Therefore, Example 12 tested the biosafety of recombinant humanized type I collagen.

[0241] The testing was commissioned to Beijing Kejian Testing Technology Group Co., Ltd. (Room 1219, 2nd Floor, Building 1, No. 4, Block B, Yongledian Development Zone, Tongzhou District, Beijing). The testing items and results are shown in Table 7. The testing period is from October 19, 2025 to November 6, 2025.

[0242] Table 7: Test results.

[0243]

[0244] The results in Table 7 indicate that the amino acid fragments obtained according to the sequence and preparation process provided by the present invention, as shown in SEQ ID NO: 1, are recombinant humanized type I collagen α1 chain fragments suitable for use as biofiller materials in medical aesthetics.

Claims

1. Recombinant humanized collagen, specifically recombinant humanized type III collagen, characterized in that: The amino acid fragment of the recombinant humanized type III collagen is shown in SEQ ID NO:

2.

2. The recombinant humanized collagen-encoding nucleic acid according to claim 1, wherein, The nucleic acid encoding the amino acids of recombinant humanized type III collagen is shown in SEQ ID NO:

4.

3. The method for preparing recombinant humanized collagen according to claim 1, characterized in that, Includes the following steps: Step 1: Construct engineered bacteria using the encoding nucleic acid of the recombinant humanized collagen as described in claim 2 to obtain a high-expression recombinant strain: Step 2: The high-expression recombinant strain obtained by screening was inoculated into BSM medium. The culture conditions were: temperature 30℃, pH ≥ 5 controlled with ammonia water, and aeration rate of 1 VVM. Step 3: After the initial carbon source is depleted, feed is added using glycerol at a concentration of 650 g / L until the bacterial culture OD600 reaches 95 to 105; Step 4: Feeding strategy during the transition period from glycerol to methanol: Add methanol to enter the transition period, with the final methanol concentration in the fermentation broth at 2 g / L. At the same time, continue feeding glycerol and control the feeding rate of glycerol so that the feeding rate of glycerol is reduced from 8 g / L / h to 0 g / L / h. Step 5: After the carbon source is exhausted, methanol is added to enter the induction period. Methanol is added to the fermentation broth to make the final concentration 8 g / L for the expression of recombinant humanized collagen fragments. Whenever the dissolved oxygen in the fermentation broth reaches 50%, the methanol addition step is repeated to make the methanol concentration reach 8 g / L. Step 6: Purify the recombinant humanized collagen fragment expressed in Step 5.

4. The method for preparing recombinant humanized collagen according to claim 3, characterized in that, In step 1, the encoding nucleic acid of the recombinant humanized collagen as described in claim 2 is ligated into an expression vector to construct a recombinant expression plasmid; the recombinant expression plasmid is then transformed into yeast. High-expression recombinant strains were obtained through screening and identification.

5. The method for preparing recombinant humanized collagen according to claim 4, characterized in that, In step 1, the yeast is Pichia pastoris.

6. The method for preparing recombinant humanized collagen according to claim 3, characterized in that, In step 3, glycerol is continuously fed at a rate of 8 g / L / h.

7. The method for preparing recombinant humanized collagen according to claim 6, characterized in that, In step 4, the method of controlling the glycerol feeding rate is to reduce the glycerol feeding rate from 8 g / L / h to 0 g / L / h within 2 hours.

8. The method for preparing recombinant humanized collagen according to claim 3, characterized in that, The bacterial growth time is 20 hours, meaning the total duration of steps 2 and 3 is 20 hours.

9. The method for preparing recombinant humanized collagen according to claim 3, characterized in that, The induction time was 96 hours, meaning that step 5 lasted for 96 hours.

10. The method for preparing recombinant humanized collagen according to claim 3, characterized in that, The fermentation system of the preparation method is 2L to 1000L.

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