Yeast heterologous expression recombinant human-derived III-type collagen and preparation thereof

Abstract

The invention discloses a yeast heterologous expression recombinant human collagen III and a preparation method thereof. The yeast heterologous expression recombinant human collagen III comprises an amino acid sequence as shown in SEQ ID No. 1. The preparation method comprises the following steps: cloning a nucleotide sequence as shown in SEQ ID No.2 into a vector to construct a recombinant expression vector plasmid I; the method comprises the following steps: cloning a nucleotide sequence shown as SEQ ID No.3 into a vector, and constructing a recombinant expression vector plasmid II; transfecting the recombinant expression vector plasmids I and II into host cells, and screening to obtain double-gene transformed recombinant bacteria; fermenting and culturing the double-gene transformation recombinant bacteria; and purifying the yeast heterologous expression recombinant human III type collagen. On the basis of retaining the hydrophobic structure and activity of the original human-derived III-type collagen, the stability and hydrophilicity of the protein structure are improved; a P4H sequence is introduced, so that the defect that a pichia pastoris expression system cannot be hydroxylated is overcome.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, and specifically to a yeast heterologous expression of recombinant human type III collagen and its preparation method. Background Technology

[0002] Collagen is a large biological protein molecule widely distributed in animal connective tissue, accounting for 25% to 30% of total protein. It is an important component of human skin tissue. Collagen not only maintains the elasticity and suppleness of human skin but also helps maintain bone strength. Common types of collagen are type I, type II, type III, type V, and type VI. Human skin mainly contains type I and type III collagen, with the content of type III collagen gradually decreasing with age, which is closely related to skin aging.

[0003] Natural collagen is generally composed of three single chains, and its amino acid composition is characterized by Gly-XY (where X and Y represent amino acids, with Y mostly being proline), and it also contains a certain proportion of hydroxyproline. Hydroxyproline plays an important role in maintaining the triple helix structure and biological properties of collagen.

[0004] Currently, there are two main methods for obtaining collagen. One is extraction from animal tissues, but the collagen extracted by this method is mostly a mixture of collagen with different molecular weights, which is not only poorly soluble in water and has poor biocompatibility, but also poses a significant risk of immunogenicity. The other method is preparation through genetic engineering. The advantage of this method is that it can avoid the disease infection risks associated with collagen extracted by traditional methods. High-density fermentation with E. coli or Pichia pastoris can achieve high expression levels, which is more conducive to industrialization.

[0005] Currently, bacterial expression systems, such as those using *E. coli*, produce pyrogens that result in excessively high levels of endotoxins in the expressed products, which are difficult to remove and hinder clinical application. Furthermore, target proteins are often expressed as inclusion bodies, leading to complex purification processes and high production costs, making industrialization difficult. Prokaryotic expression systems also suffer from imperfect post-translational modification systems, resulting in significant differences in activity between the expressed and native proteins.

[0006] Eukaryotic expression systems encounter protein degradation issues during fermentation, particularly with large collagen molecules, which are unstable during heterologous expression and more susceptible to protease degradation. Furthermore, while small collagen peptides, which are typically expressed by selectively repeating fragments, generally have low molecular weights and are easy to express, they lack the complete structure and function of collagen. Additionally, conventional expression systems such as bacteria and yeast lack the proline hydroxylase (P4H) gene, preventing the hydroxylation of proline in collagen. Consequently, the expressed recombinant collagen lacks sufficient hydroxyproline, leading to structural instability and easy degradation.

[0007] In conclusion, selecting a stable collagen fragment and P4H sequence and simultaneously transforming them into engineered bacteria for co-expression to achieve in situ hydroxylation of recombinant collagen has significant application value. Summary of the Invention

[0008] Due to the aforementioned deficiencies in existing technologies, this invention provides a co-expression system of recombinant collagen fragments and P4H, specifically a yeast heterologous expression system for recombinant human type III collagen and its preparation method. The collagen fragment has the sequence GPCCGG introduced at its C-terminus, and the sequence contains a supercoiled structure and a P4H sequence. During the expression process, proline in the collagen fragment is hydroxylated to improve the stability and hydrophilicity of the collagen, overcoming the current deficiency of insufficient hydroxyproline in recombinant collagen, which leads to the instability and easy degradation of the recombinant collagen structure.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A yeast heterologous expression of recombinant human type III collagen, wherein the yeast heterologous expression of recombinant human type III collagen comprises the amino acid sequence shown in SEQ ID No. 1.

[0011] SEQ ID NO.1 is as follows:

[0012] GKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPP GPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGHHHHHH

[0013] The original reference amino acid sequence was obtained from the human type III collagen α1 chain fragment in the GenBank database. This fragment was modified to shorten the collagen molecule size to improve transdermal efficiency, retain key hydrophobic domains to facilitate transmembrane transport, appropriately increase the proline ratio, and enhance protein hydrophilicity by introducing an intracellular hydroxylase to hydroxylate proline. The sequence GPCCGG was introduced at the C-terminus to improve protein stability. The sequence also included a large number of non-imine residues, including charged and polar residues, which helped improve the hydrophilicity of recombinant human type III collagen. In addition, the sequence contained a unique supercoiled region, which helped improve protein structural stability. A 6×His tag was added to the C-terminus. Based on the codon preferences of the recipient bacteria, the protein-coding gene was optimized to reduce rare codons.

[0014] The present invention also provides a nucleic acid molecule that encodes a yeast heterologous expression recombinant human type III collagen as described above.

[0015] As a preferred technical solution:

[0016] A nucleic acid molecule as described above, comprising a nucleotide sequence as shown in SEQ ID No. 2.

[0017] SEQ ID NO.2 (5'-3') specifically refers to:

[0018] TACGTAGGTAAAGATGGTCCACCAGGTCCAGCCGGTAACACCGGTGCTCCAGGTTCTCCTGGTGTTTCTGGTCCAAAGGGTGACGCTGGTCAACCAGGTGAAAAGGGTAGTCCAGGTGCTCAAGGTCCACCAGGCGCTCCAGGTCCATTGGGTATTGCTGGTATTACTGGTGCTAGAGGTTTGGCTGGTCCACCAGGCTGTCCAGGTCCAAGAGGTTCCCCAGGTCCTCAAGGTGTTAAGGGTGAATCTGGTAAACCAGGTGCTAATGGTTTGTCAGGTGAGAGAGGTCCTCCAGGTCCTCAAGGTTTGCCAGGTTTGGCTGGTACTGCCGGTGAACCAGGTAGAGATGGTAACCCAGGTTCTGATGGTTTGCCAGGTAGAGATGGTTCTCCTGGTGGTAAAGGTGACAGAGGTGAAAACGGTAGTCCTGGTGCTCCAGGTGCCCCTGGTCATCCAGGACCACCAGGCCCTGTTGGTCCAGCTGGCAAGTCCGGCGATAGAGGTGAATCTGGTCCAGCTGGTCCTGCTGGTGCTCCAGGCCCAGCCGGTTCCAGAGGTGCTCCTGGTCCACAAGGTCCACGTGGTGACAAGGGTGAAACTGGTGAAAGAGGTGCTGCTGGTATCAAAGGTCATAGAGGTTTTCCAGGTAACCCAGGCGCTCCAGGTTCTCCAGGCCCAGCTGGTCAACAAGGTGCTATTGGTTCTCCAGGTCCAGCTGGTCCAAGAGGTCCAGTTGGTCCATCAGGTCCACCAGGTAAAGATGGTACTTCTGGTCATCCAGGTCCAATTGGCCCACCAGGTCCTAGAGGTAATAGAGGTGAAAGAGGTTCTGAAGGTTCTCCAGGTCATCCAGGTCAACCAGGTCCACCTGGTCCTCCAGGTGCACCAGGTCCATGTTGTGGTGGTCACCATCATCATCATCATTAAGCGGCCGC

[0019] The present invention also provides a recombinant expression plasmid containing at least one nucleic acid molecule as described above.

[0020] The present invention also provides a host cell containing at least one nucleic acid molecule as described above or a recombinant expression plasmid as described above.

[0021] Furthermore, the present invention also provides a method for preparing a yeast heterologous expression recombinant human type III collagen as described above, comprising the following steps:

[0022] (1) The nucleotide sequence (recombinant collagen encoding gene) shown in SEQ ID No. 2 was cloned into a vector to construct recombinant expression plasmid I;

[0023] (2) The nucleotide sequence shown in SEQ ID No. 3 (the gene encoding hydroxylase-4-proline hydroxylase) was cloned into a vector to construct recombinant expression plasmid II;

[0024] (3) Recombinant expression plasmid I and recombinant expression plasmid II were transfected into host cells and screened (screened and verified) to obtain recombinant bacteria transformed with double genes. The recombinant collagen protein encoding gene and the hydroxylase encoding gene were simultaneously transfected into the recipient strain to construct recombinant bacteria. The recombinant bacteria expressed double proteins and simultaneously hydroxylated proline in collagen in situ. Recombinant human type III collagen was obtained by induction.

[0025] (4) After culturing the double-gene transformed recombinant bacteria and inducing expression, the fermentation broth was collected. The fermentation broth was centrifuged, filtered through a microfiltration membrane, ultrafiltered to remove impurities, and purified to obtain the yeast heterologous expression recombinant human type III collagen.

[0026] SEQ ID NO.3 (5'-3') specifically refers to:

[0027] ATGACAAATAAATTTATTTCGTACAATAAAATGGAAACCCGAGAATACTTGTTGACGATATTGTTCGTGATAGCTTGTTTCATGGTATTAAATCTCGAACGGCGAGAGGGGTTTGAAACCAGCGATCGTCCGGGTGTTTGTGATGGAAAATATTACGAAAAGATTGATGGATTTTTATCTGA TATTGAATGTGATGTATTAATCAATGCAGCAATTAAGAAAGGGTTAATAAAATCAGAGGTAGGCGGAGCAACTGAAAATGATCCTATAAAACTCGATCCGAAGAGTCGTAACTCCGAACAAACATGGTTTATGCCCGGCGAACACGAAGTTATTGATAAAATACAGAAAAAGACAAGGGAAT TTTTAAACAGTAAAAAACATTGTATTGACAAATATAATTTCGAAGATGTTCAAGTAGCTAGGTATAAACCCGGGCAATACTATTATCATCATTACGACGGAGATGACTGCGACGATGCATGCCCGAAGGACCAAAGATTAGCTACGTTGATGGTGTATCTTAAAGCTCCTGAAGAAGGTGGT GGTGGTGAGACTGATTTTCCGACACTCAAAACAAAAATAAAACCAAAGAAAGGAACTTCAATCTTTTTTTGGGTTGCGGATCCTGTAACGAGGAAATTGTACAAAGAAACTTTGCACGCAGGACTTCCCGTGAAAAGTGGAGAAAAAATCATCGCGAACCAATGGATCCGTGCTGTTAAATGA

[0028] As a preferred technical solution:

[0029] The method described above uses pPIC9K, pPICZA, pYES2, pRS413, or YEp13 as the vector; the recombinant expression vector for recombinant collagen nucleic acid molecules is preferably pPIC9K; the recombinant expression vector for 4-proline hydroxylase is preferably pPICZA.

[0030] The host cell is a yeast such as Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha, with Pichia pastoris being preferred.

[0031] The specific procedures for culturing the double-gene transformed recombinant bacteria and inducing expression, as described above, are as follows:

[0032] S1: Inoculate the double-gene transformed recombinant bacteria into basal medium and culture overnight;

[0033] S2: Transfer the bacterial cells cultivated in step S1 to seed culture medium, add carbon source during the cultivation process, and collect the bacterial cells after the bacterial cell concentration reaches the target by starving them for a period of time.

[0034] S3: The bacterial cells obtained in step S2 are added to the induction medium to induce expression. During the expression process, an inducer is added to induce the yeast to heterologously express recombinant human type III collagen.

[0035] As described above, the culture and induction temperature is 27~31℃;

[0036] The basic culture medium consists of: 1-3 wt% tryptone, 0.5-1.5 wt% yeast extract, 1-3 wt% glucose, and the remainder water;

[0037] The seed culture medium consists of: 1-3 wt% tryptone, 0.5-1.5 wt% yeast extract, 0.15-0.25 wt% dipotassium hydrogen phosphate, 1.15-1.25 wt% potassium dihydrogen phosphate, 0.5-1.5 wt% glycerol, 1.2-1.5 wt% YNB, 0.002-0.006 wt% biotin, and the balance being water;

[0038] The induction medium consists of: 1-3 wt% tryptone, 0.5-1.5 wt% yeast extract, 0.15-0.25 wt% dipotassium hydrogen phosphate, 1.15-1.25 wt% potassium dihydrogen phosphate, 0.5-1.5 wt% inducer, 1.2-1.5 wt% YNB, 0.002-0.006 wt% biotin, and the balance being water.

[0039] As described above, the culture conditions for steps S1 and S2 are pH 4.5~6.5, agitator speed of 300~700 rpm, and air flow rate of 1.0~1.5 L / min.

[0040] The culture conditions for step S3 are pH 5.0~6.5, agitator speed 400~700 rpm, and aeration rate 2~4 L / min.

[0041] The above technical solution is only one feasible technical solution of the present invention. The scope of protection of the present invention is not limited thereto. Those skilled in the art can reasonably adjust the specific design according to actual needs.

[0042] The above invention has the following advantages or beneficial effects:

[0043] (1) The yeast heterologous expression of recombinant human type III collagen of the present invention, through redesign and optimization, improves the stability and hydrophilicity of the protein structure while retaining the hydrophobic structure and activity of the original human type III collagen, and is more conducive to transdermal absorption;

[0044] (2) The yeast heterologous expression of recombinant human type III collagen of the present invention introduces the P4H sequence, which makes up for the inability of Pichia pastoris expression system to hydroxylate, and realizes intracellular in situ hydroxylation of proline in recombinant human type III collagen.

[0045] (3) The method for preparing recombinant human type III collagen by yeast heterologous expression of the present invention uses the Pichia pastoris expression system to provide a method for preparing recombinant collagen that is free of endotoxins, has low production cost and high yield, and has good application prospects. Attached Figure Description

[0046] The invention, its features, shape, and advantages will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings. Like reference numerals denote like parts throughout the drawings. The drawings are not drawn to scale; their focus is on illustrating the gist of the invention.

[0047] Figure 1 The pPIC9K-COL1 recombinant plasmid constructed in this invention is shown in the image.

[0048] Figure 2 The pPICZA-P4H recombinant plasmid constructed in this invention is shown in the map.

[0049] Figure 3 Agarose gel electrophoresis images of pPIC9K-COL1 recombinant plasmid and pPICZA-P4H recombinant plasmid;

[0050] Figure 4 SDS-PAGE electrophoresis image of the fermentation broth of Pichia pastoris GS115-pPIC9K-COL1-P4H induced expression;

[0051] Figure 5 This is a picture of collagen after crude purification and freeze-drying. Detailed Implementation

[0052] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but these are not intended to limit the scope of the invention.

[0053] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0054] The sources of the experimental materials used in the following examples are as follows:

[0055] The plasmid vector pPIC9K was purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0056] The plasmid vector pPICZA was purchased from Beijing Qingke Biotechnology Co., Ltd.

[0057] Pichia pastoris GS115 was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0058] The restriction endonuclease Sal I was purchased from BioNTech Biotechnology (Beijing) Co., Ltd.

[0059] The composition of YPD medium is: 2 wt% tryptone, 1 wt% yeast extract, 2 wt% glucose, and the balance water; the composition of BMGY medium is: 2 wt% tryptone, 1 wt% yeast extract, 0.23 wt% dipotassium hydrogen phosphate, 1.18 wt% potassium dihydrogen phosphate, 1 wt% glycerol, 1.34 wt% YNB, 0.004 wt% biotin, and the balance water; the composition of BMMY medium is: 2 wt% tryptone, 1 wt% yeast extract, 0.23 wt% dipotassium hydrogen phosphate, 1.18 wt% potassium dihydrogen phosphate, 1 wt% methanol, 1.34 wt% YNB, 0.004 wt% biotin, and the balance water.

[0060] The relevant sequences involved in this invention are as follows:

[0061] 1. SEQ ID NO.1 is as follows:

[0062] GKDGPPGPAGNTGAPGSPGVSGPKGDAGQPGEKGSPGAQGPPGAPGPLGIAGITGARGLAGPPGMPGPRGSPGPQGVKGESGKPGANGLSGERGPPGPQGLPGLAGTAGEPGRDGNPGSDGLPGRDGSPGGKGDRGENGSPGAPGAPGHPGPPGPVGPAGKSGDRGESGPAGPAGAPGPAGSRGAPGPQGPRGDKGETGERGAAGIKGHRGFPGNPGAPGSPGPAGQQGAIGSPGPAGPRGPVGPSGPPGKDGTSGHPGPIGPPGPRGNRGERGSEGSPGHPGQPGPPGPPGAPGPCCGGHHHHHH

[0063] 2. The specific sequence of SEQ ID NO.2 (5'-3') is as follows:

[0064] TACGTAGGTAAAGATGGTCCACCAGGTCCAGCCGGTAACACCGGTGCTCCAGGTTCTCCTGGTGTTTCTGGTCCAAAGGGTGACGCTGGTCAACCAGGTGAAAAGGGTAGTCCAGGTGCTCAAGGTCCACCAGGCGCTCCAGGTCCATTGGGTATTGCTGGTATTACTGGTGCTAGAGGTTTGGCTGGTCCACCAGGCTGTCCAGGTCCAAGAGGTTCCCCAGGTCCTCAAGGTGTTAAGGGTGAATCTGGTAAACCAGGTGCTAATGGTTTGTCAGGTGAGAGAGGTCCTCCAGGTCCTCAAGGTTTGCCAGGTTTGGCTGGTACTGCCGGTGAACCAGGTAGAGATGGTAACCCAGGTTCTGATGGTTTGCCAGGTAGAGATGGTTCTCCTGGTGGTAAAGGTGACAGAGGTGAAAACGGTAGTCCTGGTGCTCCAGGTGCCCCTGGTCATCCAGGACCACCAGGCCCTGTTGGTCCAGCTGGCAAGTCCGGCGATAGAGGTGAATCTGGTCCAGCTGGTCCTGCTGGTGCTCCAGGCCCAGCCGGTTCCAGAGGTGCTCCTGGTCCACAAGGTCCACGTGGTGACAAGGGTGAAACTGGTGAAAGAGGTGCTGCTGGTATCAAAGGTCATAGAGGTTTTCCAGGTAACCCAGGCGCTCCAGGTTCTCCAGGCCCAGCTGGTCAACAAGGTGCTATTGGTTCTCCAGGTCCAGCTGGTCCAAGAGGTCCAGTTGGTCCATCAGGTCCACCAGGTAAAGATGGTACTTCTGGTCATCCAGGTCCAATTGGCCCACCAGGTCCTAGAGGTAATAGAGGTGAAAGAGGTTCTGAAGGTTCTCCAGGTCATCCAGGTCAACCAGGTCCACCTGGTCCTCCAGGTGCACCAGGTCCATGTTGTGGTGGTCACCATCATCATCATCATTAAGCGGCCGC

[0065] 3. SEQ ID NO.3 (5'-3') is specifically as follows:

[0066] ATGACAAATAAATTTATTTCGTACAATAAAATGGAAACCCGAGAATACTTGTTGACGATATTGTTCGTGATAGCTTGTTTCATGGTATTAAATCTCGAACGGCGAGAGGGGTTTGAAACCAGCGATCGTCCGGGTGTTTGTGATGGAAAATATTACGAAAAGATTGATGGATTTTTATCTGA TATTGAATGTGATGTATTAATCAATGCAGCAATTAAGAAAGGGTTAATAAAATCAGAGGTAGGCGGAGCAACTGAAAATGATCCTATAAAACTCGATCCGAAGAGTCGTAACTCCGAACAAACATGGTTTATGCCCGGCGAACACGAAGTTATTGATAAAATACAGAAAAAGACAAGGGAAT TTTTAAACAGTAAAAAACATTGTATTGACAAATATAATTTCGAAGATGTTCAAGTAGCTAGGTATAAACCCGGGCAATACTATTATCATCATTACGACGGAGATGACTGCGACGATGCATGCCCGAAGGACCAAAGATTAGCTACGTTGATGGTGTATCTTAAAGCTCCTGAAGAAGGTGGT GGTGGTGAGACTGATTTTCCGACACTCAAAACAAAAATAAAACCAAAGAAAGGAACTTCAATCTTTTTTTGGGTTGCGGATCCTGTAACGAGGAAATTGTACAAAGAAACTTTGCACGCAGGACTTCCCGTGAAAAGTGGAGAAAAAATCATCGCGAACCAATGGATCCGTGCTGTTAAATGA

[0067] Example 1

[0068] A method for preparing recombinant human type III collagen by heterologous expression in yeast, comprising the following steps:

[0069] (1) Design of recombinant human type III collagen and synthesis of recombinant plasmids:

[0070] Referring to the human type III collagen α-chain fragment in the GenBank database, and using the hydrophilicity / hydrophobicity and stability of the α-chain as a reference, a protein fragment of 300 amino acid residues was designed, retaining the hydrophobic domain. The sequence GPCCGG was introduced at the C-terminus of this protein, and the sequence contains a unique supercoiled region to improve protein stability. A large number of non-imine residues, including charged and polar residues, were introduced into the sequence to improve the protein's hydrophilicity. A 6×His tag was added to the C-terminus. The amino acid sequence is shown in SEQ ID NO.1. Based on the codon preferences of the recipient bacteria, the protein-coding gene was optimized to reduce rare codons. The nucleotide sequence is shown in SEQ ID NO.2. The obtained nucleotide sequence was used to synthesize the recombinant plasmid pPIC9K-COL1, and its plasmid map is shown below. Figure 1 As shown. The cDNA sequence of the A085R protein subsegment gene of proline hydroxylase was selected, and the nucleotide sequence is shown in SEQ ID No. 3. The obtained sequence was used to synthesize the recombinant plasmid pPICZA-P4H, and its map is shown in... Figure 2 As shown, the agarose gel electrophoresis images of the two constructed recombinant plasmids are as follows. Figure 3 As shown.

[0071] (2) Construction and screening of engineered Pichia pastoris strains:

[0072] The linearized recombinant plasmid pPIC9K-COL1 was digested with restriction endonuclease Sal I and transformed into Pichia pastoris GS115 by electroporation. The electroporated Pichia pastoris GS115 recombinant bacterial culture was plated on MD plates and incubated at 30°C until single colonies appeared, thus obtaining the GS115-COL1 recombinant bacteria.

[0073] The linearized recombinant plasmid pPICZA-P4H was digested with restriction endonuclease Sal I and then transferred into the recombinant strain GS115-COL1 via electroporation. The electroporated Pichia pastoris GS115 recombinant bacterial culture was plated on MD plates containing 1‰ ampicillin antibiotic and incubated at 30°C until single colonies appeared.

[0074] The Pichia pastoris GS115 recombinant strain was screened for resistance to G418 using a gradient of concentrations of 0.5 g / L, 1.0 g / L, 3.0 g / L, and 5.0 g / L. Finally, a positive recombinant strain was obtained on a 5.0 g / L G418 plate and named GS115-COL1-P4H.

[0075] (3) Fermentation culture and induced expression of engineered Pichia pastoris:

[0076] Level I seed culture:

[0077] Pick a single colony of GS115-pPIC9K-COL and transfer it to 100mL of YPD medium. Incubate at 30℃ and 260rpm for 16-18h to obtain Grade I seed culture.

[0078] Level II seed culture:

[0079] Inoculate the Grade I seed culture at a rate of 10% into a 1L fermenter containing BMGY medium. Fermentation conditions are: temperature 28℃, pH 4.5-6.0, agitator speed 300-700 rpm, and air flow rate 1.0-1.5 L / min. Glycerol supplementation is as follows: once dissolved oxygen reaches above 90%, add 1-2% 100% glycerol to the fermenter until the cell wet weight reaches 100-150 g / L, then stop adding glycerol.

[0080] Induced expression of recombinant human type III collagen by recombinant bacteria:

[0081] After the dissolved oxygen level reaches above 90%, maintain this level for 2-3 hours to ensure glycerol depletion. Then, add methanol to induce the exocrine expression of recombinant collagen by GS115-COL1-P4H. The methanol addition method is as follows: after the dissolved oxygen level reaches above 90%, add 1-3% 100% methanol to the fermenter. The SDS-PAGE electrophoresis image of the fermentation broth is shown below. Figure 4 As shown.

[0082] (4) Crude purification of recombinant human collagen:

[0083] The fermentation broth was centrifuged to collect the supernatant, and the bacterial cells were removed. The supernatant was then microfiltered using a 0.22 μm microfiltration membrane to obtain a sterile fermentation broth. Small molecule impurities were removed using a 10 kDa ultrafiltration membrane to obtain a crude extract of recombinant collagen.

[0084] (5) Fine purification of recombinant human collagen:

[0085] Recombinant collagen was purified using affinity chromatography. After nickel column pretreatment, crude recombinant collagen extract was loaded onto the column. Impurities were then eluted using a gradient of imidazole at 0 mM, 10 mM, 30 mM, 50 mM, 100 mM, and 200 mM, respectively. The recombinant collagen was then eluted with 500 mM imidazole. After dialysis to remove imidazole, the recombinant collagen powder was lyophilized as shown in the image. Figure 5 As shown.

[0086] Those skilled in the art should understand that variations can be implemented by combining existing technology with the above embodiments, which will not be elaborated here. Such variations do not affect the essence of the present invention, and will not be elaborated here either.

[0087] The preferred embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and the devices and structures not described in detail should be understood as being implemented in a conventional manner in the art. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention using the methods and techniques disclosed above, or modify them into equivalent embodiments with equivalent changes, without departing from the scope of the present invention. This does not affect the essential content of the present invention. Therefore, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the present invention's technical solutions still fall within the protection scope of the present invention.

Claims

1. A yeast heterologous expression of recombinant human type III collagen, characterized in that, The yeast heterologous expression of recombinant human type III collagen includes the amino acid sequence shown in SEQ ID No.

1.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a yeast heterologous expression recombinant human type III collagen as described in claim 1.

3. A nucleic acid molecule according to claim 2, characterized in that, Includes the nucleotide sequence shown in SEQ ID No.

2.

4. A recombinant expression vector plasmid containing at least one nucleic acid molecule as described in claim 2 or 3.

5. A host cell containing at least one nucleic acid molecule as described in claim 2 or 3, or a recombinant expression plasmid as described in claim 4.

6. A method for preparing the yeast heterologous expression recombinant human type III collagen as described in claim 1, characterized in that, Includes the following steps: (1) The nucleotide sequence shown in SEQ ID No. 2 was cloned into a vector to construct recombinant expression plasmid I; (2) The nucleotide sequence shown in SEQ ID No. 3 was cloned into the vector to construct recombinant expression plasmid II; (3) Recombinant expression plasmid I and recombinant expression plasmid II were transfected into host cells, and double-gene transformed recombinant bacteria were screened. (4) After culturing the double-gene transformed recombinant bacteria and inducing expression, the fermentation broth was collected. The fermentation broth was centrifuged, filtered through a microfiltration membrane, ultrafiltered to remove impurities, and purified to obtain the yeast heterologous expression recombinant human type III collagen.

7. The method according to claim 6, characterized in that, The carrier is pPIC9K, pPICZA, pYES2, pRS413 or YEp13; The host cell is Pichia pastoris, Saccharomyces cerevisiae, or Hansenula polymorpha.

8. The method according to claim 6, characterized in that, The specific procedures for culturing the dual-gene transformed recombinant bacteria and inducing expression are as follows: S1: Inoculate the double-gene transformed recombinant bacteria into basal medium and culture overnight; S2: Transfer the bacterial cells cultivated in step S1 to seed culture medium, add carbon source during the cultivation process, and collect the bacterial cells after the bacterial cell concentration reaches the target by starving them for a period of time. S3: The bacterial cells obtained in step S2 are added to the induction medium to induce expression. During the expression process, an inducer is added to induce the yeast to heterologously express recombinant human type III collagen.

9. The method according to claim 8, characterized in that, The temperature for cultivation and induction is 27~31℃; The basic culture medium consists of: 1-3 wt% tryptone, 0.5-1.5 wt% yeast extract, 1-3 wt% glucose, and the remainder water; The seed culture medium consists of: 1-3 wt% tryptone, 0.5-1.5 wt% yeast extract, 0.15-0.25 wt% dipotassium hydrogen phosphate, 1.15-1.25 wt% potassium dihydrogen phosphate, 0.5-1.5 wt% glycerol, 1.2-1.5 wt% YNB, 0.002-0.006 wt% biotin, and the balance being water; The induction medium consists of: 1-3 wt% tryptone, 0.5-1.5 wt% yeast extract, 0.15-0.25 wt% dipotassium hydrogen phosphate, 1.15-1.25 wt% potassium dihydrogen phosphate, 0.5-1.5 wt% inducer, 1.2-1.5 wt% YNB, 0.002-0.006 wt% biotin, and the balance being water.

10. The method according to claim 8, characterized in that, The culture conditions for steps S1 and S2 are: pH 4.5-6.5, agitator speed 300-700 rpm, and air flux 1.0-1.5 L / min. The culture conditions for step S3 are pH 5.0~6.5, agitator speed 400~700 rpm, and aeration rate 2~4 L / min.

Images