Recombinant yeast type XVII humanized collagen and its preparation method

Abstract

The present invention provides a yeast recombinant type XVII humanized collagen and its preparation method, in particular, a recombinant humanized collagen expressing the 15th helical region of type XVII collagen, a recombinant engineering bacterium and its preparation method, belonging to the technical fields of genetic engineering and synthetic biology. The present invention provides the full-length sequence of the 15th helical region of type XVII collagen, and mutants are obtained by mutating amino acid residues based on this sequence; for the first time, the present invention expresses the full-length sequence of the 15th helical region of type XVII collagen in Pichia pastoris, establishes a complete set of fermentation processes and purification processes, improves the protein expression level and reduces degradation, and through experiments, it is verified that the obtained protein has biological activities such as cell adhesion activity and cell migration promotion activity that reach or exceed those of natural human collagen, can achieve the purpose of real product application, and can be widely applied in the fields of medicine, medical devices, biological materials, tissue engineering, cosmetics, etc.

Description

Technical Field

[0001] The present invention relates to a recombinant humanized type XVII collagen of yeast and its preparation method, in particular to a recombinant humanized collagen expressing the 15th helical region of type XVII collagen, a recombinant engineering bacterium and its preparation method, belonging to the technical fields of genetic engineering and synthetic biology. Background Art

[0002] There are 28 different collagens in humans. Among them, human type XVII collagen is a transmembrane non-fibroblast collagen and is a component of hemidesmosomes in cells, playing an important role in the function of epithelial cells-basement membrane; it can regulate the adhesion, separation, development and differentiation of epithelial cells, and has an important role in the differentiation and regeneration of keratinized cells; it can maintain the activity of hair follicle stem cells and epidermal stem cells, and is important for cell aging and skin differentiation. Human type XVII collagen is composed of three identical α1(XVII) chains, which are divided into three major structural domains: intracellular, transmembrane, and extracellular. It can also be divided into 16 non-triple helix regions and 15 triple helix regions according to whether it has a typical (Gly-X-Y)n amino acid repeat sequence and can form a triple helix region. Among them, the 15th helical region is 242 amino acids in length and is the longest triple helix region in type XVII collagen, with typical (Gly-X-Y)n amino acid repeat sequence characteristics. The current research on human type XVII collagen mainly focuses on developmental biology, gene research and disease pathogenesis, and the understanding of its protein structure, function and other aspects is extremely limited. The most research and understanding is on the 15th helical region: existing research shows that it can bind to uncommon integrins α5β1 and αVβ1, has special integrin binding sites, has good cell adhesion activity for various cells, and still maintains its biological activity in the single-chain state after heat denaturation. The 15th helical region is the most promising functional region in human type XVII collagen.

[0003] XVII collagen exists in extremely small amounts in the human body and in animals, making it very difficult to extract. It cannot be obtained by relying on traditional acid, alkali, or enzymatic methods to process animal tissues. A small amount of extraction only meets the needs of scientific research and cannot be mass-produced, so there is no possibility of large-scale application. At the same time, there are inevitably immunogenicity and potential biosafety hazards such as viruses and diseases. Currently, the main way to solve such problems is to obtain recombinant collagen through biotechnology such as genetic engineering. Among the existing several recombinant protein expression systems: the mammalian cell expression system and the insect cell (baculovirus) expression system have high costs and low yields, and these two methods are generally not used for the large-scale production and application of collagen. The recombinant proteins expressed by the prokaryotic (Escherichia coli) system have no post-translational modification, and intracellular expression requires cell lysis, resulting in a large amount of impurity proteins. Moreover, they naturally carry pyrogenic substances such as endotoxin and peptidoglycan, which are components of the bacterial cell wall. As a eukaryotic microorganism, Pichia pastoris has complete organelles of eukaryotic cells, can perform certain post-translational modifications on recombinant proteins, strongly supports the realization of the biological functions of proteins, and has the advantages of high-density, low-cost, short-cycle, and high-expression large-scale fermentation industrial production of the microbial expression system; the recombinant protein can be secreted extracellularly without impurity proteins from cell lysis; the cell wall components do not contain endotoxin and peptidoglycan. Pichia pastoris has a clear genetic background, and a variety of genetically engineered drugs and vaccines have been approved for market, with less difficulty in regulatory approval. It is the most ideal collagen expression system.

[0004] However, the Pichia pastoris expression system still has its disadvantages. The most important one is that the extracellular recombinant proteins secreted and expressed are often degraded by the protease system in Pichia pastoris, especially when expressing recombinant proteins with natural sequences (such as collagen) by high-density fermentation, the degradation situation will be relatively serious. In response to this situation, researchers often modify the corresponding amino acid sequences of collagen to achieve the purpose of reducing degradation. However, the amino acid sequence of a protein is the cornerstone of all its physical and chemical properties, biological functions, etc. Therefore, the amino acid sequence cannot only aim to reduce degradation but also ensure its physical and chemical properties, especially the stability of its biological functions, which requires a lot of research to achieve. In addition, the amino acid sequence of a protein is the result of natural evolution over hundreds of millions of years, and our current understanding of collagen is still limited. Maintaining the stability of its amino acid sequence is a strategy with the least potential risks (beyond the current scientific level of understanding) in the production and development of recombinant human collagen.

[0005] In existing research, through prokaryotic (E. coli) expression, Application No. CN201911051106.3 used a PET vector and an E. coli expression system to separately select a 69 - amino - acid sequence (named 17A in the patent) and another 63 - amino - acid sequence (named 17B in the patent) from the amino - acid sequence of the 15th helix region of human XVII collagen, and their three - repeat sequences (named 17A3 and 17B3 in the patent) for expression, but none of them covered the entire 15th helix region sequence. The prerequisite for the application of recombinant collagen is the ability to carry out large - scale, high - density, and high - expression fermentation production and purification. Pichia pastoris is an ideal expression host. However, as a eukaryote, when a large amount of heterologous protein is expressed and secreted, that is, when a large amount of intracellular biological resources are occupied, Pichia pastoris will inevitably carry out corresponding regulation on the expression of heterologous proteins, and its prominent manifestation is actually the degradation of the expressed recombinant protein. This is more prominent in the amino - acid sequence of natural collagen without mutation or modification. For specific types of recombinant proteins, determining a complete set of fermentation and purification processes to reduce degradation is the key technology for obtaining recombinant collagen with a complete natural amino - acid sequence, especially when carrying out large - scale preparation production of high - density and high - expression fermentation production and purification, it is even more indispensable. Summary of the Invention

[0006] The purpose of the present invention is to overcome some technical problems existing in the prior art and provide a yeast - recombinant humanized type XVII collagen, a recombinant engineering bacterium, and a preparation method thereof, especially to provide a recombinant humanized collagen expressing the 15th helix region of type XVII collagen, a recombinant engineering bacterium, and a preparation method thereof.

[0007] To achieve the above - mentioned purpose, the present invention adopts the following technical solutions:

[0008] The present invention first provides a recombinant humanized type XVII collagen, and the recombinant humanized type XVII collagen includes an amino - acid sequence having 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more identity with the 567 - 808 positions of SEQ ID NO.1 and maintains the biological activity of the 15th helix region of type XVII collagen.

[0009] In certain embodiments, the recombinant humanized type XVII collagen comprises an amino acid sequence having an identity of more than 80%, more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98%, or more than 99% with SEQ ID NO.2 (1703NT), SEQ ID NO.4 (1703), SEQ ID NO.6 (1703MNT), or SEQ ID NO.8 (1703M) and maintaining the biological activity of the 15th helical region of type XVII collagen.

[0010] In certain embodiments, the recombinant humanized type XVII collagen comprises the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6, or SEQ ID NO.8.

[0011] The present invention also provides an isolated polynucleotide encoding the recombinant humanized type XVII collagen of the present invention.

[0012] In certain embodiments, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7, or SEQ ID NO.9, or a degenerate sequence thereof.

[0013] The present invention also provides a vector comprising the polynucleotide of the present invention.

[0014] In certain embodiments, the vector is a eukaryotic vector or a prokaryotic vector.

[0015] In certain embodiments, the vector is pPIC9K.

[0016] The present invention also provides a host cell or a recombinant engineering bacterium, which comprises the nucleic acid of the present invention or the vector of the present invention.

[0017] In certain embodiments, the host cell or the recombinant engineering bacterium is a eukaryotic cell or a prokaryotic cell.

[0018] In certain embodiments, the host cell or the recombinant engineering bacterium is a Pichia pastoris engineering bacterium.

[0019] In certain embodiments, the Pichia pastoris engineering bacterium is deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms, with the deposit numbers of CGMCC No.21889, CGMCC No.21888, CGMCC No.21890, and CGMCC No.21884.

[0020] The present invention also provides a composition, which comprises the recombinant humanized collagen type XVII of the present invention, or the polynucleotide of the present invention, or the vector of the present invention, or the host cell or recombinant engineering bacterium of the present invention.

[0021] The present invention also provides a product, which comprises the recombinant humanized collagen type XVII of the present invention, or the polynucleotide of the present invention, or the vector of the present invention, or the host cell or recombinant engineering bacterium of the present invention, or the composition of the present invention; preferably, the product is selected from drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0022] The present invention also provides the use of the recombinant humanized collagen type XVII of the present invention, the polynucleotide of the present invention, the vector of the present invention, the host cell or recombinant engineering bacterium of the invention, the composition of the present invention or the product of the present invention in the preparation of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

[0023] The present invention also provides a preparation method for improving the production level of recombinant humanized collagen type XVII and with low protein degradation, comprising the following steps:

[0024] (1) Inoculate the recombinant engineering bacterium into a seed medium and culture overnight to prepare a bacterial seed solution;

[0025] (2) Set the fermentation temperature and pH value, inoculate the bacterial seed solution into a fermentation medium, adjust the stirring speed, air flow rate, tank pressure and DO value, culture until the carbon source is exhausted and the DO rapidly rebounds, and start to feed a feeding medium until the OD 600 value reaches a certain value, and stop feeding the feeding medium;

[0026] (1) After the glycerol is exhausted and the DO≥70%, start to feed an induction medium and enter the methanol induction stage, and adjust the rotation speed, ventilation volume, tank pressure and feeding speed to make the DO≥30%;

[0027] (4) After the induced fermentation is completed, take the supernatant of the fermentation broth and detect the protein;

[0028] Preferably, the recombinant humanized collagen type XVII is a recombinant humanized collagen expressing the 15th helix region of collagen type XVII; more preferably, the recombinant humanized collagen type XVII comprises that described in claim 1 or 2, or comprises the amino acid sequence shown in SEQ ID NO.2.

[0029] In some embodiments, the seed medium in the step (1) is YPG.

[0030] In some embodiments, the fermentation temperature in step (2) is set to 25°C - 35°C, preferably 30°C, and the pH value is set to 3.5 - 6, preferably 4.0; the feeding medium is 50% W / V glycerol, and 12 mL of PTM is added per liter. 1 .

[0031] In some embodiments, in step (2), when the OD 600 value of the bacteria is 50 - 150, preferably the OD 600 value of the bacteria is 50, the feeding of the feeding medium is stopped.

[0032] In some embodiments, the components of the fermentation medium in step (2) include: NH 4 H 2 PO 4 11.9 - 47.6 g / L, KH 2 PO 4 2.515 - 10.06 g / L, CaSO 4 ·2H 2 O 0.295 - 1.18 g / L, K 2 SO 4 4.55 - 18.2 g / L, MgSO 4 ·7H 2 O 3.725 - 14.9 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L; preferably, the components of the fermentation medium include NH 4 H 2 PO 4 11.9 g / L, KH 2 PO 4 2.515 g / L, CaSO 4 ·2H 2 O 0.295 g / L, K 2 SO 4 4.55 g / L, MgSO 4 ·7H 2 O 3.725 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L.

[0033] In some embodiments, the components of the induction medium in step (3) include: pure methanol, 50% glycerol, PTM 1 ; wherein the volume ratio of pure methanol to 50% glycerol is 10 - 7:0 - 3, and 12 mL of PTM is added per liter 1 ; preferably, the volume ratio of pure methanol to 50% glycerol is 8:2.

[0034] In some embodiments, after the step (4), there is further a step of purifying the fermentation product; preferably, the purification includes successively performing hydrophobic chromatography and cation exchange chromatography steps.

[0035] In some embodiments, the hydrophobic chromatography is equilibrated with buffer A, the impurities are washed with buffer B, and the elution is performed with buffer C. The cation exchange chromatography is equilibrated with buffer D and eluted with buffer E; preferably, buffer A includes: 20 mM KH 2 PO 4 , 2 M ammonium sulfate, pH 5.0; buffer B includes: 20 mM KH 2 PO 4 , 0.6 M ammonium sulfate, pH 5.0; buffer C includes: 20 mM KH 2 PO 4 , pH 5.0; buffer D includes: 20 mM tartaric acid, 100 mM sodium chloride, pH 4.0; buffer E includes: 20 mM tartaric acid, 500 mM sodium chloride, pH 4.0.

[0036] The present invention further provides a method for purifying recombinant humanized collagen type XVII, comprising the following steps:

[0037] (1) Hydrophobic chromatography: Prepare buffer A: 20 mM KH 2 PO 4 , 2 M ammonium sulfate, pH 5.0; buffer B: 20 mM KH 2 PO 4 , 0.6 M ammonium sulfate, pH 5.0; buffer C: 20 mM KH 2 PO 4 , pH 5.0; Collect the supernatant of the fermentation broth of recombinant humanized collagen type XVII, equilibrate the hydrophobic chromatography medium with buffer A, after sample loading, re-equilibrate with buffer A; then wash the impurities with buffer B and perform elution with buffer C, and start collecting eluate 1;

[0038] (2) Cation exchange chromatography: Prepare buffer D: 20 mM tartaric acid, 100 mM sodium chloride, pH 4.0; buffer E: 20 mM tartaric acid, 500 mM sodium chloride, pH 4.0; Equilibrate the cation exchange chromatography medium with buffer D, load eluate 1, after sample loading, re-equilibrate with buffer D, perform elution with buffer E, collect eluate 2, and ultrafilter and lyophilize eluate 2 to obtain the purified protein lyophilized product.

[0039] Preferably, the recombinant humanized collagen type XVII is a recombinant humanized collagen expressing the 15th helical region of collagen type XVII; more preferably, the recombinant humanized collagen type XVII is as described in claim 1 or 2, or comprises the amino acid sequence shown in SEQ ID NO.2.

[0040] Advantages of the present invention:

[0041] (1) The present invention has achieved for the first time the Pichia pastoris recombinant expression of the 15th helical region of natural collagen type XVII (named 1703NT, the sequence is shown in SEQ ID NO.2).

[0042] In view of the possible easy degradation property of the natural collagen sequence, the present invention has mutated a few amino acid residues in the 15th helical region sequence 1703NT of human collagen type XVII, named 1703MNT (the sequence is shown in SEQ ID NO.6). 1703MNT significantly reduces the situation of more degradation of the natural protein during Pichia pastoris expression, and it has been detected and verified that its basic physical and chemical properties remain unchanged and its biological activity has not decreased (even more excellent).

[0043] The application of the 15th helical region of human collagen type XVII of the present invention to the human body will not cause immune rejection and allergic reactions, and has biological activities such as cell adhesion activity and cell migration promoting activity that reach or even exceed those of natural human collagen, and can achieve the purpose of real product application, and can be widely applied to fields such as medicine, medical devices, biological materials, tissue engineering, and cosmetics.

[0044] (2) Based on the amino acid sequences of 1703NT and 1703MNT, the present invention calculates and optimizes the codon preference of the DNA sequence and related optimization parameters during its transcription and translation processes, and synthesizes a DNA sequence more suitable for high-efficiency expression in Pichia pastoris. The present invention modifies the DNA sequences of 1703NT and 1703MNT, adds a DNA sequence encoding a Strep-Tag II tag to the amino terminus and a DNA sequence encoding a 6×His Tag tag to the carboxyl terminus respectively, so that it contains a bispecific affinity purification label, which can be used for affinity chromatography purification and is also convenient for immunological antibody detection based on the two tag sequences, named 1703 (the sequence is shown in SEQ ID NO.4) and 1703M (the sequence is shown in SEQ ID NO.8) respectively. The exogenous DNAs encoding 1703, 1703M, 1703NT, and 1703MNT are cloned into the expression vector pPIC9K to construct recombinant expression vectors pPIC9K-1703, pPIC9K-1703M, pPIC9K-1703NT, and pPIC9K-1703MNT. And further induce expression to screen engineering bacteria with high expression levels.

[0045] (3) Based on the requirements of high-density large-scale fermentation production (rather than the laboratory research stage), the present invention has established a complete set of methods and technological processes for the fermentation expression, extraction, and purification of the natural full-length 15th helix region with low degradation in Pichia pastoris. While increasing the expression level, the degradation of the natural full-length 15th helix region is greatly reduced, and the recombinant collagen of the natural full-length 15th helix region is obtained.

[0046] The present invention optimizes the fermentation medium formula, the optimal fermentation pH during fermentation, the initial induction bacterial concentration OD 600 , the induction medium formula for the fed-batch of mixed carbon sources, etc., and has established a complete set of fermentation processes. The collagen expression level of the engineered strain of Pichia pastoris (preservation number CGMCC No. 21888) expressing recombinant humanized collagen type XVII 1703 is increased from about 11 g / L to about 17 g / L, and the engineered strain of Pichia pastoris (preservation number CGMCC No. 21889) expressing recombinant humanized collagen type XVII 1703NT can reach >15 g / L. At the same time, the protein degradation during fermentation is significantly improved. In particular, there is only one main dominant electrophoresis band (with the largest optical density), and the previous main degradation band with a relatively small molecular weight and a relatively large proportion (about 40%) basically disappears, indicating that the fermentation conditions at this time play a role in inhibiting the degradation of recombinant collagen, and its effect is similar to the anti-degradation effect brought by mutants such as 1703M that change the amino acid sequence.

[0047] At the same time, a two-step purification method is established in the present invention. Using a tartaric acid buffer system, the method is simple and effective. High-purity recombinant humanized collagen type XVII 1703 and 1703NT can be obtained through hydrophobic chromatography and cation exchange chromatography. The freeze-dried products of 1703 and 1703NT after purification show a single band by electrophoresis detection. After liquid phase analysis and calculation (area normalization method), the single peak is obvious and the purity is high. 1703 can reach 94%, and 1703NT can reach 95%.

[0048] The fermentation and purification processes of the present invention are applicable to the fermentation and purification of high-density bioreactors, and have the conditions for large-scale industrial production.

[0049] (3) The present invention has verified the biological activity of the recombinantly expressed 15th helix region through experiments. The recombinant collagen of the 15th helix region obtained in the present invention has good adhesion activity and cell migration promotion activity.

[0050] In the present invention, LC-MS detection was performed on the freeze-dried products of 1703 and 1703NT, demonstrating that their molecular weights are consistent with the theoretically predicted values. N-terminal and C-terminal sequencing verification and protein full-sequence analysis based on LC-MS / MS were carried out on the highly pure freeze-dried products of 1703 and 1703NT, proving that their N-terminal and C-terminal are complete, and the amino acid sequences expressed by 1703 and 1703NT are correct. Infrared spectrum scanning was performed on the highly pure freeze-dried products of 1703, 1703M, 1703NT, and 1703MNT, and the wave numbers of amide A, amide B, amide I, amide II, and amide III all conform to the structural characteristics of recombinant collagen.

[0051] In the present invention, in vitro cultured NIH / 3T3 cells were used for cell adhesion and cell migration experiments of 1703, 1703NT, and 1703M. The experiments showed that the cell adhesion activities of 1703, 1703NT, and 1703M are significantly superior to those of commercially available natural human collagen, and there is no significant difference in the cell adhesion activities of 1703, 1703NT, and 1703M, indicating that 1703M with amino acid sequence mutation did not significantly change the cell adhesion activity of the sequence before mutation; the cell migration promoting activities of the purified freeze-dried products of 1703, 1703NT, and 1703M are significantly superior to those of natural human collagen, and the cell migration promoting activity of 1703M is superior to that of 1703 and 1703NT. Description of the Drawings

[0052] Figure 1 It is the SDS-PAGE detection result of the supernatant of the bacterial liquid after 24 hours of induced expression of 1703, 1703MNT, 1703NT, and 1703MNT.

[0053] Figure 2 It is the WB diagram of the supernatant of the bacterial liquid after 24 hours of induced expression of 1703 and 1703M. In the figure, the left figure is the WB diagram of the anti-6×His Tag antibody, and the right figure is the WB diagram of the anti-Strep-Tag II antibody.

[0054] Figure 3 It is the mass spectrometry analysis result of the target band in the SDS-PAGE detection result of the supernatant of the bacterial liquid after 24 hours of induced expression of 1703.

[0055] Figure 4 It is the mass spectrometry analysis result of the target band in the SDS-PAGE detection result of the supernatant of the bacterial liquid after 24 hours of induced expression of 1703M.

[0056] Figure 5 It is the SDS-PAGE diagram of the supernatant of the fermentation broth obtained after 48 hours of induced expression with different basal salt fermentation media.

[0057] Figure 6SDS-PAGE diagrams of the supernatant of the fermentation broth obtained after 48 h of induced expression under different pH conditions.

[0058] Figure 7 For the supernatant of the fermentation broth obtained after 48 h of induced expression with different initial OD 600 values.

[0059] Figure 8 SDS-PAGE diagrams of the supernatant of the fermentation broth obtained after 48 h of induced expression with different induction media.

[0060] Figure 9 SDS-PAGE diagrams of the supernatant of the fermentation broth obtained after induced expression in the 1703 fermentation parallel experiment.

[0061] Figure 10 SDS-PAGE diagrams of the supernatant of the fermentation broth obtained after induced expression of 1703NT.

[0062] Figure 11 HPLC spectra of the freeze-dried products of purified 1703 (Figure a) and 1703NT (Figure b).

[0063] Figure 12 SDS-PAGE electrophoresis diagrams of the freeze-dried products of purified 1703 (Figure a) and 1703NT (Figure b).

[0064] Figure 13 Deconvoluted molecular weights of the purified freeze-dried 1703 sample.

[0065] Figure 14 Deconvoluted molecular weights of the purified freeze-dried 1703NT sample.

[0066] Figure 15 Secondary mass spectra of the C-terminal peptide segments of the purified freeze-dried 1703 sample.

[0067] Figure 16 Secondary mass spectra of the C-terminal peptide segments of the purified freeze-dried 1703NT sample.

[0068] Figure 17 Infrared spectrum scans of the purified freeze-dried 1703 sample.

[0069] Figure 18 Infrared spectrum scans of the purified freeze-dried 1703NT sample.

[0070] Figure 19 Infrared spectrum scans of the purified freeze-dried 1703M sample.

[0071] Figure 20 Infrared spectrum scans of the purified freeze-dried 1703MNT sample.

[0072] Figure 21 Cell adhesion activity test results of 1703, 1703NT, 1703M obtained in the present invention, natural human collagen, and BSA.

[0073] Figure 22 Actual comparison chart of cell migration states of 1703, 1703NT, 1703M obtained in the present invention, natural human collagen, and BSA.

[0074] Figure 23 Cell migration rates of 1703, 1703NT, 1703M obtained in the present invention, natural human collagen, and BSA after NIH / 3T3 cells were cultured for 24 h and 48 h. Detailed implementation manners

[0075] In order to enable those skilled in the art to better understand the technical solutions of the present invention, the preferred embodiments of the present invention are described in detail below. However, the following embodiments do not limit the protection scope of the present invention.

[0076] In the embodiments of the present invention, those not described in detail are all completed by conventional experimental methods. Those not described in detail in the processes involved in the embodiments can be understood and easily implemented by those skilled in the art according to the product specifications or basic knowledge in the art, and thus will not be described in detail.

[0077] Example 1: Design and expression of recombinant collagen amino acid sequence

[0078] (1) Design of amino acid sequence and optimization of DNA sequence

[0079] Select the human XVII collagen sequence for optimization. The specific sequence reference:

[0080] Uniprot Q9UMD9-1 sequence (https: / / www.uniprot.org / uniprot / Q9UMD9), NCBI reference sequence Q9UMD9.3 (https: / / www.ncbi.nlm.nih.gov / protein / Q9UMD9.3), and the two sequences are the same, as shown in SEQ ID NO.1:

[0081] SEQ ID NO.1:

[0082]

[0083] In the present invention, the 15th helix region of human type XVII collagen is expressed. The sequence selected is the bold and underlined part in SEQ ID NO.1. This recombinant humanized type XVII collagen, that is, the recombinant humanized collagen expressing the 15th helix region of type XVII collagen, is named 1703NT, which has a total of 242 amino acids, and its amino acid sequence is shown in SEQ ID NO.2:

[0084] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKI

[0085] The DNA sequence encoding 1703NT is codon-optimized using Pichia pastoris as the host, and the optimized sequence is shown in SEQ ID NO.3:

[0086] GGTTCTCCTGGTCCAAAAGGAGATATGGGTTCACCCGGTCCCAAAGGAGATAGAGGATTCCCTGGTACTCCAGGTATCCCCGGTCCCCTGGGTCACCCTGGACCTCAAGGTCCTAAAGGTCAAAAGGGTTCTGTAGGAGATCCAGGTATGGAGGGTCCCATGGGTCAGAGAGGTAGAGAAGGTCCCATGGGACCAAGAGGTGAAGCTGGACCTCCCGGAAGTGGTGAAAAAGGAGAAAGAGGAGCAGCAGGAGAACCTGGACCCCATGGACCTCCAGGAGTTCCTGGATCAGTCGGACCCAAAGGTTCATCCGGTTCTCCTGGACCTCAAGGTCCACCAGGACCCGTCGGATTGCAAGGATTGAGAGGAGAAGTTGGACTTCCCGGAGTTAAGGGTGACAAGGGTCCTATGGGTCCTCCTGGTCCAAAGGGAGATCAGGGTGAAAAGGGTCCTAGAGGTCTGACTGGTGAACCAGGAATGAGAGGACTTCCCGGTGCCGTGGGTGAACCCGGTGCAAAAGGAGCAATGGGTCCTGCCGGTCCTGATGGACACCAGGGACCCAGAGGAGAGCAGGGATTAACAGGAATGCCTGGTATCAGAGGTCCCCCAGGTCCCTCAGGAGACCCAGGAAAGCCAGGACTTACTGGTCCCCAGGGTCCTCAAGGTCTGCCTGGAACTCCCGGAAGACCCGGAATCAAAGGTGAACCAGGAGCCCCAGGAAAAATC

[0087] Add a Strep-Tag II tag to the amino terminus and a 6×His Tag tag to the carboxyl terminus of SEQ ID NO.2. The sequence after adding the tags contains 260 amino acids. This recombinant type XVII humanized collagen, that is, the recombinant humanized collagen expressing the 15th helix region of type XVII collagen, is named 1703. Its amino acid sequence (the underlined part is the tag sequence) is shown in SEQ ID NO.4:

[0088] YVEFWSHPQFEKGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPMGPRGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPRGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPRGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKI HHHHHH

[0089] The DNA sequence corresponding to 1703 is shown in SEQ ID NO.5 (the underlined part is the sequence corresponding to the tag):

[0090]

[0091] In the present invention, for the mutant of recombinant humanized collagen XVII 1703NT, M at the 63rd position, R at the 66th position, R at the 152nd position, and R at the 188th position in the amino acid sequence shown in SEQ ID NO.2 are all changed to P, named 1703MNT. The mutated sequence has a total of 242 amino acids and is shown in SEQ ID NO.6:

[0092] GSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPPGPPGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPPGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKI

[0093] The DNA sequence encoding 1703MNT is shown in SEQ ID NO.7:

[0094] GGTTCTCCAGGTCCTAAAGGAGATATGGGTTCTCCAGGACCAAAGGGAGATAGAGGTTTTCCAGGTACTCCTGGTATTCCAGGTCCTTTGGGTCATCCAGGTCCTCAAGGTCCAAAGGGTCAAAAAGGTTCTGTTGGAGATCCAGGAATGGAAGGTCCAATGGGTCAAAGAGGTAGAGAAGGTCCACCTGGTCCACCTGGAGAAGCTGGTCCACCTGGTTCTGGTGAAAAGGGAGAGAGAGGTGCTGCTGGAGAGCCAGGTCCTCACGGTCCACCTGGTGTTCCTGGTTCTGTTGGTCCAAAAGGTTCTTCTGGTTCTCCAGGACCACAAGGTCCACCTGGTCCAGTTGGTTTGCAAGGTTTGAGAGGTGAAGTTGGTTTGCCAGGTGTTAAGGGAGATAAAGGTCCTATGGGTCCACCTGGTCCAAAGGGAGATCAAGGTGAAAAAGGTCCACCTGGTTTGACTGGAGAGCCTGGTATGAGAGGTTTGCCAGGTGCTGTTGGTGAACCTGGTGCTAAGGGTGCTATGGGTCCAGCTGGTCCTGATGGTCATCAAGGTCCACCTGGAGAGCAAGGTTTGACTGGTATGCCAGGTATTAGAGGTCCACCTGGACCTTCTGGAGATCCAGGTAAACCTGGTTTGACTGGTCCACAAGGTCCTCAAGGTTTGCCAGGTACTCCTGGTAGACCAGGTATTAAGGGAGAGCCTGGTGCTCCAGGTAAAATT

[0095] The variant of SEQ ID NO.6 with a Strep-Tag II tag added to the amino terminus and a 6×His Tag tag added to the carboxyl terminus, named 1703M, has an amino acid sequence of 260 amino acids in total (the underlined part is the tag sequence), as shown in SEQ ID NO.8:

[0096] YVEFWSHPQFEKGSPGPKGDMGSPGPKGDRGFPGTPGIPGPLGHPGPQGPKGQKGSVGDPGMEGPMGQRGREGPPGPPGEAGPPGSGEKGERGAAGEPGPHGPPGVPGSVGPKGSSGSPGPQGPPGPVGLQGLRGEVGLPGVKGDKGPMGPPGPKGDQGEKGPPGLTGEPGMRGLPGAVGEPGAKGAMGPAGPDGHQGPPGEQGLTGMPGIRGPPGPSGDPGKPGLTGPQGPQGLPGTPGRPGIKGEPGAPGKI HHHHHH

[0097] The DNA sequence corresponding to 1703M is as follows in SEQ ID NO.9 (the underlined part is the sequence corresponding to the label):

[0098] TACGTAGaattcTGGTCACATCCACAATTTGAGAAGGGTTCTCCAGGTCCTAAAGGAGATATGGGTTCTCCAGGACCAAAGGGAGATAGAGGTTTTCCAGGTACTCCTGGTATTCCAGGTCCTTTGGGTCATCCAGGTCCTCAAGGTCCAAAGGGTCAAAAAGGTTCTGTTGGAGATCCAGGAATGGAAGGTCCAATGGGTCAAAGAGGTAGAGAAGGTCCACCTGGTCCACCTGGAGAAGCTGGTCCACCTGGTTCTGGTGAAAAGGGAGAGAGAGGTGCTGCTGGAGAGCCAGGTCCTCACGGTCCACCTGGTGTTCCTGGTTCTGTTGGTCCAAAAGGTTCTTCTGGTTCTCCAGGACCACAAGGTCCACCTGGTCCAGTTGGTTTGCAAGGTTTGAGAGGTGAAGTTGGTTTGCCAGGTGTTAAGGGAGATAAAGGTCCTATGGGTCCACCTGGTCCAAAGGGAGATCAAGGTGAAAAAGGTCCACCTGGTTTGACTGGAGAGCCTGGTATGAGAGGTTTGCCAGGTGCTGTTGGTGAACCTGGTGCTAAGGGTGCTATGGGTCCAGCTGGTCCTGATGGTCATCAAGGTCCACCTGGAGAGCAAGGTTTGACTGGTATGCCAGGTATTAGAGGTCCACCTGGACCTTCTGGAGATCCAGGTAAACCTGGTTTGACTGGTCCACAAGGTCCTCAAGGTTTGCCAGGTACTCCTGGTAGACCAGGTATTAAGGGAGAGCCTGGTGCTCCAGGTAAAATT CATCATCACCATCATCAC

[0099] (2) Synthesis of DNA sequence and construction of recombinant expression vector

[0100] Using the expression vector pPIC9K-col17a1 in the inventor's authorized patent CN113185604B as a template, primers P1 and P2 were designed to amplify the 15th helix region sequence of type XVII collagen. After purification of the PCR product, double digestion was performed with EcoRI and NotI, and after the digestion was completed, the target band was recovered by gel cutting. The plasmid pPIC9K was digested with EcoRI and NotI, and the digested plasmid was recovered by column. The digested and recovered target fragment and plasmid were mixed at a molecular weight ratio of 3:1, and DNA Ligation Kit from Takara was used for ligation. The ligation product was transformed into DH5α, and the transformants grown overnight were subjected to colony PCR using the universal primers 5’AOX and 3’AOX. Five transformants of the positive clones were randomly selected to extract plasmids and send them for sequencing. The results were as expected, and this plasmid was named pPIC9K-1703.

[0101] The sequence of P1 is shown in SEQ ID NO.10:

[0102] (EcoRI)

[0103] cgGAATTCTGGAGTCATCCTCAATTCGAAAAAGGTTCTCCTGGTCCAAAAGGAGA

[0104] The sequence of P2 is shown in SEQ ID NO.11:

[0105] ATAGTTTAGCGGCCGCTTAGTGATGATGGTGATGGTGGATTTTTCCTGGGGCTCCTGGT(NotI)

[0106] The sequence of 5'AOX is shown in SEQ ID NO.12: 5’-GACTGGTTCCAATTGACAAGC-3’

[0107] The sequence of 3'AOX is shown in SEQ ID NO.13: 5’-GGCAAATGGCATTCTGACAT-3’

[0108] After commissioning Nanjing Genscript Biotech Co., Ltd. to synthesize SEQ ID NO.9 and clone it into the EcoRI and NotI digestion sites of pPIC9K, the expression plasmid pPIC9K-1703M was obtained. Nanjing Genscript Biotech Co., Ltd. was commissioned to construct subclones by removing the tag DNA sequences at both ends of 1703 and 1703M, and the cloning sites were EcoRI and NotI of pPIC9K, and the expression plasmids pPIC9K-1703NT and pPIC9K-1703MNT were obtained.

[0109] (3) Construction and screening of recombinant engineering strains

[0110] Separate 10 μg of each of the above recombinant expression vector plasmids (pPIC9K-1703, pPIC9K-1703NT, pPIC9K-1703MNT, and pPIC9K-1703M) was digested overnight at 37°C with SacⅠ (purchased from TaKaRa Dalian Co., Ltd., and the specific operation was carried out according to the kit instructions) to linearize it, and then the linearized plasmid was recovered using a PCR product purification kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) to control the volume to about 10 μL.

[0111] The linearized plasmid was electrotransformed into the competent cells of the empty host strain Pichia pastoris GS115 (purchased from the China Center for Industrial Culture Collection of Microorganisms). The electrotransformed bacterial solution was spread on an MD plate, with 100 μL - 200 μL spread on each plate, left standing at room temperature for 10 min, and then incubated upside down at 30°C for 2 - 5 days until single colonies (positive transformants) appeared.

[0112] Add 2 mL of sterile double-distilled water to the surface of the MD plate, and then gently scrape the His + transformants on the plate surface with a sterile triangular spreader and transfer them to a 50 mL centrifuge tube. Dilute the bacterial suspension with sterile double-distilled water, and spread 10 5 cells on a YPD plate containing 0.5 mg / mL G418, incubate upside down, and after 3 - 4 d of incubation at 30°C until single colonies appear. Pick colonies from the YPD plate into a sterile 96-well plate (200 μL YPD / well), mix well, and incubate at 30°C for 48 h; mix the bacterial solution in the wells, take 10 μL from each and transfer it to a new sterile 96-well plate, and repeat this operation after 24 h of incubation at 30°C; after 24 h, take 1 μL from the third 96-well plate and spot it on YPD plates containing 1.0 mg / mL and 4 mg / mL G418 respectively, and continue to incubate at 30°C for 96 h - 120 h. If the Pichia pastoris transformants can grow on the plate containing a high concentration of G418, it indicates that the transformants contain multiple copies of the target gene, that is, multiple recombinant fragments have entered the yeast cells and integrated into the yeast chromosome through homologous recombination. Through this step of screening, high-copy and highly expressible recombinant yeast engineering strains can be obtained.

[0113] Samples of the 4 constructed engineering bacteria were all sent to the China General Microbiological Culture Collection Center for preservation, and the corresponding strain preservation numbers are:

[0114] The strain expressing protein 1703M, preservation number: CGMCC No.21884;

[0115] The strain expressing protein 1703, preservation number: CGMCC No.21888;

[0116] The strain expressing protein 1703NT, with the preservation number: CGMCC No. 21889;

[0117] The strain expressing protein 1703MNT, with the preservation number: CGMCC No. 21890.

[0118] The preservation address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; The preservation date is: March 11, 2021. The taxonomic nomenclature is: Pichia pastoris.

[0119] (4) Induced expression and identification of recombinant collagen

[0120] Separate single colonies were selected and placed in 100 mL Erlenmeyer flasks containing 10 mL of BMGY medium, and cultured at 28 - 30 °C and 220 rpm until the OD 600 reached 2 - 6 (16 - 18 h). Centrifuge at 1500 - 3000 g for 5 min at room temperature, collect the bacterial cells, resuspend the bacterial cells with BMMY medium to make the OD 600 about 2, and place it on a shaker at 28 - 30 °C and 220 rpm to continue growing for 3 days. Add 100% methanol to the medium every 24 h to a final concentration of 1.0%. Take the bacterial liquid samples at time points (sampling once every 24 h after the start of induction), the sampling volume is 1 mL, place it in a 1.5 mL EP tube, centrifuge at the maximum speed for 2 - 3 min, collect the supernatant, add 5× loading buffer (250 mM Tris-HCl, pH 6.8, 10% SDS, 0.5% bromophenol blue, 50% glycerol, 5% β-mercaptoethanol), and place it in a 100 °C metal bath for heating for 10 min for SDS-PAGE detection. Since there are Srtep-TagⅡ tags at the amino terminus and 6×His tags at the carboxyl terminus of 1703 and 1703M, antibodies against Srtep-TagⅡ and anti-6×His Tag (purchased from Nanjing Genscript Biotech Corporation) were used for Western Blot detection (the specific operation refers to the instruction manual).

[0121] As Figure 1 shown, 1703 and 1703M can be highly secreted and expressed in the extracellular culture supernatant. The theoretical molecular weights of 1703 and 1703M are 24968.16 Da and 24756.88 Da respectively; The theoretical molecular weights of 1703NT and 1703MNT are 22566.54 Da and 22355.26 Da respectively, and the apparent molecular weight is about 32 kDa. It can be seen from the electrophoresis pattern that:

[0122] (1) There are two main bands in the 1703 and 1703NT lanes (with the largest optical density values), which are consistent with the expected size of the apparent molecular weight. The larger molecular weight is the full-length band (measured by Image Lab software, accounting for 41.5%), and the second-largest degradation band with a smaller molecular weight is the main degradation band and accounts for a large proportion (measured by Image Lab software, accounting for 40.2%). The proportions of the two are close;

[0123] (2) The degradation bands of 1703M were significantly less than those of 1703, and the degradation bands of 1703MNT were significantly less than those of 1703NT. The main degradation bands accounting for about 40% of the 1703 and 1703NT lanes basically disappeared, indicating that the amino acid mutation performed in the present invention can achieve the purpose of reducing the degradation of the amino acid sequence.

[0124] from Figure 2 (ECL chemiluminescence color development, fully automatic chemiluminescence image analysis system Tanon 5200 synthesizes protein molecular weight standards into the image) It can be seen that the amino-terminal Srtep-TagⅡ tag and carboxyl-terminal 6×His tag of 1703 and 1703M can be detected, and the maximum bands are the same as the apparent molecular weight in SDS-PAGE. When detected with anti-His antibody, the 1703M band is significantly less than 1703, which is consistent with the SDS-PAGE results.

[0125] Figure 1 and Figure 2 It can be seen that the variant 1703M after the amino acid sequence was modified is more stable and protein degradation is alleviated.

[0126] The expected bands of 1703 and 1703M on SDS-PAGE were cut out and digested with trypsin. The peptides of recombinant collagen after trypsin digestion were detected by Nano-HPLC-MS / MS mass spectrometry (completed by Suzhou Putai Biotechnology Co., Ltd.), and the detected peptides were sequenced (Uniprot database). The results are as follows Figure 3 and Figure 4 As shown: the peptides detected after enzymatic hydrolysis of 1703 and 1703M all belong to the relevant regions of the human XVII collagen sequence selected during the amino acid sequence selection and design, indicating that the collagen of the present invention was successfully expressed.

[0127] Example 2. Fermentation process for improving the production level of humanized collagen while reducing degradation

[0128] In a specific embodiment of the present invention, the strain used is Pichia pastoris expressing protein 1703 and 1703NT, with the preservation numbers being 1703: CGMCC No. 21888, 1703NT: CGMCC No. 21889, the preservation date being March 11, 2021, and the preservation unit being the General Microbiology Center of the China Committee for Culture Collection of Microorganisms.

[0129] The components of conventional general culture media and solution formulations are as follows. Unless otherwise specified, only the abbreviations are written in the following text, and the formulations will not be elaborated further; unless otherwise specified for the manufacturer and reagent grade, each component in the formulation can be of domestic analytical pure or chemical pure grade.

[0130] YPG medium: 10 g / L yeast extract, 20 g / L peptone, 10 g / L anhydrous glycerol.

[0131] PTM 1 : CuSO 4 ·5H 2 O 6 g / L, MnSO 4 ·H 2 O 3 g / L, NaCl 0.08 g / L, Na 2 MoO 4 ·2H 2 O 0.2 g / L, H 3 BO 3 0.02 g / L, CoCl 2 ·6H 2 O 0.5 g / L, ZnCl 2 20 g / L, FeSO 4 ·7H 2 O 65 g / L, biotin (USP Grade) 0.2 g / L, concentrated H 2 SO 4 5 mL / L, filtered and sterilized with a 0.22 μm sterile filter membrane, and stored at 4°C.

[0132] Alkali solution formulation: 300 ml concentrated ammonia water, add 700 ml sterile purified water, and mix evenly.

[0133] UV method protein quantification formula: C (mg / mL) = (A215 - A225) * 0.144.

[0134] (1) Optimization of the fermentation medium formulation

[0135] The classic BSM medium formulation (Invitrogen) contains 85% H 3 PO 4, KOH is not convenient to operate in actual production. The present invention explores and optimizes the basic medium formula for the types of proteins expressed, and determines a fermentation medium prepared from exclusive basic salts. The strain is an engineered strain of Pichia pastoris expressing protein 1703, with the preservation number of CGMCC No. 21888.

[0136] The general media used in the 1# to 4# fermenters in this part are as follows: (1) Seed medium, YPG; (2) Induction medium: pure methanol, with 12 mL of PTM added per liter 1 ; (3) Feed medium: 50% W / V glycerol, sterilized by high-pressure moist heat, and 12 mL of PTM is added per liter in the sterile solution 1 .

[0137] 1# fermenter: Use fermentation medium A, formula: NH 4 H 2 PO 4 47.6 g / L, KH 2 PO 4 10.06 g / L, CaSO 4 ·2H 2 O 1.18 g / L, K 2 SO 4 18.2 g / L, MgSO 4 ·7H 2 O 14.9 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L. After preparing the components except PTM 1 , sterilize by high-temperature moist heat, and add PTM 1 when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water.

[0138] 2# fermenter: Use fermentation medium B, formula: NH 4 H 2 PO 4 35.7 g / L, KH 2 PO 4 7.545 g / L, CaSO 4 ·2H 2 O 0.885 g / L, K 2 SO 4 13.65 g / L, MgSO 4 ·7H 2 O 11.175 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L. After preparing the components except PTM 1 , sterilize by high-temperature moist heat, and add PTM 1, adjust the pH to 5.0 with ammonia water.

[0139] Fermenter No. 3: Use fermentation medium C, formula: NH 4 H 2 PO 4 23.8 g / L, KH 2 PO 4 5.03 g / L, CaSO 4 ·2H 2 O 0.59 g / L, K 2 SO 4 9.1 g / L, MgSO 4 ·7H 2 O 7.45 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L. After preparing the components except PTM, sterilize by high-temperature and moist heat, and add PTM after the temperature drops to room temperature 1 , adjust the pH to 5.0 with ammonia water. 1 Fermenter No. 4: Use fermentation medium D, formula: NH

[0140] 11.9 g / L, KH 4 H 2 PO 4 2.515 g / L, CaSO 2 PO 4 0.295 g / L, K 4 ·2H 2 O 0.295 g / L, K 2 SO 4 4.55 g / L, MgSO 4 ·7H 2 O 3.725 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L. After preparing the components except PTM, sterilize by high-temperature and moist heat, and add PTM after the temperature drops to room temperature 1 , adjust the pH to 5.0 with ammonia water. 1 The fermentation processes in Fermenters No. 1 to No. 4 are all controlled uniformly: Inoculate the strain into the seed medium YPG, and culture overnight at 30 °C and 220 rpm to prepare the strain solution. Set the fermentation temperature at 30 °C and pH 5.0. Add the strain solution to a 5 L fermenter (Baoxing Biology) containing 3 L of fermentation medium at an inoculation amount of 10%, adjust the stirring speed to 300 r / min - 700 r / min, the air flow rate to 2 VVM, the tank pressure to 0 - 0.05 MPa, DO ≥ 30%, culture until the carbon source is exhausted, DO rises rapidly, and start to feed the feeding medium until the cell OD

[0141] 126 600= 150, wet weight 200 g / L, stop feeding the supplementary medium. After the glycerol is exhausted and DO ≥ 70%, start feeding the induction medium and enter the methanol induction stage. Adjust the rotation speed, ventilation volume, tank pressure and feeding rate to make DO ≥ 30%. Take samples every 4 h to measure OD 600 , wet weight and UV protein content. After 48 h of induction, end the fermentation, discharge the tank, collect the fermentation broth, centrifuge at 7000 rpm for 20 minutes, take the supernatant of the fermentation broth, detect the UV protein content, and perform SDS-PAGE electrophoresis.

[0142] By UV detection, the final recombinant collagen concentration in the supernatant of the fermentation broth: for No. 1, 9.99 g / L; for No. 2, 10.17 g / L; for No. 3, 10.75 g / L; for No. 4, 11.30 g / L. The SDS-PAGE electrophoresis diagram of the supernatant of the fermentation broth is as Figure 5 shown. Combining the recombinant collagen concentration with the SDS-PAGE electrophoresis diagram, it can be seen that the expression level of fermentation medium D is relatively high, so fermentation medium D is selected as the fermentation medium. However, it can be seen from the SDS-PAGE electrophoresis diagram that in each lane, there are mainly two main bands (the ones with the largest optical density value). The band with a larger molecular weight is the full-length band (accounting for about 40%), and the band with a relatively smaller molecular weight is the degradation band, and the proportion is relatively large (about 40%). Optimization of the simple medium can obtain a relatively high yield, but there is still a situation of protein degradation, so the subsequent process optimization will continue on this basis.

[0143] (2) Optimization of the fermentation pH process to reduce the degradation of recombinant collagen during fermentation

[0144] The general media used in fermenters No. 1 to No. 4 in this part are as follows: (1) Seed medium, YPG; (2) Induction medium: pure methanol, add 12 mL of PTM per liter 1 ; (3) Supplementary medium: 50% W / V glycerol, autoclaved by high-pressure moist heat, add 12 mL of PTM per liter in the sterile solution 1 ; (4) Fermentation medium, fermentation medium D: NH 4 H 2 PO 4 11.9 g / L, KH 2 PO 4 2.515 g / L, CaSO 4 ·2H 2 O 0.295 g / L, K 2 SO 4 4.55 g / L, MgSO 4 ·7H 2 O 3.725 g / L, glycerol 20 g / L, PTM 10.45 mL / L. The strain is an engineered strain of Pichia pastoris expressing recombinant collagen 1703, and the preservation number is CGMCC No. 21888.

[0145] Fermenter 1: Use fermentation medium D, remove components other than PTM 1 After preparation by removing components other than PTM, it is sterilized by high-temperature and high-humidity sterilization. After the temperature drops to room temperature, add PTM 1 , and adjust the pH to 6.0 with ammonia water. Set the fermentation temperature at 30 °C and pH 6.0.

[0146] Fermenter 2: Use fermentation medium D, remove components other than PTM 1 After preparation by removing components other than PTM, it is sterilized by high-temperature and high-humidity sterilization. After the temperature drops to room temperature, add PTM 1 , and adjust the pH to 5.0 with ammonia water. Set the fermentation temperature at 30 °C and pH 5.0.

[0147] Fermenter 3: Use fermentation medium D, remove components other than PTM 1 After preparation by removing components other than PTM, it is sterilized by high-temperature and high-humidity sterilization. After the temperature drops to room temperature, add PTM 1 , and adjust the pH to 4.0 with ammonia water. Set the fermentation temperature at 30 °C and pH 4.0.

[0148] Fermenter 4: Use fermentation medium D, remove components other than PTM 1 After preparation by removing components other than PTM, it is sterilized by high-temperature and high-humidity sterilization. After the temperature drops to room temperature, add PTM 1 , and adjust the pH to 3.5 with ammonia water. Set the fermentation temperature at 30 °C and pH 3.5.

[0149] The fermentation processes of Fermenters 1 to 4 are all controlled basically the same: Inoculate the strain into the seed medium YPG and culture it overnight at 30 °C and 220 rpm to prepare the strain solution. Set the fermentation temperature at 30 °C (the pH values set for Fermenters 1 to 4 are different). Add the strain solution to a 5 L fermenter (Baoxing Biology) containing 3 L of fermentation medium D (the pH values of the medium in Fermenters 1 to 4 are different) at an inoculation amount of 10%, adjust the stirring speed to 300 r / min - 700 r / min, the air flux to 2 VVM, the tank pressure to 0 - 0.05 MPa, and DO ≥ 30%. Cultivate until the carbon source is exhausted and DO rapidly rebounds, then start to feed the feeding medium. When the OD of the bacteria 600 = 150 and the wet weight is 200 g / L, stop feeding the feeding medium. After the glycerol is exhausted and DO ≥ 70%, start to feed the induction medium and enter the methanol induction stage. Adjust the rotation speed, ventilation volume, tank pressure, and feeding speed to make DO ≥ 30%. Take samples every 4 h and measure the OD 600, wet weight, and UV protein content. After 48 h of induction, the fermentation was terminated, and the fermentation broth was collected by discharging the tank and centrifuged at 7000 rpm for 20 minutes. The supernatant of the fermentation broth was taken to detect the UV protein content and perform SDS-PAGE electrophoresis.

[0150] After UV detection, the final recombinant collagen concentration in the supernatant of the fermentation broth: for No. 1, 9.70 g / L; for No. 2, 12.00 g / L; for No. 3, 12.50 g / L; for No. 4, 9.50 g / L. The SDS-PAGE electrophoresis pattern of the supernatant of the fermentation broth is as Figure 6 shown. Maintaining the pH at 4.0 during fermentation in fermenter No. 3 can effectively reduce the degradation of the target protein, and at the same time, the protein content detected by UV is the highest, indicating that the fermentation pH of 4.0 is the optimal fermentation pH. At the same time, it can be found that in the lane of the electrophoresis pattern of fermenter No. 3, there is only one main electrophoresis band (with the largest optical density), and the previous main degradation band with a relatively small molecular weight and a large proportion has basically disappeared, indicating that the optimization of the fermentation conditions at this time plays a role in inhibiting the degradation of recombinant collagen.

[0151] (3) Optimization of the initial OD 600 value process

[0152] The initial bacterial concentration value OD 600 directly affects the final protein expression level.

[0153] The general media used in fermenters No. 1 to No. 3 in this part are as follows: (1) Seed medium, YPG; (2) Induction medium: pure methanol, with 12 mL of PTM added per liter 1 ; (3) Feed medium: 50% W / V glycerol, sterilized by high-pressure moist heat, and 12 mL of PTM is added per liter in the sterile solution 1 ; (4) Fermentation medium, fermentation yeast medium D: NH 4 H 2 PO 4 11.9 g / L, KH 2 PO 4 2.515 g / L, CaSO 4 ·2H 2 O 0.295 g / L, K 2 SO 4 4.55 g / L, MgSO 4 ·7H 2 O 3.725 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L. After preparing the components except PTM 1 and sterilizing by high-temperature moist heat, PTM is added after the temperature drops to room temperature. 1, adjust the pH to 4.0 with ammonia water. Set the fermentation temperature at 30°C and the pH at 4.0. The strain is the engineered strain of Pichia pastoris expressing protein 1703, with the preservation number of CGMCC No. 21888.

[0154] Fermenter 1: Set the fermentation temperature at 30°C and the pH at 4.0. Add the seed culture solution into a 5L fermenter (Baoxing Biology) containing 3L of fermentation medium D at an inoculation amount of 10%. Adjust the stirring speed to 300 r / min - 700 r / min, the air flow rate to 2 VVM, the tank pressure to 0 - 0.05 MPa, and the DO ≥ 30%. Cultivate until the carbon source is exhausted and the DO rapidly rebounds, then start to feed the feeding medium until the initial OD of the bacteria for induction 600 = 50 (wet weight 74 g / L), and stop feeding the feeding medium. After the glycerol is exhausted and the DO ≥ 70%, start to feed the induction medium and enter the methanol induction stage. Adjust the rotation speed, ventilation volume, tank pressure, and feeding speed to make the DO ≥ 30%. Take samples every 4 h to measure the OD 600 , wet weight, and UV protein content. After 48 h of induction, end the fermentation, discharge the fermenter, collect the fermentation broth, centrifuge it at 7000 rpm for 20 minutes, take the supernatant of the fermentation broth, detect the UV protein content, and perform SDS-PAGE electrophoresis.

[0155] Fermenter 2: Set the fermentation temperature at 30°C and the pH at 4.0. Add the seed culture solution into a 5L fermenter (Baoxing Biology) containing 3L of fermentation medium D at an inoculation amount of 10%. Adjust the stirring speed to 300 r / min - 700 r / min, the air flow rate to 2 VVM, the tank pressure to 0 - 0.05 MPa, and the DO ≥ 30%. Cultivate until the carbon source is exhausted and the DO rapidly rebounds, then start to feed the feeding medium until the initial OD of the bacteria for induction 600 = 100 (wet weight 121 g / L), and stop feeding the feeding medium. After the glycerol is exhausted and the DO ≥ 70%, start to feed the induction medium and enter the methanol induction stage. Adjust the rotation speed, ventilation volume, tank pressure, and feeding speed to make the DO ≥ 30%. Take samples every 4 h to measure the OD 600 , wet weight, and UV protein content. After 48 h of induction, end the fermentation, discharge the fermenter, collect the fermentation broth, centrifuge it at 7000 rpm for 20 minutes, take the supernatant of the fermentation broth, detect the UV protein content, and perform SDS-PAGE electrophoresis.

[0156] Fermenter 3: Set the fermentation temperature at 30°C and the pH at 4.0. Add the seed culture solution into a 5L fermenter (Baoxing Biology) containing the fermentation medium D at an inoculation amount of 10%. Adjust the stirring speed to 300 r / min - 700 r / min, the air flow rate to 2 VVM, the tank pressure to 0 - 0.05 MPa, and the DO ≥ 30%. Cultivate until the carbon source is exhausted and the DO rapidly rebounds, then start to feed the feeding medium until the initial OD of the bacteria for induction 600= 150 (wet weight 203 g / L), stop the fed-batch medium feeding. After the glycerol is exhausted and DO ≥ 70%, start feeding the induction medium to enter the methanol induction stage, and adjust the rotation speed, ventilation volume, tank pressure and feeding rate to make DO ≥ 30%. Take samples every 4 h to measure OD 600 , wet weight and UV protein content. After 48 h of induction, end the fermentation, discharge the fermenter, collect the fermentation broth, centrifuge at 7000 rpm for 20 minutes, take the supernatant of the fermentation broth, detect the UV protein content, and perform SDS-PAGE electrophoresis.

[0157] By UV detection, the final recombinant collagen concentration in the supernatant of the fermentation broth: for No. 1, 14.60 g / L; for No. 2, 13.00 g / L; for No. 3, 12.30 g / L. The SDS-PAGE electrophoresis pattern of the supernatant of the fermentation broth is as Figure 7 shown. In the electrophoresis lanes of the electrophoresis pattern, there is only one main electrophoresis band (the largest optical density). The initial OD of fermentation induction in the No. 1 fermenter 600 = 50, and the protein expression level is significantly higher than that of OD 600 = 100 and OD 600 = 150. OD 600 = 50 is the optimal initial OD for induction. 600 Optimal.

[0158] (4) Fed-batch of mixed carbon sources, optimization of the induction medium

[0159] Methanol in the induction medium can effectively induce the expression of recombinant collagen. However, using only methanol as the main component of the induction medium to provide carbon source for Pichia pastoris may inhibit the cell growth state. Appropriate addition of a certain amount of glycerol or other carbon sources can effectively promote the expression of recombinant collagen during fermentation. In this part, the components of the induction medium were optimized and the fed-batch of mixed carbon sources was adjusted. The strain is the engineered strain of Pichia pastoris expressing protein 1703, and the preservation number is CGMCC No. 21888.

[0160] The general media used in fermenters No. 1 to No. 4 in this part are as follows: (1) Seed medium, YPG; (2) Fed-batch medium: 50% W / V glycerol, autoclaved by high-pressure moist heat, add 12 mL of PTM per liter in the sterile solution 1 ; (3) Fermentation medium, fermentation yeast medium D: NH 4 H 2 PO 4 11.9 g / L, KH 2 PO 4 2.515 g / L, CaSO 4 ·2H 2 O 0.295 g / L, K 2 SO 44.55 g / L, MgSO 4 ·7H 2 O 3.725 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L. After preparing the components except PTM and subjecting them to high-temperature and high-humidity sterilization, add PTM after the temperature drops to room temperature 1 . Adjust the pH to 4.0 with ammonia water 1 .

[0161] For fermenter #1, use induction medium A, pure methanol: 50% glycerol (sterile) = 7:3, add 12 mL of PTM per liter 1 .

[0162] For fermenter #2, use induction medium B, pure methanol: 50% glycerol (sterile) = 9:1, add 12 mL of PTM per liter 1 .

[0163] For fermenter #3, use induction medium C, pure methanol: 50% glycerol (sterile) = 8:2, add 12 mL of PTM per liter 1 .

[0164] For fermenter #4, use induction medium D, pure methanol: 50% glycerol (sterile) = 10:0, add 12 mL of PTM per liter 1 .

[0165] The fermentation processes of fermenters #1 to #4 are all controlled basically the same: Inoculate the strain into the seed medium YPG and culture it overnight at 30°C and 220 rpm to prepare the strain solution. Set the fermentation temperature at 30°C and pH 4.0. Add the strain solution to a 5 L fermenter (Baoxing Biology) containing fermentation medium D at an inoculation amount of 10%, adjust the stirring speed to 300 r / min - 700 r / min, the air flow rate to 2 VVM, the tank pressure to 0 - 0.05 MPa, and DO ≥ 30%. Culture until the carbon source is exhausted and DO rapidly rebounds, then start to feed the feeding medium. When the cell OD 600 = 50 and the wet weight is 74 g / L, stop feeding the feeding medium. After the glycerol is exhausted and DO ≥ 70%, start to feed the induction medium (the induction media for fermenters #1 to #4 are different), enter the methanol induction stage, and adjust the rotation speed, ventilation volume, tank pressure, and feeding speed to make DO ≥ 30%. Take samples every 4 h to measure OD 600 , wet weight, and UV protein content. After 48 h of induction, end the fermentation, discharge the fermenter, collect the fermentation broth, centrifuge it at 7000 rpm for 20 minutes, take the supernatant of the fermentation broth, detect the UV protein content, and perform SDS-PAGE electrophoresis

[0166] After UV detection, the final recombinant collagen concentration in the supernatant of the fermentation broth: No. 1, 12.30 g / L; No. 2, 15.90 g / L; No. 3, 16.70 g / L; No. 4, 14.30 g / L. The SDS-PAGE electrophoresis pattern of the supernatant of the fermentation broth is as shown in Figure 8 shown. In the electrophoresis lanes of the electrophoresis pattern, there is only one main electrophoresis band (with the maximum optical density). When the induction medium in the No. 3 fermenter is pure methanol: 50% glycerol = 8:2, the protein expression level is the highest.

[0167] (5) Verification of the optimized fermentation process

[0168] The optimized and stable fermentation process is a complete set of processes, and several key points are as follows:

[0169] a. The fermentation medium used is fermentation medium D: NH 4 H 2 PO 4 11.9 g / L, KH 2 PO 4 2.515 g / L, CaSO 4 ·2H 2 O 0.295 g / L, K 2 SO 4 4.55 g / L, MgSO 4 ·7H 2 O 3.725 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L. After preparing by removing components other than PTM 1 and subjecting to high-temperature and high-humidity sterilization, add PTM after the temperature drops to room temperature 1 , and adjust the pH to 4.0 with ammonia water;

[0170] b. The optimal fermentation pH is 4.0 during fermentation;

[0171] c. The initial induced bacterial concentration OD 600 = 50;

[0172] d. The induction medium used for the fed-batch of the mixed carbon source is induction medium C: pure methanol: 50% glycerol (sterile) = 8:2, and add 12 mL of PTM per liter 1 ;

[0173] The rest are general media: seed medium (YPG), feeding medium (50% W / V glycerol, high-pressure and high-humidity sterilized, add 12 mL of PTM per liter in the sterile solution 1 ).

[0174] The fermentation process is as follows: The strain is inoculated into the seed medium YPG and cultured overnight at 30 °C and 220 rpm to prepare the strain solution. Set the fermentation temperature at 30 °C and pH at 4.0. Add the strain solution to a 5 L fermenter (Baoxing Biology) containing the fermentation medium at an inoculation amount of 10%, adjust the stirring speed to 300 r / min - 700 r / min, the air flux to 2 VVM, the tank pressure to 0 - 0.05 MPa, and DO ≥ 30%. Cultivate until the carbon source is exhausted and DO rapidly rebounds, then start to feed the feeding medium until the bacterial OD 600 = 50, the wet weight is 74 g / L, and stop feeding the feeding medium. After the glycerol is exhausted and DO ≥ 70%, start to feed the induction medium and enter the methanol induction stage. Adjust the rotation speed, ventilation volume, tank pressure, and feeding speed to make DO ≥ 30%. Take samples every 4 h to measure OD 600 , wet weight, and UV protein content. After induction for 48 h, end the fermentation. Centrifuge the fermentation broth at 7000 rpm for 20 minutes, take the supernatant of the fermentation broth, detect the UV protein content, and perform SDS-PAGE electrophoresis.

[0175] According to the above fermentation conditions, parallel experiments in multiple batches of fermenters were carried out using the engineered strain of Pichia pastoris expressing protein 1703 (deposit number CGMCC No. 21888). At the end of fermentation, there were no significant differences in the relevant parameters, and the protein expression level was stable at > 16 g / L. At the same time, the goal of low degradation of recombinant collagen was achieved. See Table 1 below and Figure 9 .

[0176] Table 1. Results of parallel experiments in multiple batches of fermenters under the fermentation conditions in this section

[0177] Fermenter Total feeding volume (mL) <![CDATA[OD 600 > Wet weight (g / L) Collagen content (g / L) 1# 368 16 212 16.3 2# 375 192 222 16.2 3# 381 186 229 17.2 4# 388 181 227 16

[0178] At the same time, the same fermentation process can also be applied to the fermentation expression of the engineered strain of Pichia pastoris expressing protein 1703NT (strain deposit number CGMCC No. 21889), which has fewer degradation bands and the protein expression level is also stable at > 15 g / L. The results are as Figure 10 shown.

[0179] After a series of fermentation process optimizations, the collagen expression level of the engineered Pichia pastoris strain expressing Protein 1703 (preservation number CGMCC No. 21888) in a 5L tank increased from about 11g / L to about 17g / L, and the engineered Pichia pastoris strain expressing Protein 1703NT (preservation number CGMCC No. 21889) could reach >15g / L. At the same time, protein degradation during fermentation was significantly improved. In particular, there was only one main electrophoresis band (with the largest optical density), and the previous main degradation band with a relatively small molecular weight and a relatively large proportion (about 40%) basically disappeared, indicating that the fermentation conditions at this time played a role in inhibiting the degradation of recombinant collagen, and its effect was similar to the anti-degradation effect brought by mutants such as 1703M that changed the amino acid sequence.

[0180] Example 3. Establishment of a purification process for obtaining high-purity protein

[0181] Proteins 1703 and 1703NT only differ in the tag sequences at the N-terminus and C-terminus in the amino acid sequence, and the amino acid sequence of the recombinant humanized collagen as the main part is exactly the same. The present invention has developed a two-step purification method, which can obtain high-purity proteins 1703 and 1703NT without using affinity chromatography (the tag sequence can bind to the corresponding affinity chromatography medium).

[0182] (1) Hydrophobic chromatography

[0183] Each of the above buffers is prepared with deionized water:

[0184] Buffer A includes: 20 mM KH 2 PO 4 , 2 M ammonium sulfate, pH 5.0; Buffer B includes: 20 mM KH 2 PO 4 , 0.6 M ammonium sulfate, pH 5.0; Buffer C includes: 20 mM KH 2 PO 4 , pH 5.0.

[0185] The fermentation broth was collected and centrifuged (Thermo Fisher Scientific, Lynx 6000) to separate the bacterial sludge and the supernatant. 20 mM potassium dihydrogen phosphate and 2 M ammonium sulfate were added to the supernatant to fully dissolve it. The pH of the supernatant was adjusted to 5.0 and filtered. The filter membrane (Shanghai Xingya Purification Material Factory) was 0.45 μm. The flow rate was set to 30 mL / min for buffer A (0.45 μm filter membrane filtration) to balance the hydrophobic chromatography medium (column: Lisui Technology Co., Ltd., XK50 / 30. Hydrophobic filler: Capto Phenyl, Si Tuo Fan Biotechnology Co., Ltd., loaded on Si Tuo Fan Biotechnology Co., Ltd., AKTA pure 150M) until the A215 absorbance value drops to 30Mau and the conductivity remains at about 192ms / cm, set the flow rate of 20mL / min for sample loading, and the sample volume is 300mL / time. After the sample loading is completed, set the flow rate of 30mL / min, and use buffer A for rebalancing until the A215 absorbance value drops to 30Mau and the conductivity remains at about 192ms / cm. Under the condition of constant flow rate, buffer B is used for washing (filtered with a 0.45μm filter membrane) until the A215 absorbance value drops to 30Mau, and buffer C is set for elution. When the A215 absorbance value rises, open the sample collection valve and start collecting eluent 1 until the A215 absorbance value drops to 50Mau, then stop collecting. Eluent 1 is placed in a refrigerator at 4 degrees Celsius for use.

[0186] (2) Cationic chromatography

[0187] Each buffer solution is prepared with deionized water:

[0188] Buffer D includes: 20 mM tartaric acid, 100 mM sodium chloride, pH 4.0; Buffer E includes: 20 mM tartaric acid, 500 mM sodium chloride, pH 4.0.

[0189] Equilibrate the cation exchange chromatography medium (column tube: Lisui Technology Co., Ltd., XK50 / 30. Cation exchanger: Cytiva, SP Sepharose Fast Flow, loaded on Cytiva, AKTA pure 150M) with buffer D (filtered through a 0.45 μm filter membrane) at a flow rate of 30 mL / min until the absorbance at A215 drops to 30 Mau and the conductivity remains at about 11 ms / cm. Take out eluate 1, adjust the pH to 4.0, load eluate 1 at a flow rate of 20 mL / min. After loading, re-equilibrate with buffer D at a flow rate of 30 mL / min until the absorbance at A215 drops to 30 Mau and the conductivity remains at about 11 ms / cm. While keeping the flow rate constant, elute with buffer E. When the absorbance at A215 rises, open the sample collection valve and start collecting eluate 2 until the absorbance at A215 drops to 100 Mau, then stop collection. Ultrafilter and lyophilize eluate 2 (ultrafiltration equipment: Cytiva, AKTA Flux), and finally collect the lyophilized product.

[0190] Dissolve the lyophilized products of 1703 and 1703NT in ultrapure water to 2 mg / mL, filter through a 0.22 μm filter membrane, and inject 10 μL (Sepax Bio-C18 chromatographic column, high performance liquid chromatograph is Waters2695 or Agilent LC1260) to analyze the purity.

[0191] Dissolve the lyophilized products of 1703 and 1703NT in ultrapure water to 1 mg / mL, filter through a 0.22 μm filter membrane, and inject 5 μL for SDS-PAGE electrophoresis detection.

[0192] The purification results are as Figure 11 shown. The purified 1703 ( Figure 11 a), 1703NT ( Figure 11 b) lyophilized products, after liquid phase analysis and calculation (area normalization method), have obvious single peaks and high purity. 1703 can reach 94% and 1703NT can reach 95%. Figure 12 In the electrophoresis diagram, the purified 1703 ( Figure 12 a), 1703NT ( Figure 12 b) lyophilized products both show a single band.

[0193] In summary, the present invention has developed a two-step purification method using a tartaric acid-containing buffer system. The method is simple and effective, and high-purity proteins 1703 and 1703NT can be obtained through hydrophobic chromatography and cation exchange chromatography.

[0194] Example 4. Characterization of the properties and detection of the biological activities of recombinant humanized collagen type XVII 1703 and 1703NT

[0195] (1) Molecular weight detection

[0196] The theoretically predicted molecular weight of protein 1703 is 24968.16 Da, and the theoretically predicted molecular weight of protein 1703NT is 22566.54 Da. LC-MS analysis was performed on the highly purified lyophilized products of 1703 and 1703NT (capillary high-performance liquid chromatograph ThermoFisher Scientific Ultimate 3000, electrospray-quadrupole time-of-flight mass spectrometer AB SCIEXTripleTOF 5600Mass Spectrometer, chromatographic column ACQUITY UPLC Protein BEH C4Column) to obtain their deconvoluted molecular weights, and the detection was entrusted to Beijing Bio-Tech Pack Technology Co., Ltd. As Figure 13 shown, the molecular weight of 1703 is mainly 24967.82 Da, which is basically consistent with the theoretical molecular weight (24967.81 Da); as Figure 14 shown, the molecular weight of 1703NT is mainly 22566.12 Da, which is basically consistent with the theoretical molecular weight (22566.22 Da). (2) N-terminal, C-terminal sequencing, and full-sequence sequencing verification

[0197] Beijing Bio-Tech Pack Technology Co., Ltd. was entrusted to perform N-terminal and C-terminal sequencing verification and LC-MS / MS-based protein full-sequence analysis on the highly purified lyophilized products of 1703 and 1703NT.

[0198] N-terminal sequencing was performed to analyze the N-terminal sequence of the sample (Edman degradation method) using a Shimadzu fully automatic protein and polypeptide sequencer (PPSQ-33A): An appropriate amount of the 1703 and 1703NT freeze-dried samples was dissolved. The sample solution was dropped onto a PVDF membrane and placed in a reactor. After assembling the reactor, it was placed at a fixed position on the instrument. Through the software PPSQ-30 Analysis, the sample name, sample number, number of test cycles, and selection of the method file were set. After the settings were completed, the test was started. The raw data and spectra generated by the PPSQ-33A were identified and peak-labeled by the PPSQ-30 Data Processing software, and the corresponding spectra were exported. After data analysis, the protein N-terminal sequence was determined. The N-terminal sequence detected for the 1703NT freeze-dried sample was: NH2-Tyr-Val-Glu-Phe-Trp-Ser-His-Pro-Glu-Phe-Glu-Lys-Gly-Ser-Pro, i.e., YVEFWSHPQFEKGSP, which was consistent with the theoretical N-terminal amino acid sequence (YVEFWSHPQFEKGSP). The N-terminal sequence detected for the 1703NT freeze-dried sample was: NH2-Gly-Ser-Pro-Gly-Pro-Lys-Gly-Asp-Met-Gly, i.e., GSPGPKGDMG, which was consistent with the theoretical N-terminal amino acid sequence (GSPGPKGDMG).

[0199] C-terminal sequencing: An appropriate amount of the 1703 and 1703NT high-purity freeze-dried samples was subjected to enzymatic hydrolysis with trypsin and pepsin. Then, the processed samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to obtain the raw file of the original mass spectrometry results. After analysis with the software Byonic and matching of the data, the identification results were obtained. After the mass spectrometry data were retrieved from the database, the secondary mass spectrometry spectrum of the C-terminal peptide of the 1703 freeze-dried sample is shown as Figure 15 follows, and its sequence was: PGTPGRPGIKGEPGAPGKIHHHHHH, which was consistent with the theoretical C-terminal amino acid sequence (PGTPGRPGIKGEPGAPGKIHHHHHH). The secondary mass spectrometry spectrum of the C-terminal peptide of the 1703NT freeze-dried sample is shown as Figure 16 follows, and its sequence was: PGAPGKI, which was consistent with the theoretical C-terminal amino acid sequence (PGAPGKI).

[0200] LC-MS / MS-based full protein sequence analysis: Further, highly pure freeze-dried samples of 1703 and 1703NT were subjected to enzymatic digestion with trypsin, chymotrypsin, pepsin, trypsin&Glu-C protease, and trypsin&Asp-N protease. After that, the processed samples were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS / MS) to obtain the raw file of the original mass spectrometry results. Through software Byonic analysis and data matching, the results of full sequence sequencing verification were obtained. Based on the comprehensive analysis of the detection results, the amino acid sequences and their total coverage rates of the 1703 and 1703NT freeze-dried samples were both 100%, and the amino acid sequence of the sample protein was consistent with the theoretical amino acid sequence.

[0201] Protein biosynthesis starts from the N-terminus and ends at the C-terminus. Whether the amino acid sequences at the N- and C-termini are correct directly indicates whether the amino acid sequence is correct and complete. LC-MS / MS-based full protein sequence analysis can verify whether the expressed amino acid sequence is correct. The above detection results show that the amino acid sequences expressed by 1703 and 1703NT are correct.

[0202] (3) Fourier transform infrared spectroscopy (FT-IR) analysis

[0203] The characteristic absorption peaks of collagen groups can be detected by infrared spectroscopy analysis. A small amount of highly pure freeze-dried samples of 1703, 1703NT, 1703M, and 1703MNT were ground into powder with KBr and pressed into tablets respectively. At room temperature, they were scanned in the range of 4000 - 400 cm -1 (Thermo Scientific, Nicolet TM iS TM 10 FT-IR spectrometer). The methods and result analysis refer to (Jeong, H., J. Venkatesan and S. Kim, Isolation and characterization of collagen from marine fish (Thunnus obesus). Biotechnology and Bioprocess Engineering, 2013. 18(6): p. 1185 - 1191.).

[0204] From the infrared spectrum scan maps of the highly pure freeze-dried samples of 1703, 1703NT, 1703M, and 1703MNT (corresponding to Figure 17 , 18 , 19, 20 in sequence), it can be seen that their characteristic absorption wavenumbers all conform to the structural characteristics of recombinant collagen: amide A (3299 cm-1 around), amide B (3081 cm -1 around), amide I (1650 cm -1 around), amide II (1530 - 1550 cm -1 around), amide III (1240 cm -1 around) (see References [1]. Chen Jingtao et al., Infrared Spectroscopy Study of Recombinant Collagen and Bovine-derived Type I Collagen. Materials Review, 2008(03): pp. 119 - 121. [2]. Doyle, B.B., E.G. Bendit and E.R. Blout, Infrared spectroscopy of collagen and collagen-like polypeptides. Biopolymers, 1975. 14(5): p. 937 - 957. [3]. Zhou Aimei et al., Isolation, Purification and Structural Characterization of Recombinant Human-derived Collagen. Food and Fermentation Industries, 2015(03): pp. 46 - 52.).

[0205] (4) Recombinant Collagen Cell Adhesion Activity Experiment

[0206] The detection method for the cell adhesion activity of recombinant collagen refers to Juming Yao, Satoshi Yanagisawa, Tetsuo Asakura. Design, Expression and Characterization of Collagen-Like Proteins Based on the Cell Adhesive and Crosslinking Sequences Derived from Native Collagens, J Biochem. 136, 643 - 649 (2004). It was commissioned to be completed by the Functional Nanomaterials and Biomedical Detection Laboratory of the School of Pharmacy, Changzhou University.

[0207] Specific implementation method: NIH / 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNM6, and the culture and subculture methods are carried out according to the cell instructions) were cultured normally. Take 1703, 1703NT, 1703M purified freeze-dried products, control human collagen (Sigma, catalog number C7774), and bovine serum albumin (BSA, purchased from Sangon Biotech (Shanghai) Co., Ltd.) and dissolve them (ultrapure water or 1M HCl solution). Use the UV protein quantification empirical formula: C (mg / mL) = 0.144 * (A215 - A225) to measure the protein concentration, and then dilute it to 0.5 mg / mL with PBS (pH 7.4). Add 100 μL of various protein solutions and blank PBS solution controls to a 96-well cell culture plate, and let it stand at room temperature for 60 min; then add 10 5 well-maintained 3T3 cells to each well, and incubate at 37 °C and 5% CO 2 for 60 min. Wash the cells in the wells 4 times with PBS. Use an LDH detection kit (Roche, 04744926001) to detect the OD 492nm absorbance value (the specific operation is carried out according to the instructions), analyze the data and perform a significant difference analysis (SPSS 22 software, Duncan method, P < 0.05).

[0208] The OD 492nm absorbance correspondingly characterizes the cell adhesion activity of the collagen sample: the higher the adhesion activity, the more cells the protein adheres to, and the more the collagen can help cells adhere to the wall or adhere to the extracellular matrix in a short time, which is more conducive to constructing a better extracellular environment. As Figure 21 shown, the cell adhesion activities of 1703, 1703NT, and 1703M are significantly better than those of commercial natural human collagen, and there is no significant difference in the cell adhesion activities of 1703, 1703NT, and 1703M, indicating that 1703M after amino acid sequence mutation does not significantly change the cell adhesion activity of the pre-mutation sequence.

[0209] (5) Scratch assay to detect the cell migration activity of recombinant collagen

[0210] The method for detecting and analyzing the cell migration activity of recombinant collagen refers to Bobadilla, A., et al., In vitro cell migration quantification method for scratch assays. J R Soc Interface, 2019.16(151): p. 20180709. It was completed by the Functional Nanomaterials and Biomedical Detection Laboratory of the School of Pharmacy, Changzhou University.

[0211] Specific implementation method: Take the purified and freeze-dried products of 1703, 1703NT, and 1703M, control human collagen (Sigma, catalog number C7774), and bovine serum albumin (BSA, purchased from Sangon Biotech (Shanghai) Co., Ltd.) and dissolve them (in ultrapure water or 1M HCl solution). Use the UV protein quantification empirical formula: C (mg / mL) = 0.144 * (A215 - A225) to measure the protein concentration, and then dilute it to 0.5 mg / mL with serum-free DMEM culture medium (GIBCO, catalog number 12800017, pH 7.4) (after dilution, adjust its pH to be stable at 7.0 - 7.4). Normally culture and passage NIH / 3T3 cells (purchased from the Cell Bank of the Chinese Academy of Sciences, catalog number GNM6, and the culture and passage methods are carried out according to the cell instruction manual). Seed well-conditioned cells into a 6-well plate, and inoculate 2 mL of cell suspension at a density of 20,000 cells / mL per well, and culture for 36 h. Use a 200 μL pipette tip to make scratches, wash the cells 3 times with PBS to remove the scratched cells. Add the protein solution diluted with serum-free DMEM culture medium to the wells, and continue to culture in an incubator at 37 °C and 5% CO 2 2 incubator. Sample and take pictures at 0 h, 24 h, and 48 h. Use Image J software to process the pictures of cell migration to obtain the data of the initial scratch area and the cell-free blank area, and calculate: Migration rate = (1 - cell-free blank area / initial scratch area) * 100%, analyze the data and perform a significant difference analysis (SPSS 22 software, Duncan method, P < 0.05).

[0212] The in vitro cell migration experiment simulates the process of cell migration in vivo to a certain extent and directly reflects the interaction between cells and the extracellular matrix and between cells under the influence of the matrix. Cell migration activity is a more effective indicator to characterize the biological activity of collagen. The higher the migration rate and the faster the speed, the better the biological activity of collagen. As Figure 22 shown in the actual comparison pictures of cell migration taken at different times (the area between the two red lines is the initial and the scratched wound area after cell migration. In the lower right corner of each picture in the figure, the red horizontal line part is the scale, and the scale size is 100 μm for all pictures) and Figure 23 shown in the comparison of calculating the cell migration rate (Image J calculates the cell-free blank area), it can be seen that the cell migration activities of the purified and freeze-dried products of 1703, 1703NT, and 1703M are significantly better than those of natural human collagen, and the cell migration-promoting activity of 1703M is also better than that of 1703 and 1703NT.

Claims

1. A preparation method for improving the production level of recombinant humanized collagen type XVII and with low protein degradation, characterized in that, it comprises the following steps: (1) Inoculate the recombinant engineering bacteria into the seed culture medium and culture overnight to prepare a bacterial seed solution; the recombinant engineering bacteria are preserved in the General Microbiology Center of the China Microbial Culture Collection Management Committee, with the preservation numbers of CGMCC No. 21889 and CGMCC No. 21888; (2) Set the fermentation temperature and pH value, inoculate the bacterial seed solution into the fermentation medium, adjust the stirring speed, air flux, tank pressure, and DO value, culture until the carbon source is exhausted and the DO rapidly rebounds, then start to feed the feeding medium, and stop feeding the feeding medium when the OD600 value of the bacteria reaches a certain value; The components of the fermentation medium include NH 4 H 2 PO 4 11.9 g / L, KH 2 PO 4 2.515 g / L, CaSO 4 ·2H 2 O 0.295 g / L, K 2 SO 4 4.55 g / L, MgSO 4 ·7H 2 O 3.725 g / L, glycerol 20 g / L, PTM 1 0.45 mL / L; The pH value is set to 4.0; When the OD600 value of the bacteria is 50, stop feeding the feeding medium; (3) After the glycerol is exhausted and DO≥70%, start to feed the induction medium and enter the methanol induction stage, adjust the rotation speed, ventilation volume, tank pressure, and feeding speed to make DO≥30%; The components of the induction medium in step (3) include: pure methanol, 50% glycerol, and PTM 1; wherein the volume ratio of pure methanol to 50% glycerol is 8:2, and 12 mL of PTM is added per liter. 1 ; (4) After the induced fermentation ends, take the supernatant of the fermentation broth and detect the protein; The recombinant humanized collagen type XVII is a recombinant humanized collagen expressing the 15th helical region of collagen type XVII; The recombinant humanized collagen type XVII has the amino acid sequences shown in SEQ ID NO. 2 and SEQ ID NO.

4.

2. The preparation method according to claim 1, characterized in that, after the step (4), it further includes the step of purifying the fermentation product; the purification includes the steps of hydrophobic chromatography and cation exchange chromatography carried out in sequence.

3. The preparation method according to claim 2, characterized in that, the hydrophobic chromatography is balanced with buffer A, washed with buffer B to remove impurities, and eluted with buffer C; the cation exchange chromatography is balanced with buffer D and eluted with buffer E; Buffer A contains: 20 mM KH 2 PO 4 , 2 M ammonium sulfate, pH 5.0; Buffer B contains: 20 mM KH 2 PO 4 , 0.6 M ammonium sulfate, pH 5.0; Buffer C contains: 20 mM KH 2 PO 4 , pH 5.0; Buffer D contains: 20 mM tartaric acid, 100 mM sodium chloride, pH 4.0; Buffer E contains: 20 mM tartaric acid, 500 mM sodium chloride, pH 4.

0.

4. The preparation method according to claim 3, characterized in that, the purification steps include: (1) Hydrophobic chromatography: Prepare buffer A: 20 mM KH 2 PO 4 , 2 M ammonium sulfate, pH 5.0; buffer B: 20 mM KH 2 PO 4 , 0.6 M ammonium sulfate, pH 5.0; buffer C: 20 mM KH 2 PO 4 , pH 5.0; Collect the supernatant of the fermentation broth of recombinant humanized collagen type XVII, balance the hydrophobic chromatography medium with buffer A, after the sample loading is completed, rebalance with buffer A; then wash with buffer B to remove impurities and elute with buffer C, and start to collect the eluate 1; (2) Cation exchange chromatography: Prepare buffer D: 20 mM tartaric acid, 100 mM sodium chloride, pH 4.0; buffer E: 20 mM tartaric acid, 500 mM sodium chloride, pH 4.0; Balance the cation exchange chromatography medium with buffer D, load the eluate 1, after the sample loading is completed, rebalance with buffer D, elute with buffer E, collect the eluate 2, and ultrafilter and freeze-dry the eluate 2 to obtain the freeze-dried product of the purified protein.

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