A novel type I recombinant collagen, its production method and use

Abstract

The present invention relates to a novel type I recombinant collagen protein, a production method and use thereof. The type I recombinant collagen protein of the present invention is composed of a total of 753 amino acids at positions 305-1057 in the amino acid sequence of the α1 chain of natural human type I collagen protein, the secretion yield of the type I recombinant collagen protein in a commercial Pichia pastoris expression system can be as high as about 23 g / L, the extracellular secreted protein is basically not degraded during the fermentation process, and the purity can reach more than 80% by simple ultrafiltration and decolorization, and industrial mass production can be achieved.

Description

Technical Field

[0001] The invention belongs to the field of biochemical engineering and relates to a novel genetically engineered recombinant collagen, a production method and application thereof. Background Art

[0002] Collagen is a biological macromolecular protein, the main component of animal connective tissue, and the most abundant and widely distributed functional protein in mammals, accounting for 25% to 30% of the total protein. It is closely related to the formation and maturation of tissues, the transmission of information between cells, joint lubrication, wound healing, calcification, blood coagulation and aging, and is one of the most critical raw materials in the biotechnology industry. It is widely used in medical materials, cosmetics, and food industries.

[0003] The collagen currently used in industry is mainly extracted from animal skin or bones through acid, alkali or enzymatic methods. Its main source is animal connective tissue. However, collagen extracted from animal tissues has risks such as animal-derived diseases. At the same time, large-scale preparation puts tremendous pressure on animal breeding on the supply side.

[0004] With the large-scale application of genetic engineering technology, genetic engineering recombinant expression of collagen has successfully solved the bottleneck of large-scale preparation of collagen by using modeled expression hosts to express exogenous proteins. Compared with traditional animal collagen extraction, genetic engineering recombinant preparation of collagen has the following advantages: 1. A wide variety of collagen is produced, which can be collagen from common sources such as cattle, pigs, and fish, and human collagen can also be produced, which has better safety and immune efficacy in the fields of medical devices. 2. It can effectively avoid animal-derived diseases. Recombinant collagen usually uses simple prokaryotic or eukaryotic cells as expression hosts. Its cell pathogens cannot be transmitted to each other due to the huge difference in cell structure with humans. 3. The production cycle is short, cost-saving, can be quickly amplified, and is suitable for industrial large-scale production. Compared with the long animal culture of 3-5 months, the microbial culture cycle only takes 2 or 3 days and the culture is simple. Large-scale culture can be carried out by providing simple C source and N source, which is easy to prepare at industrial level. Therefore, the preparation of recombinant collagen, especially recombinant human collagen, is currently a hot spot in collagen production research.

[0005] Recombinant human collagen is considered to be the collagen closest to the body's natural synthesis because its amino acid composition is similar or consistent with that of the human body. It has good biocompatibility and high safety while also having the efficacy and functions of animal collagen. There are a large number of literature reports on it, and commercial products have been successfully developed for use in the fields of beauty, cosmetics and medical devices.

[0006] At present, the main problems of recombinant collagen in production are as follows: 1) easy to degrade. Because recombinant collagen cannot form a stable triple helix structure like natural collagen under the existing expression system, it is easy to be degraded by the protease in the production system; 2) difficult to purify. Collagen is easy to degrade, resulting in the need to remove a large amount of degraded proteins in addition to host protein interference during the purification process of the prepared recombinant collagen, which increases the difficulty of purification and makes it difficult to prepare high-purity target proteins; 3) low yield. Due to the existence of degradation problems, the yield of recombinant collagen is low, far from people's expectations. It is generally believed that when using commercial expression hosts and expression vectors, the yield of recombinant collagen with a molecular weight of more than 70,000 reaches 10g / L, which is already quite high. Therefore, the types of mass-produced recombinant collagen are relatively small, which seriously restricts the further application of recombinant collagen.

[0007] As the market acceptance of recombinant collagen gradually increases, the market demand for recombinant collagen is gradually increasing. Therefore, recombinant collagen that can be mass-produced is urgently needed. Recombinant collagen that can be mass-produced must have the characteristics of high single-batch yield, good stability of expressed protein, and easy purification. Therefore, high-yield, good stability, and easy purification recombinant collagen is urgently needed in the current market.

[0008] Recombinant collagen can be obtained by constructing truncated proteins or truncated peptides of natural collagen. The uncertainty of the position and length of the truncated protein in natural collagen makes it possible to have tens of millions of recombinant collagen types in theory. However, there has been a lack of effective theoretical guidance on which of these recombinant collagens can have a higher secretion yield. The best results must be screened out through a large number of experimental verifications. This is the main reason why there are currently few types of recombinant collagen in mass production. Summary of the invention

[0009] In view of the technical problems existing in the above-mentioned prior art, the object of the present invention is to provide a new type I recombinant collagen which is substantially not degraded during the fermentation process and thus has a high secretion yield.

[0010] The present invention provides a novel type I recombinant collagen protein, which consists of 753 amino acids at positions 305-1057 in the amino acid sequence of the α1 chain of natural human type I collagen protein. The secretion yield of the type I recombinant collagen protein in a commercial Pichia pastoris expression system can be as high as about 23 g / L, the extracellular secreted protein is basically not degraded during the fermentation process, and the purity can reach more than 80% through simple ultrafiltration and decolorization, so that industrial mass production can be achieved.

[0011] The present invention comprises:

[0012] 1. A type I recombinant collagen, the amino acid sequence of which is shown in SEQ ID No.: 1.

[0013] SEQ ID No.: 1

[0014]

[0015] 2. The recombinant type I collagen according to item 1, further comprising a tag for facilitating purification.

[0016] 3. The type I recombinant collagen according to item 2, wherein the tag is a His tag, a Flag tag or a c-Myc tag.

[0017] 4. A nucleic acid encoding the recombinant type I collagen according to any one of items 1 to 3.

[0018] 5. An expression vector comprising the nucleic acid described in item 4.

[0019] 6. A host cell into which the expression vector described in item 5 is introduced. The host cell may be a prokaryotic cell or a eukaryotic cell, including bacterial hosts such as Escherichia coli, Bacillus subtilis, and Bacillus licheniformis, eukaryotic hosts such as Pichia pastoris, Saccharomyces cerevisiae, animal cells, and plant cells, preferably Escherichia coli and Pichia pastoris, and more preferably Pichia pastoris.

[0020] 7. A method for producing type I recombinant collagen according to items 1 to 3, comprising culturing the host cell according to item 6 to secrete the type I recombinant collagen into a culture medium, and separating and purifying the protein in the culture medium to obtain the type I recombinant collagen.

[0021] The expression may be one of constitutive expression and induced expression or a combination of the two, wherein the inducer for inducing expression may be an inducer such as IPTG, β-galactoside, methanol, ethanol, etc. The expression strain may be a commonly used genetic engineering bacterial expression host such as Escherichia coli, Saccharomyces cerevisiae, Pichia pastoris, Bacillus licheniformis, etc., preferably Escherichia coli and Pichia pastoris, more preferably Pichia pastoris.

[0022] 8. The production method according to item 7, wherein the separation and purification adopts one or a combination of methods such as salting out, chromatography, affinity chromatography, acid-base precipitation, membrane separation, etc., preferably a combination of chromatography and membrane separation, and more preferably a combination of ion exchange chromatography and membrane separation.

[0023] 9. Use of the recombinant type I collagen according to items 1 to 3 in skin care products, health products, tissue engineering materials, medical devices, etc., preferably in tissue engineering materials and medical devices.

[0024] 10. The use according to item 9, wherein the tissue engineering material is selected from subcutaneous fillers, artificial bones, artificial skin, oral absorbable biomembranes, bone implants, vascular scaffolds, interstitial scaffolds and collagen sponges.

[0025] The beneficial effect of the present invention is that: in the technical field, it is generally believed that when using commercial expression hosts and expression vectors, the yield of recombinant collagen with a molecular weight of more than 70,000 reaches 10g / L, which is already quite high. The present invention provides a new type I recombinant collagen composed of amino acids, which is a truncated protein of the α1 chain of human type I collagen. The yield of the type I recombinant collagen can be as high as 23g / L through extracellular secretion expression of a commercial Pichia yeast expression system, which is significantly higher than the high yield level of recombinant collagen considered in the technical field. The results of extracellular protein SDS-PAGE show that the target protein accounts for more than 70%, and the purity can reach 80% after simple ultrafiltration decolorization. The secretion yield of the type I recombinant collagen is significantly better than the full-length α1 chain of human type I collagen or other truncated proteins. At the same time, the type I recombinant collagen is significantly better than the full-length chain of the α1 chain of human type I collagen in terms of adhesion promotion properties, and shows good safety. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The electrophoresis diagram shows the fermentation results of recombinant collagen type I-α1 (2ul loading amount). Lanes 1 to 8 represent the sampling time of 0h, 5h, 10h, 15h, 20h, 25h, 30h, and 40h after induction, respectively.

[0027] Figure 2A The electrophoresis diagram shows the fermentation results of recombinant collagen I-1 (2ul loading amount). Lanes 1 to 3 represent the sampling time of 25h, 30h, and 40h after induction, respectively.

[0028] Figure 2B The electrophoresis diagram shows the fermentation results of recombinant collagen I-2 (2ul loading amount). Lanes 1 to 3 represent the sampling time of 25h, 30h, and 40h after induction, respectively.

[0029] Figure 2C The electrophoresis diagram shows the fermentation results of recombinant collagen I-full length (2ul loading amount). Lanes 1 to 3 represent the sampling time of 25h, 30h, and 40h after induction, respectively. DETAILED DESCRIPTION

[0030] Example 1 Preparation of recombinant collagen type I-α1 in a yeast expression system

[0031] 1. Experimental Methods

[0032] 1. Preparation of shuttle plasmid

[0033] According to the amino acid sequence shown in SEQ ID No.1, the codon optimization of the yeast expression system was carried out to obtain the target gene sequence of type I-α1. The obtained target gene sequence was entrusted to Qingke Biotechnology Co., Ltd. for gene synthesis. The synthesized gene was connected to the pPicZαA plasmid to obtain the pPicZαA-I-α1 plasmid.

[0034] 2) Preparation of yeast expression strains

[0035] pPicZαA-I-α1 was linearized with Pme I and then transformed into Pichia pastoris X-33 competent cells. Bleomycin resistance was used as a selection marker to screen transformants to obtain a yeast expression strain.

[0036] 2. Induced expression of target protein

[0037] (1) Pick a single colony of the constructed yeast expression strain and add it to 5 ml of YPD liquid medium (1% yeast extract, 2% peptone and 2% glucose), and culture it at 30°C and 200 rpm for 48 h for activation;

[0038] (2) Transfer the inoculum to a 500 ml Erlenmeyer flask (containing 200 ml of YPD culture medium) at a 1% inoculum volume and culture at 30°C, 200 rpm for 24 h to serve as the seed for the tank;

[0039] (3) Prepare 3 L of BSM medium and add it to a 5 L fermenter, sterilize at 121° C. for 20 min, and after cooling to 30° C., adjust the pH to 5.0, and add the seeds prepared in (2) to the fermenter by flame inoculation for fermentation;

[0040] (4) Cultivate until OD 600 When OD was 70, 50% glycerol was added until 600 When the temperature reaches about 120, stop adding, and wait until the dissolved oxygen rebounds to 100%, then start adding methanol for induction;

[0041] (5) During the induction process, the dissolved oxygen was controlled to be not less than 30% and the pH was about 5.0. The induction was continued for 40 h, and the fermentation was terminated. The culture medium was centrifuged at 12,000 rpm for 2 min, and the supernatant was taken for detection of protein yield and purity using the BCA method and SDS-PAGE method.

[0042] 3. Collagen purification

[0043] The collected supernatant was passed through a 30KD ultrafiltration membrane and then through a hollow fiber column and then freeze-dried to obtain recombinant collagen type I-α1.

[0044] 2. Results

[0045] The BCA test result of the recombinant collagen type I-α1 fermentation tank prepared by the above method is as high as 23g / L. The test result of SDS-PAGE electrophoresis shows that the target protein accounts for more than 70%. The purity of the protein after ultrafiltration through the hollow fiber column can reach more than 80%. The protein electrophoresis results are as follows Figure 1 shown.

[0046] Example 2 Preparation of recombinant collagen type I-α1 control protein and full-length type I collagen α1 chain in yeast expression system

[0047] The same design concept as that of recombinant collagen type I-α1 was adopted to design protein sequences with similar molecular weights, and the target gene was obtained after codon optimization. After the target gene was fully synthesized, the same shuttle plasmid and host bacteria were used to express the protein sequence. At the same time, the full-length type I collagen α1 chain was expressed in the same way. The results of sequence design and expression are shown in the table below (Table 1), and the protein electrophoresis is shown in Figure 2.

[0048] Table 1 Different amino acid sequences and expression results

[0049]

[0050]

[0051] The results of Examples 1 and 2 show that the yield of recombinant collagen composed of the amino acid sequence provided by the present invention is much higher than that of similarly designed sequences intercepted at different positions and of different sizes, and is also much higher than that of the full-length type I amino acid sequence. Figure 1 The results of Figure 2 show that the protein electrophoresis purity of the amino acid sequence provided by the present invention is higher than that of other amino acid sequences used as controls. Therefore, the amino acid sequence provided by the present invention is easier to prepare on a large scale and has greater potential for mass production.

[0052] For a long time, there has been a lack of effective theoretical guidance on which recombinant collagen can have a higher secretion yield, and a large number of experiments must be used to screen and verify. Therefore, high-yield recombinant collagen is extremely rare. In the technical field, it is generally believed that when a commercial expression host and expression vector are used, the yield of recombinant collagen with a molecular weight of more than 70,000 reaches 10g / L, which is already quite high. The yield of the type I recombinant collagen of the present invention can be as high as 23g / L, which is significantly higher than the high yield level of recombinant collagen considered in the technical field.

Claims

1. A type I recombinant collagen, the amino acid sequence of which is shown in SEQ ID No:

1.

2. The type I recombinant collagen according to claim 1, further comprising a tag for facilitating purification.

3. The recombinant type I collagen according to claim 2, wherein The tag is a His tag, a Flag tag or a c-Myc tag. 4 . A nucleic acid encoding the recombinant type I collagen according to claim 1 .

5. An expression vector comprising the nucleic acid of claim 4.

6. A host cell into which the expression vector according to claim 5 has been introduced.

7. A method for producing the type I recombinant collagen according to claim 1, comprising culturing the host cell according to claim 6 to secrete the type I recombinant collagen into a culture medium, and separating and purifying the protein in the culture medium to obtain the type I recombinant collagen.

8. The method for producing type I recombinant collagen according to claim 7, wherein: The separation and purification adopts one or a combination of salting out, chromatography, acid-base precipitation and membrane separation.

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