WO2024087784A1

WO2024087784A1 - Recombinant type xvii humanized collagen expressed in yeast and preparation method therefor

Info

Publication number: WO2024087784A1
Application number: PCT/CN2023/111030
Authority: WO
Inventors: 王丽萍; 李佳佳; 钱晨明; 程鹏飞; 刘慧敏; 蒋雯雯; 魏雪花; 徐园; 钱松; 凡孝菊; 李海航
Original assignee: 江苏创健医疗科技股份有限公司
Priority date: 2022-10-26
Filing date: 2023-08-03
Publication date: 2024-05-02
Also published as: CN116375847A

Abstract

The present invention belongs to the technical field of genetic engineering and synthetic biology. Provided are a recombinant type XVII humanized collagen expressed in yeast and a preparation method therefor, and particularly provided are a recombinant humanized collagen that the 15th helical region of type XVII collagen is expressed, and a recombinant engineering bacterium and a preparation method therefor. Provided is a full-length sequence of the 15th helical region of type XVII collagen, and a mutant obtained by means of the mutation of amino acid residues on the basis of the sequence. The full-length sequence of the 15th helical region of type XVII collagen is expressed in Pichia pastoris for the first time, and a complete set of fermentation and purification processes are established to improve the protein expression level and reduce the degradation. It is verified by means of experiments that the obtained protein has biological activities such as a cell adhesion activity and cell-migration-promoting activity reaching or exceeding those of natural human collagen, can achieve the purpose of real product application of the product, and can be widely used in fields such as medicine, medical devices, biomaterials, tissue engineering and cosmetics.

Description

Yeast recombinant type XVII humanized collagen and preparation method thereof

Technical Field

The present invention relates to yeast recombinant humanized type XVII collagen and a preparation method thereof, in particular to a recombinant humanized collagen expressing the 15th helical region of type XVII collagen, a recombinant engineering bacterium and a preparation method thereof, and belongs to the technical field of genetic engineering and synthetic biology.

Background technique

Humans have 28 different collagens, among which human XVII collagen is a transmembrane non-fibroblastic collagen, a component of hemidesmosomes in cells, and plays an important role in the function of epithelial cells and basement membranes; it can regulate the adhesion, separation and developmental differentiation of epithelial cells, and plays an important role in the differentiation and regeneration of keratinocytes; it can maintain the activity of hair follicle stem cells and epidermal stem cells, and plays an important role in cell aging and skin differentiation. Human 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 helical regions and 15 triple helical regions according to whether it has a typical (Gly-X-Y)n amino acid repeating sequence and whether it can form a triple helical region. Among them, the 15th helical region is 242 amino acids in length, which is the longest triple helical region in XVII collagen and has the typical (Gly-X-Y)n amino acid repeating sequence characteristics. The current research on human collagen XVII mainly focuses on developmental biology, genetic research and disease mechanisms. The research on the structure and function of the protein itself is extremely limited. The research on the 15th helical region is the most recognized: existing studies have shown that it can bind to uncommon integrins α5β1 and αVβ1, has special integrin binding sites, has good cell adhesion activity to a variety of cells, and still maintains its biological activity in a single-chain state after heat denaturation. The 15th helical region is the most promising functional region in human collagen XVII.

Human collagen XVII is present in very low amounts in the human body and animals, and is difficult to extract. It cannot be obtained by treating animal tissues with traditional acid, alkali, or enzymatic methods. A small amount of extraction only meets the needs of scientific research, and cannot be mass-produced. There is no possibility of large-scale application, and there are inevitably immunogenicity and potential biosafety risks such as viruses and epidemics. The main way to solve such problems now is to obtain recombinant collagen through biotechnology such as genetic engineering. Among the existing recombinant protein expression systems: mammalian cell expression systems and insect cell (baculovirus) expression systems have high costs and low yields, and these two methods are generally not used for mass production and application of collagen. The recombinant protein expressed by the prokaryotic (Escherichia coli) system has no post-translational modification, and intracellular expression requires bacterial lysis, which will result in a large number of impurity proteins, and naturally carries endotoxins, peptidoglycans, and other pyrogenic substances as components of bacterial cell walls. As a eukaryotic microorganism, Pichia pastoris has complete organelles of eukaryotic cells, can perform certain post-translational modifications on recombinant proteins, and strongly supports the realization of protein biological functions. It has the advantages of large-scale fermentation industrial production such as high density, low cost, short cycle, and high expression of microbial expression system; recombinant proteins can be secreted outside the cell, without impurity proteins from bacterial lysis; cell wall components do not contain endotoxins and peptidoglycan. Pichia pastoris has a clear genetic background, and a variety of genetically engineered drugs and vaccines have been marketed. It is easy to obtain regulatory approval, and is the most ideal collagen expression system.

However, the Pichia pastoris expression system still has its disadvantages. The most important one is that the secreted exogenous recombinant proteins are often degraded by the protease system in Pichia pastoris, especially when the recombinant proteins with natural sequences (such as collagen) are expressed in high-density fermentation. The degradation is more serious. In response to this situation, researchers often modify the corresponding amino acid sequence of collagen to reduce degradation. However, the amino acid sequence of a protein is the cornerstone of all its properties, such as its physical and chemical properties and biological functions. Therefore, the amino acid sequence cannot only be used to reduce degradation, but also to ensure its physical and chemical properties. In addition, the amino acid sequence of proteins is the result of natural evolution over hundreds of millions of years. People's current understanding of collagen is still limited. Maintaining the stability of its amino acid sequence is a strategy with the least hidden dangers in the production and development of recombinant human collagen (which cannot be recognized at the current scientific level).

In existing studies, through prokaryotic (E. coli) expression, application number CN201911051106.3 uses a PET vector and an E. coli expression system to 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 helical region of human XVII collagen, and express them with three repeats of these two sequences (named 17A3 and 17B3 in the patent), but none of them covers the entire 15th helical region sequence. The premise of the application of recombinant collagen is that it can be fermented and purified on a large scale, with high density and high expression. Pichia pastoris is an ideal expression host. However, as a eukaryotic organism, when a large amount of heterologous proteins are expressed and secreted, that is, when the intracellular biological resources are occupied in large quantities, Pichia pastoris will inevitably regulate the expression of heterologous proteins accordingly, and its outstanding performance is actually the degradation of the expressed recombinant protein. This is more prominent in the amino acid sequence of natural collagen that has not been mutated or modified. For specific types of recombinant proteins, determining a complete set of fermentation and purification processes to reduce degradation is the key technology to obtain recombinant collagen with a complete natural amino acid sequence, especially for high-density, high-expression fermentation production and purification large-scale preparation and production.

Summary of the invention

The purpose of the present invention is to overcome some technical problems existing in the prior art, provide yeast recombinant type XVII humanized collagen, recombinant engineering bacteria and preparation methods thereof, and in particular, provide recombinant humanized collagen expressing the 15th helical region of type XVII collagen, recombinant engineering bacteria and preparation methods thereof.

To achieve the above object, the present invention adopts the following technical solutions:

The present invention first provides a recombinant type XVII humanized collagen, which includes an amino acid sequence that is 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% identical to positions 567-808 of SEQ ID NO.1, and maintains the biological activity of the 15th helical region of type XVII collagen.

In certain embodiments, the recombinant type XVII humanized collagen comprises an amino acid sequence that has 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% identity with SEQ ID NO.2 (1703NT), SEQ ID NO.4 (1703), SEQ ID NO.6 (1703MNT), or SEQ ID NO.8 (1703M) and maintains the biological activity of the 15th helical region of type XVII collagen.

In certain embodiments, the recombinant type XVII humanized 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.

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

In some embodiments, the polynucleotide includes 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.

The present invention also provides a vector, wherein the vector comprises the polynucleotide of the present invention.

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

In certain embodiments, the vector is pPIC9K.

The present invention also provides a host cell or a recombinant engineered bacterium, wherein the host cell or the recombinant engineered bacterium comprises the present invention. The nucleic acid or the vector of the present invention.

In certain embodiments, the host cell or recombinant engineered bacteria is a eukaryotic cell or a prokaryotic cell.

In certain embodiments, the host cell or recombinant engineered bacteria is Pichia pastoris engineered bacteria.

In certain embodiments, the Pichia pastoris engineered bacteria is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit numbers being CGMCC No. 21889, CGMCC No. 21888, CGMCC No. 21890, and CGMCC No. 21884.

The present invention also provides a composition, which includes the recombinant humanized type XVII collagen described in the present invention, or the polynucleotide described in the present invention, or the vector described in the present invention, or the host cell or recombinant engineered bacteria described in the present invention.

The present invention also provides a product, which includes the recombinant humanized type XVII collagen described in the present invention, or the polynucleotide described in the present invention, or the vector described in the present invention, or the host cell or recombinant engineered bacteria described in the present invention, or the composition described in the present invention; preferably, the product is selected from drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.

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

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

(1) inoculating the recombinant engineered bacteria into a seed culture medium and culturing overnight to prepare a seed solution;

(2) Setting the fermentation temperature and pH value, inoculating the bacterial seed solution into the fermentation medium, adjusting the stirring speed, air flux, tank pressure, and DO value, culturing until the carbon source is exhausted and the DO recovers rapidly, starting to feed the feed medium, and stopping the feeding of the feed medium when the bacterial OD ₆₀₀ value reaches a certain value;

(1) After the glycerol is exhausted and DO ≥ 70%, the induction medium is fed and the methanol induction stage is entered. The rotation speed, ventilation volume, tank pressure and flow acceleration are adjusted to make DO ≥ 30%;

(4) After the induction fermentation is completed, the supernatant of the fermentation liquid is taken and the protein is detected;

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

In certain embodiments, the seed culture medium in step (1) is YPG.

In certain embodiments, in step (2), the fermentation temperature is set at 25°C-35°C, preferably 30°C, and the pH is set at 3.5-6, preferably 4.0; the feed medium is 50% W/V glycerol, with 12 mL PTM ₁ added per liter.

In certain embodiments, in step (2), when the bacterial OD ₆₀₀ value is 50-150, preferably when the bacterial OD ₆₀₀ value is 50, the feeding of the feed medium is stopped.

In certain embodiments, the fermentation medium components in step (2) include: 11.9-47.6 g/L NH ₄ H ₂ PO ₄ , 2.515-10.06 g/L KH ₂ PO ₄ , 0.295-1.18 g/L CaSO ₄ ·2H ₂ O , 4.55-18.2 g/L K ₂ SO _{4 ,} 3.725-14.9 g/L MgSO ₄ ·7H ₂ O , 20 g/L glycerol, and 0.45 mL/L PTM ₁ . Preferably, the fermentation medium components include 11.9 g/L NH ₄ H ₂ PO ₄ , 2.515 g/L KH ₂ PO ₄ , 0.295 g/L CaSO ₄ ·2H ₂ O , 4.55 g/L K ₂ SO ₄ , 3.725-14.9 g/L MgSO ₄ ·7H ₂ O 3.725g/L, glycerol 20g/L, PTM ₁ 0.45mL/L.

In certain embodiments, the induction medium components 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 ₁ is added per liter; preferably, the volume ratio of pure methanol to 50% glycerol is 8:2.

In certain embodiments, step (4) further includes a step of purifying the fermentation product; preferably, the purification includes sequentially performing hydrophobic chromatography and cationic chromatography steps.

In certain embodiments, the hydrophobic chromatography uses buffer A for balancing, buffer B for washing, and buffer C for elution. The cationic chromatography uses buffer D for balancing and buffer E for elution; preferably, buffer A includes: 20mM KH ₂ PO ₄ , 2M ammonium sulfate, pH 5.0; buffer B includes: 20mM KH ₂ PO ₄ , 0.6M ammonium sulfate, pH 5.0; buffer C includes: 20mM KH ₂ PO ₄ , pH 5.0; buffer D includes: 20mM tartaric acid, 100mM sodium chloride, pH 4.0; buffer E includes: 20mM tartaric acid, 500mM sodium chloride, pH 4.0.

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

(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 fermentation supernatant of recombinant type XVII humanized collagen, balance the hydrophobic chromatography medium with buffer A, and re-balance with buffer A after loading; then wash with buffer B and elute with buffer C, and start collecting eluate 1;

(2) Cationic 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; use buffer D to balance the cationic chromatography medium, load the eluate 1, and after loading, rebalance with buffer D, elute with buffer E, collect eluate 2, and ultrafilter and freeze-dry the eluate 2 to obtain a purified protein freeze-dried product.

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

Beneficial effects of the present invention:

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

In view of the possible degradation of natural collagen sequences, the present invention mutated a few amino acid residues in the 15th helical region sequence 1703NT of human XVII collagen, and named it 1703MNT (the sequence is shown in SEQ ID NO.6). 1703MNT significantly reduces the degradation of natural proteins when expressed in Pichia pastoris, and its basic physical and chemical properties have not changed, and its biological activity has not decreased (even more excellent) through testing.

The 15th helical region of the human XVII collagen of the present invention will not produce immune rejection and allergic reactions when applied to the human body, and has biological activities such as cell adhesion activity and cell migration promotion activity that reach or even exceed those of natural human collagen, can achieve the purpose of true product application, and can be widely used in the fields of medicine, medical devices, biomaterials, tissue engineering, cosmetics, etc.

(2) The present invention uses the amino acid sequences of 1703NT and 1703MNT as the basis, calculates and optimizes the codon preference of the DNA sequence and the related optimization parameters in the transcription and translation process, and synthesizes a DNA sequence that is more suitable for efficient expression in Pichia pastoris. The present invention modifies the DNA sequences of 1703NT and 1703MNT, respectively adding 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, so that they contain a bispecific affinity purification tag, which can be purified by affinity chromatography and is also convenient for immunological antibody detection based on the two tag sequences, and are named 1703 (sequence as shown in SEQ ID NO.4) and 1703M (sequence as shown in SEQ ID NO.8). The exogenous DNA encoding 1703, 1703M, 1703NT, and 1703MNT was cloned into the expression vector pPIC9K to construct the recombinant expression vectors pPIC9K-1703, pPIC9K-1703M, pPIC9K-1703NT, and pPIC9K-1703MNT. The expression was further induced to screen the engineered bacteria with high expression levels.

(3) Based on the needs of high-density large-scale fermentation production (rather than the laboratory research stage), the present invention has established a complete set of methods and process flows for fermentation expression, extraction and purification of the natural full-length 15th helical region with low degradation in Pichia pastoris, which greatly reduces the degradation of the natural full-length 15th helical region while increasing the expression level, thereby obtaining the natural full-length 15th helical region recombinant collagen.

The present invention optimizes the fermentation medium formula, the most suitable fermentation pH during fermentation, the initial induction bacteria concentration _OD600 , the induction medium formula used in the mixed carbon source feed and other conditions, and establishes a complete set of fermentation processes. The collagen expression amount of the Pichia pastoris engineering strain (preservation number CGMCC No. 21888) expressing the recombinant XVII humanized collagen 1703 is increased from about 11 g/L to about 17 g/L, and the Pichia pastoris engineering strain (preservation number CGMCC No. 21889) expressing the recombinant XVII humanized collagen 1703NT can reach> 15 g/L. At the same time, protein degradation was significantly improved during the fermentation process, especially there was only one dominant electrophoresis band (with the highest optical density), and the 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 about by mutants such as 1703M that changed the amino acid sequence.

At the same time, the present invention establishes a two-step purification method, using a tartaric acid buffer system, the method is simple and effective, and high-purity recombinant type XVII humanized collagen 1703 and 1703NT can be obtained through hydrophobic chromatography and cationic chromatography. The purified 1703 and 1703NT freeze-dried products both show a single band in electrophoresis detection, and through liquid phase analysis and measurement (area normalization method), a single peak is obvious, and the purity is high, 1703 can reach 94%, and 1703NT can reach 95%.

The fermentation and purification process of the present invention is suitable for fermentation and purification in a high-density bioreactor and has the conditions for large-scale industrial production.

(3) The present invention verifies the biological activity of the recombinantly expressed 15th helical region through experiments. The 15th helical region recombinant collagen obtained by the present invention has good adhesion activity and cell migration promoting activity.

In the present invention, 1703 and 1703NT freeze-dried products were subjected to LC-MS detection, proving that their molecular weights were consistent with theoretical predictions. Sequencing verification of the N-terminus and C-terminus of the high-purity freeze-dried products of 1703 and 1703NT and protein full sequence analysis based on LC-MS/MS proved that their N-terminus and C-terminus were complete, and the amino acid sequences expressed by 1703 and 1703NT were correct. Infrared spectroscopy scanning of the high-purity freeze-dried products of 1703, 1703M, 1703NT, and 1703MNT showed that the wave numbers of their amide A, amide B, amide I, amide II, and amide III all met the structural characteristics of recombinant collagen.

In the present invention, in vitro cultured NIH/3T3 cells are used to carry out cell adhesion and cell migration experiments of 1703, 1703NT and 1703M. The experiments show that the cell adhesion activity of 1703, 1703NT and 1703M is significantly better than that of commercial natural human collagen, and there is no significant difference in the cell adhesion activity of 1703, 1703NT and 1703M, indicating that the cell adhesion activity of 1703M after the amino acid sequence mutation does not significantly change the cell adhesion activity of the sequence before the mutation; the cell migration promoting activity of the purified lyophilized products of 1703, 1703NT and 1703M is significantly better than that of natural human collagen, and the cell migration promoting activity of 1703M is better than that of 1703 and 1703NT.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the SDS-PAGE detection results of the bacterial supernatant of 1703, 1703MNT, 1703NT, and 1703MNT after 24 hours of induction expression.

Figure 2 is a WB image of the supernatant of the bacterial solution after 1703 and 1703M were induced for 24 hours. In the figure, the left image is anti-6×His Tag The right picture is the WB picture of the antibody. The right picture is the WB picture of the anti-Strep-Tag II antibody.

FIG3 is the mass spectrometry analysis result of the target band in the SDS-PAGE detection result of the bacterial supernatant after 1703 was induced to express for 24 hours.

FIG. 4 is the mass spectrometry analysis result of the target band in the SDS-PAGE detection result of the bacterial supernatant after 1703M induction expression for 24 hours.

FIG. 5 is an SDS-PAGE image of the fermentation supernatant obtained after 48 h of expression induction in different basal salt fermentation medium.

FIG. 6 is an SDS-PAGE image of the fermentation broth supernatant obtained after 48 h of induction expression under different pH conditions.

FIG. 7 is an SDS-PAGE image of the fermentation supernatant obtained after 48 h of induction expression at different initial OD ₆₀₀ values.

FIG8 is an SDS-PAGE image of the fermentation supernatant obtained after 48 h of expression induction in different induction media.

FIG. 9 is an SDS-PAGE image of the fermentation supernatant obtained after induced expression in the 1703 fermentation parallel experiment.

FIG. 10 is an SDS-PAGE image of the fermentation supernatant obtained after 1703NT induced expression.

FIG. 11 is the HPLC spectrum of the lyophilized products of 1703 (FIG. a) and 1703NT (FIG. b) after purification.

FIG. 12 is the SDS-PAGE electrophoresis of the lyophilized products of 1703 (FIG. a) and 1703NT (FIG. b) after purification.

FIG. 13 shows the deconvoluted molecular weight of the purified 1703 lyophilized sample.

FIG. 14 shows the deconvoluted molecular weight of the purified 1703NT lyophilized sample.

FIG15 is a secondary mass spectrum of the purified C-terminal peptide of the lyophilized sample 1703.

FIG. 16 is a secondary mass spectrum of the C-terminal peptide of the purified 1703NT freeze-dried sample.

FIG17 is an infrared spectrum scan of the purified lyophilized sample 1703.

FIG. 18 is an infrared spectrum scan of the purified 1703NT freeze-dried sample.

FIG. 19 is an infrared spectrum scan of the purified 1703M freeze-dried sample.

FIG. 20 is an infrared spectrum scan of the purified 1703MNT freeze-dried sample.

FIG. 21 shows the cell adhesion activity test results of 1703, 1703NT, 1703M, natural human collagen and BSA obtained in the present invention.

FIG. 22 is a comparison diagram of the cell migration status of 1703, 1703NT, 1703M obtained by the present invention and natural human collagen and BSA.

FIG. 23 shows the cell migration rates of 1703, 1703NT, 1703M, natural human collagen and BSA after culturing NIH/3T3 cells for 24 h and 48 h.

Detailed ways

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

In the embodiments of the present invention, those that are not described in detail are all completed by conventional experimental methods. Those processes involved in the embodiments that are not described in detail are all understandable and easily implemented by those skilled in the art based on the product instructions or basic knowledge in the field, and therefore are not described in detail again.

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

(1) Amino acid sequence design and DNA sequence optimization

Select human XVII collagen sequence for optimization, specific sequence reference:

Uniprot Q9UMD9-1 sequence (https://www.uniprot.org/uniprot/Q9UMD9) and NCBI reference sequence Q9UMD9.3 (https://www.ncbi.nlm.nih.gov/protein/Q9UMD9.3) are identical. As shown in SEQ ID NO.1:

SEQ ID NO.1:

The 15th helical region of human type XVII collagen expressed in the present invention is the selected sequence of the bold underlined portion in SEQ ID NO.1. This recombinant type XVII humanized collagen, i.e., the recombinant humanized collagen expressing the 15th helical region of type XVII collagen, is named 1703NT, with a total of 242 amino acids, and its amino acid sequence is shown in SEQ ID NO.2:

The DNA sequence encoding 1703NT was codon optimized using Pichia pastoris as the host. The optimized sequence is shown in SEQ As shown in ID NO.3:

A Strep-Tag II tag was added to the amino terminus of SEQ ID NO.2, and a 6×His Tag tag was added to the carboxyl terminus. The sequence after adding the tags had 260 amino acids in total. This recombinant humanized collagen type XVII, i.e., the recombinant humanized collagen expressing the 15th helical region of type XVII collagen, was named 1703, and its amino acid sequence (the underlined part is the tag sequence) is shown in SEQ ID NO.4:

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

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

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

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

The DNA sequence corresponding to 1703M is as follows, such as SEQ ID NO.9 (the underlined part is the sequence corresponding to the tag):

(2) Synthesis of DNA sequences and construction of recombinant expression vectors

Primers P1 and P2 were designed using the expression vector pPIC9K-col17a1 in the patent CN113185604B that the inventor has authorized as a template to amplify the 15th helical region sequence of type XVII collagen. After the PCR product was purified, it was double-digested with EcoRI and NotI. After the digestion was completed, the target band was recovered by cutting the gel. The plasmid pPIC9K was double-digested with EcoRI and NotI, and the digested plasmid was recovered by column. The target fragment recovered by digestion and the plasmid were mixed at a molecular weight ratio of 3:1 and ligated using Takara's DNA Ligation Kit. The ligation product was transformed into DH5α, and the transformants grown overnight were subjected to colony PCR using universal primers 5'AOX and 3'AOX as primers. Five transformants were randomly selected from the positive clones for plasmid extraction and sequencing. The results were consistent with expectations and the plasmid was named pPIC9K-1703.

The P1 sequence is shown in SEQ ID NO.10:

(EcoRI)

The P2 sequence is shown in SEQ ID NO.11:

The 5'AOX sequence is shown in SEQ ID NO.12: 5'-GACTGGTTCCAATTGACAAGC-3'

The 3'AOX sequence is shown in SEQ ID NO.13: 5'-GGCAAATGGCATTCTGACAT-3'

Nanjing GenScript Biotech Co., Ltd. was commissioned to synthesize SEQ ID NO.9 and cloned into the EcoRI and NotI restriction sites of pPIC9K to obtain the expression plasmid pPIC9K-1703M. Nanjing GenScript Biotech Co., Ltd. was commissioned to construct a subclone that removed the tag DNA sequences at both ends of 1703 and 1703M, with the cloning sites being EcoRI and NotI of pPIC9K to obtain the expression plasmids pPIC9K-1703NT and pPIC9K-1703MNT.

(3) Construction of recombinant engineering strains and screening of strains

10 μg of the above-mentioned recombinant expression vector plasmids (pPIC9K-1703, pPIC9K-1703NT, pPIC9K-1703MNT and pPIC9K-1703M) were respectively digested with SacⅠ (purchased from Dalian TaKaRa Company, the specific operation was carried out according to the kit instructions) at 37°C overnight to linearize them, 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.

The linearized plasmid was electrotransformed into the competent cells of the empty host strain Pichia pastoris GS115 (purchased from China Industrial Microbiological Culture Collection Center), and the electrotransformed bacterial solution was spread on the MD plate, with 100 μL to 200 μL spread on one plate, and allowed to stand at room temperature for 10 min. Inverted culture was performed at 30°C for 2-5 days until a single colony (positive transformant) appeared.

Add 2 mL of sterile double distilled water to the surface of the MD plate, then use a sterile triangular applicator to gently scrape off the His ⁺ transformants on the surface of the plate and transfer them to a 50 mL centrifuge tube. Dilute the bacterial suspension with sterile double distilled water, spread 10 ⁵ cells on a YPD plate containing 0.5 mg/mL G418, invert, and culture at 30°C for 3 to 4 days until a single colony appears. Pick colonies from the YPD plate to a sterile 96-well plate (200 μL YPD/well), mix, and culture at 30°C for 48 hours; mix the bacterial solution in the well, take 10 μL each and inoculate it into a new sterile 96-well plate, culture at 30°C for 24 hours, and repeat this operation again; after 24 hours, 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, and continue to culture at 30°C for 96 hours to 120 hours. If the Pichia transformant can grow on a plate containing high concentration of G418, it means that the transformant contains multiple copies of the target gene, that is, multiple recombinant fragments have entered the yeast and integrated into the yeast chromosome through homologous recombination. This step of screening can obtain high-copy, highly efficient recombinant yeast engineering strains.

The four engineered bacteria samples constructed were sent to the General Microbiology Center of China Microbiological Culture Collection Administration for preservation. The corresponding strain preservation numbers are:

The strain expressing protein 1703M has the deposit number: CGMCC No.21884;

The strain expressing protein 1703 has the deposit number: CGMCC No. 21888;

The strain expressing protein 1703NT has the deposit number: CGMCC No.21889;

The strain expressing protein 1703MNT has the deposit number: CGMCC No.21890.

The deposit addresses are all: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is all: March 11, 2021. The classification name is all: Pichia pastoris.

(4) Induced expression and identification of recombinant collagen

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 OD ₆₀₀ was 2-6 (16-18 h). The cells were collected by centrifugation at 1500-3000 g for 5 min at room temperature, and the cells were resuspended in BMMY medium to an OD ₆₀₀ of about 2. The cells were placed on a shaker at 28-30°C and 220 rpm and continued to grow for 3 days. 100% methanol was added to the culture medium every 24 h to a final concentration of 1.0%. Take bacterial samples at different time points (sampling once every 24 hours after induction), the sample volume is 1 mL, placed in a 1.5 mL EP tube, centrifuged at 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), place in a 100°C metal bath for heating for 10 min, and perform SDS-PAGE detection. Because 1703 and 1703M have Srtep-TagⅡ tags at the amino terminus and 6×His tags at the carboxyl terminus, Western Blot detection can be performed with anti-Srtep-TagⅡ and anti-6×His Tag antibodies (purchased from Nanjing GenScript Biotech Co., Ltd.) (refer to the instructions for specific operations).

As shown in Figure 1, 1703 and 1703M can be efficiently secreted and expressed in the extracellular culture supernatant. The theoretical molecular weights of 1703 and 1703M are 24968.16Da and 24756.88Da respectively; the theoretical molecular weights of 1703NT and 1703MNT are 22566.54Da and 22355.26Da respectively, and the apparent molecular weight is about 32kDa. From the electrophoresis diagram, we can see:

(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 one is the degradation band with a smaller molecular weight and a larger proportion (measured by Image Lab software, accounting for 40.2%). The proportions of the two are close;

(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 mutations performed in the present invention can achieve the purpose of reducing the degradation of amino acid sequences.

As can be seen from Figure 2 (ECL chemiluminescence color development, the fully automatic chemiluminescence image analysis system Tanon 5200 synthesizes the protein molecular weight standard into the image), the amino-terminal Srtep-TagⅡ tag and the carboxyl-terminal 6×His tag of 1703 and 1703M can be detected, and the largest 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.

As can be seen from Figures 1 and 2, the variant 1703M after the amino acid sequence was modified was more stable and protein degradation was alleviated.

The expected bands of 1703 and 1703M on SDS-PAGE were cut off 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 shown in Figures 3 and 4: the peptides detected after 1703 and 1703M were digested belonged to the relevant regions of the human XVII collagen sequence selected during the amino acid sequence selection design, indicating that the collagen of the present invention was successfully expressed.

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

In a specific embodiment of the present invention, the strain used is Pichia pastoris expressing proteins 1703 and 1703NT, with the preservation numbers 1703: CGMCC No.21888 and 1703NT: CGMCC No.21889, the preservation date is March 11, 2021, and the preservation unit is the General Microbiology Center of China Culture Collection Administration.

The ingredients of conventional culture media and solution formulas are shown below. Unless otherwise specified, only the abbreviation will be written in the following text, and the formula will not be described in detail. Unless otherwise specified for the manufacturer and reagent grade, the components in the formula can be domestic analytical grade or chemical grade.

YPG medium: yeast extract 10 g/L, protein 20 g/L, anhydrous glycerol 10 g/L.

PTM ₁ : CuSO ₄ ·5H ₂ O 6 g/L, MnSO ₄ ·H ₂ O 3 g/L, NaCI 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, sterilized by filtration with a 0.22 μm sterile filter membrane and stored at 4°C.

Alkali solution formula: 300ml of concentrated ammonia water, add 700ml of sterile purified water and mix well.

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

(1) Optimization of fermentation medium formula

The classic BSM medium formula (Invitrogen) contains 85% H ₃ PO ₄ and KOH, which is not convenient for operation in actual production. The present invention explores and optimizes the basic medium formula according to the type of protein expressed, and determines the fermentation medium prepared by the exclusive basic salt. The strain is an engineered strain of Pichia pastoris expressing protein 1703, and the deposit number is CGMCC No.21888.

The general culture medium used in fermenters 1# to 4# in this section is as follows: (1) Seed culture medium, YPG; (2) Induction culture medium: pure methanol, add 12 mL PTM ₁ per liter; (3) Feed culture medium: 50% W/V glycerol, autoclaved, add 12 mL PTM ₁ per liter in the sterile solution.

1# fermentation tank: use fermentation medium A, formula: NH ₄ H ₂ PO ₄ 47.6g/L, KH ₂ PO ₄ 10.06g/L, CaSO ₄ ·2H ₂ O 1.18g/L, K ₂ SO ₄ 18.2g/L, MgSO ₄ ·7H ₂ O 14.9g/L, glycerol 20g/L, PTM ₁ 0.45mL/L. After removing the ingredients other than PTM ₁ , sterilize with high temperature and wet heat, add PTM ₁ when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water.

Fermentation tank #2: Use fermentation medium B, formula: NH ₄ H ₂ PO ₄ 35.7g/L, KH ₂ PO ₄ 7.545g/L, CaSO ₄ ·2H ₂ O 0.885g/L, K ₂ SO ₄ 13.65g/L, MgSO ₄ ·7H ₂ O 11.175g/L, glycerol 20g/L, PTM ₁ 0.45mL/L. After removing the ingredients other than PTM ₁ , sterilize with high temperature and wet heat, add PTM ₁ when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water.

3# fermentation tank: use fermentation medium C, formula: NH ₄ H ₂ PO ₄ 23.8g/L, KH ₂ PO ₄ 5.03g/L, CaSO ₄ ·2H ₂ O 0.59g/L, K ₂ SO ₄ 9.1g/L, MgSO ₄ ·7H ₂ O 7.45g/L, glycerol 20g/L, PTM ₁ 0.45mL/L. After removing the ingredients other than PTM ₁ , sterilize with high temperature and wet heat, add PTM ₁ when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water.

Fermentation tank #4: Use fermentation medium D, formula: NH ₄ H ₂ PO ₄ 11.9g/L, KH ₂ PO ₄ 2.515g/L, CaSO ₄ ·2H ₂ O 0.295g/L, K ₂ SO ₄ 4.55g/L, MgSO ₄ ·7H ₂ O 3.725g/L, glycerol 20g/L, PTM ₁ 0.45mL/L. After removing the ingredients other than PTM ₁ , sterilize with high temperature and wet heat, add PTM ₁ when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water.

The fermentation process of 1#~4# fermentation tanks was controlled in the same way: the strain was inoculated into the seed culture medium YPG, and cultured overnight at 30℃ and 220rpm to prepare the strain solution. The fermentation temperature was set at 30℃ and pH5.0. The strain solution was added to a 5L fermentation tank (Baoxing Biotechnology) containing 3L fermentation medium at a 10% inoculation rate, and the stirring speed was adjusted to 300r/min-700r/min, the air flux was 2VVM, the tank pressure was 0-0.05MPa, and DO was ≥30%. The culture was cultured until the carbon source was exhausted and DO recovered rapidly. The feed medium was started to be fed until the bacterial OD ₆₀₀ =150 and the wet weight was 200g/L, and the feed medium was stopped. After the glycerol was exhausted and DO was ≥70%, the induction medium was started to be fed, and the methanol induction stage was entered. The speed, ventilation volume, tank pressure and flow acceleration were adjusted to make DO ≥30%. Samples were taken every 4h to measure OD ₆₀₀ , wet weight and UV protein content. After 48 hours of induction, the fermentation was terminated, the fermentation broth was collected and centrifuged at 7000 rpm for 20 minutes, the supernatant of the fermentation broth was taken, the UV protein content was detected, and SDS-PAGE electrophoresis was performed.

After UV detection, the final recombinant collagen concentration in the fermentation supernatant was: 1#, 9.99g/L; 2#, 10.17g/L; 3#, 10.75g/L; 4#, 11.30g/L. The SDS-PAGE electrophoresis of the fermentation supernatant is shown in Figure 5. The recombinant collagen concentration combined with the SDS-PAGE electrophoresis shows that the expression level of fermentation medium D is higher, so fermentation medium D is selected as the fermentation medium. However, it can be seen from the SDS-PAGE electrophoresis that there are two main bands (with the largest optical density value) in each lane. The larger molecular weight is the full-length band (accounting for about 40%) and the smaller molecular weight is the degradation band, which accounts for a large proportion (about 40%). The optimization of the simple culture medium can obtain a higher yield, but protein degradation still occurs, so the subsequent process optimization will continue on this basis.

(2) Optimize the fermentation pH process to reduce the degradation of recombinant collagen during fermentation

The general culture medium used in fermentation tanks 1# to 4# in this section is as follows: (1) Seed culture medium, YPG; (2) Induction culture medium: pure methanol, 12 mL PTM ₁ per liter; (3) Feed culture medium: 50% W/V glycerol, sterilized by high pressure and heat, 12 mL PTM ₁ per liter of sterile solution; (4) Fermentation culture medium, fermentation culture 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. The strain is an engineering strain of Pichia pastoris expressing recombinant collagen 1703, and its deposit number is CGMCC No. 21888.

1# Fermentation tank: Use fermentation medium D, remove the ingredients other than PTM ₁ , sterilize with high temperature and wet heat, add PTM ₁ when the temperature drops to room temperature, and adjust the pH to 6.0 with ammonia water. Set the fermentation temperature to 30℃ and pH 6.0.

2# Fermentation tank: Use fermentation medium D, remove the ingredients other than PTM ₁ , sterilize with high temperature and moist heat, add PTM ₁ when the temperature drops to room temperature, and adjust the pH to 5.0 with ammonia water. Set the fermentation temperature to 30℃ and pH 5.0.

3# Fermentation tank: Use fermentation medium D, remove the ingredients other than PTM ₁ , sterilize with high temperature and wet heat, add PTM ₁ when the temperature drops to room temperature, and adjust the pH to 4.0 with ammonia water. Set the fermentation temperature to 30℃ and pH 4.0.

4# Fermentation tank: Use fermentation medium D, remove the ingredients other than PTM ₁ , sterilize with high temperature and moist heat, add PTM ₁ when the temperature drops to room temperature, and adjust the pH to 3.5 with ammonia water. Set the fermentation temperature to 30℃ and pH 3.5.

The fermentation process of 1#~4# fermentation tanks is basically controlled in the same way: the bacteria are inoculated into the seed culture medium YPG, and cultured overnight at 30℃ and 220rpm to prepare the bacterial liquid. The fermentation temperature is set at 30℃ (the pH value of 1#~4# fermentation tanks is different). The bacterial liquid is added to a 5L fermentation tank (Baoxing Biology) containing 3L fermentation medium D (the pH value of the culture medium in 1#~4# fermentation tanks is different) at a 10% inoculation amount, and the stirring speed is adjusted to 300r/min-700r/min, the air flux is 2VVM, the tank pressure is 0-0.05MPa, and DO≥30%. Cultivate until the carbon source is exhausted and DO recovers rapidly, and start to feed the supplementary medium until the bacterial OD ₆₀₀ =150 and the wet weight is 200g/L, and stop feeding the supplementary medium. After the glycerol is exhausted and DO≥70%, start to feed the induction medium, enter the methanol induction stage, and adjust the speed, ventilation volume, tank pressure and flow acceleration to make DO≥30%. Samples were taken every 4 hours to determine OD ₆₀₀ , wet weight and UV protein content. After 48 hours of induction, the fermentation was terminated, the fermentation broth was collected and centrifuged at 7000 rpm for 20 minutes, the supernatant of the fermentation broth was taken, the UV protein content was detected, and SDS-PAGE electrophoresis was performed.

After UV detection, the final recombinant collagen concentration in the fermentation supernatant was: 1#, 9.70g/L; 2#, 12.00g/L; 3#, 12.50g/L; 4#, 9.50g/L. The SDS-PAGE electrophoresis of the fermentation supernatant is shown in Figure 6. Maintaining pH 4.0 during fermentation in the 3# fermenter can effectively reduce the degradation of the target protein. At the same time, the protein content detected by UV is the highest, indicating that pH 4.0 is the most suitable fermentation pH during fermentation. At the same time, it can be found that in the electrophoresis lane of the 3# fermenter, there is only one main electrophoretic band (with the highest optical density), and the 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 has played a role in inhibiting the degradation of recombinant collagen.

(3) Optimization of induction initial _OD600 value process

The bacterial concentration value _OD600 at the beginning of induction directly affects the final expression amount of subsequent protein.

The general culture medium used in fermentation tanks 1# to 3# in this section is as follows: (1) Seed culture medium, YPG; (2) Induction culture medium: pure methanol, 12 mL PTM ₁ is added per liter; (3) Feed culture medium: 50% W/V glycerol, sterilized by high pressure wet heat, 12 mL PTM ₁ is added per liter of sterile solution; (4) Fermentation culture medium, yeast culture 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 removing the ingredients other than PTM ₁ , it is sterilized by high pressure wet heat, and PTM ₁ is added when the temperature drops to room temperature, and the pH is adjusted to 4.0 with ammonia water. The fermentation temperature was set at 30°C and pH 4.0. The strain was an engineered strain of Pichia pastoris expressing protein 1703, with a deposit number of CGMCC No.21888.

1# Fermentation tank: set the fermentation temperature to 30℃ and pH 4.0. Add the bacterial liquid to a 5L fermentation tank (Baoxing Biotechnology) containing 3L fermentation medium D at a 10% inoculation rate, adjust the stirring speed to 300r/min-700r/min, the air flux to 2VVM, the tank pressure to 0-0.05MPa, and the DO≥30%. Cultivate until the carbon source is exhausted and the DO recovers rapidly. Start to feed the supplementary medium until the initial OD ₆₀₀ of bacterial induction is 50 (wet weight 74g/L), and stop feeding the supplementary medium. After the glycerol is exhausted and DO≥70%, start to feed the induction medium, enter the methanol induction stage, and adjust the speed, ventilation volume, tank pressure and flow acceleration to make DO≥30%. Take samples every 4h, and measure OD ₆₀₀ , wet weight and UV protein content. After 48h of induction, end the fermentation, collect the fermentation liquid in the tank and centrifuge it at 7000rpm for 20 minutes, take the fermentation liquid supernatant, detect the UV protein content, and perform SDS-PAGE electrophoresis.

2# fermenter: set the fermentation temperature to 30℃ and pH 4.0. Add the bacterial liquid to a 5L fermenter (Baoxing Biotechnology) containing 3L fermentation medium D at a 10% inoculum, adjust the stirring speed to 300r/min-700r/min, the air flux to 2VVM, the tank pressure to 0-0.05MPa, and the DO≥30%. Cultivate until the carbon source is exhausted and the DO recovers rapidly. Start to feed the feed medium until the initial OD ₆₀₀ of bacterial induction is 100 (wet weight 121g/L), and stop feeding the feed medium. After the glycerol is exhausted and DO≥70%, start to feed the induction medium, enter the methanol induction stage, and adjust the speed, ventilation volume, tank pressure and flow acceleration to make DO≥30%. Take samples every 4h, and measure OD ₆₀₀ , wet weight and UV protein content. After 48h of induction, end the fermentation, collect the fermentation liquid in the tank and centrifuge it at 7000rpm for 20 minutes, take the fermentation liquid supernatant, detect the UV protein content, and perform SDS-PAGE electrophoresis.

3# Fermentation tank: set the fermentation temperature to 30℃ and pH 4.0. Add the bacterial liquid to a 5L fermentation tank (Baoxing Biotechnology) containing fermentation medium D at a 10% inoculation rate, adjust the stirring speed to 300r/min-700r/min, the air flux to 2VVM, the tank pressure to 0-0.05MPa, and the DO≥30%. Cultivate until the carbon source is exhausted and the DO recovers rapidly. Start to feed the supplementary medium until the initial OD ₆₀₀ of bacterial induction is 150 (wet weight 203g/L), and stop feeding the supplementary medium. After the glycerol is exhausted and DO≥70%, start to feed the induction medium, enter the methanol induction stage, and adjust the speed, ventilation volume, tank pressure and flow acceleration to make DO≥30%. Take samples every 4h, and measure OD ₆₀₀ , wet weight and UV protein content. After 48h of induction, end the fermentation, collect the fermentation liquid in the tank and centrifuge it at 7000rpm for 20 minutes, take the fermentation liquid supernatant, detect the UV protein content, and perform SDS-PAGE electrophoresis.

After UV detection, the final recombinant collagen concentration in the fermentation supernatant was: 1#, 14.60 g/L; 2#, 13.00 g/L; 3#, 12.30 g/L. The SDS-PAGE electrophoresis of the fermentation supernatant is shown in Figure 7. In the electrophoresis lane, there is only one main electrophoresis band (maximum optical density). The protein expression level of the initial OD ₆₀₀ = 50 induced by fermentation in the 1# fermenter is significantly higher than that of OD ₆₀₀ = 100 and OD ₆₀₀ = 150, and OD ₆₀₀ = 50 is the optimal initial OD ₆₀₀ for induction.

(4) Mixed carbon source feeding and optimization of induction medium

Methanol in the induction medium can effectively induce the expression of recombinant collagen, but using methanol as the main component of the induction medium to provide a carbon source for Pichia may inhibit the growth of cells. Adding a certain amount of glycerol or other carbon sources can effectively promote the expression of recombinant collagen during fermentation. This section optimizes the components of the induction medium and performs mixed carbon source addition. The strain is an engineered strain of Pichia pastoris expressing protein 1703, and the deposit number is CGMCC No. 21888.

The universal culture medium used in fermentation tanks 1# to 4# in this section is as follows: (1) Seed culture medium, YPG; (2) Feed culture medium: 50% W/V glycerol, sterilized by high pressure wet heat, 12 mL PTM ₁ is added to each liter of the sterile solution; (3) Fermentation culture medium, yeast culture 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 O3.725 g/L, glycerol 20 g/L, PTM ₁ 0.45 mL/L, after removing the ingredients other than PTM ₁ and sterilizing by high pressure wet heat, PTM ₁ is added when the temperature drops to room temperature, and the pH is adjusted to 4.0 with aqueous ammonia.

In the 1# fermentation tank, induction medium A was used, pure methanol: 50% glycerol (sterile) = 7:3, and 12 mL PTM ₁ was added per liter.

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

In the fermentation tank #3, the induction medium C was used, pure methanol: 50% glycerol (sterile) = 8:2, and 12 mL PTM ₁ was added per liter.

In the 4# fermentation tank, the induction medium D was used, pure methanol: 50% glycerol (sterile) = 10:0, and 12 mL PTM ₁ was added per liter.

The fermentation process of 1#~4# fermenters was basically controlled in the same way: the strain was inoculated into the seed culture medium YPG, and cultured overnight at 30℃ and 220rpm to prepare the strain solution. The fermentation temperature was set at 30℃ and pH 4.0. The strain solution was added to a 5L fermenter containing fermentation medium D (Baoxing Biology) at a 10% inoculation amount, and the stirring speed was adjusted to 300r/min-700r/min, the air flux was 2VVM, the tank pressure was 0-0.05MPa, and DO was ≥30%. The culture was cultured until the carbon source was exhausted and DO recovered rapidly. The feed medium was started to be fed until the bacterial OD ₆₀₀ =50 and the wet weight was 74g/L, and the feed medium was stopped. After the glycerol was exhausted and DO was ≥70%, the induction medium was started (the induction medium of 1#~4# fermenters was different), and the methanol induction stage was entered. The speed, ventilation volume, tank pressure and flow acceleration were adjusted to make DO ≥30%. Samples were taken every 4 hours to measure OD ₆₀₀ , wet weight and UV protein content. After 48 hours of induction, the fermentation was terminated, the fermentation broth was collected and centrifuged at 7000 rpm for 20 minutes, the supernatant of the fermentation broth was taken, the UV protein content was detected, and SDS-PAGE electrophoresis was performed.

After UV detection, the final recombinant collagen concentration in the fermentation broth supernatant was: 1#, 12.30g/L; 2#, 15.90g/L; 3#, 16.70g/L; 4#, 14.30g/L. The SDS-PAGE electrophoresis of the fermentation broth supernatant is shown in Figure 8. In the electrophoresis lane, there is only one main electrophoresis band (with the highest optical density). When the induction medium of the 3# fermenter is pure methanol: 50% glycerol = 8:2, the protein expression is the highest.

(5) Validation of optimized fermentation process

The optimized and stabilized fermentation process is a complete set of processes, including several key points:

a. The fermentation medium used was 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, the ingredients other than PTM ₁ were prepared and sterilized by high temperature and moist heat, and PTM ₁ was added when the temperature dropped to room temperature, and the pH was adjusted to 4.0 with aqueous ammonia;

b. pH 4.0 is the most suitable fermentation pH;

c. Initial bacterial concentration of induction OD ₆₀₀ = 50;

d. The induction medium used for the mixed carbon source flow addition is the induction medium C: pure methanol: 50% glycerol (sterile) = 8:2, and 12 mL PTM ₁ is added per liter;

The rest are general culture media: seed culture medium (YPG), feed culture medium (50% W/V glycerol, sterilized by high pressure moist heat, 12 mL PTM ₁ per liter of sterile solution).

The fermentation process is as follows: the strain is inoculated into the seed culture medium YPG, and cultured overnight at 30°C and 220rpm to prepare the strain solution. The fermentation temperature is set at 30°C and pH 4.0. The strain solution is added to a 5L fermenter (Baoxing Biological) containing the fermentation medium at a 10% inoculation amount, and the stirring speed is adjusted to 300r/min-700r/min, the air flux is 2VVM, the tank pressure is 0-0.05MPa, and the DO is ≥30%. Culture until the carbon source is exhausted and the DO recovers rapidly, and the feed medium is started to be fed until the bacterial OD ₆₀₀ =50 and the wet weight is 74g/L, and the feed medium is stopped. After the glycerol is exhausted and DO ≥70%, the induction medium is started to be fed, and the methanol induction stage is entered. The speed, ventilation volume, tank pressure and flow acceleration are adjusted to make DO ≥30%. Samples are taken every 4h to measure OD ₆₀₀ , wet weight and UV protein content. After 48 hours of induction, the fermentation was terminated, the fermentation broth was collected and centrifuged at 7000 rpm for 20 minutes, the supernatant of the fermentation broth was taken, the UV protein content was detected, and SDS-PAGE electrophoresis was performed.

According to the above fermentation conditions, multiple batches of parallel experiments were carried out in fermentation tanks using the protein expression 1703 Pichia pastoris engineered strain (CGMCC No. 21888). At the end of the fermentation, there was no significant difference in the relevant parameters, and the protein expression level was It is stabilized at >16 g/L, while achieving the goal of low degradation of recombinant collagen, as shown in Table 1 and Figure 9 below.

Table 1. Results of parallel experiments in multiple batches of fermenters according to the fermentation conditions in this section

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

After a series of fermentation process optimizations, the collagen expression of the Pichia pastoris engineering strain (CGMCC No. 21888) expressing protein 1703 in a 5L tank increased from about 11g/L to about 17g/L, and the Pichia pastoris engineering strain (CGMCC No. 21889) expressing protein 1703NT could reach >15g/L. At the same time, protein degradation was significantly improved during the fermentation process, especially the main dominant electrophoresis band (with the highest optical density) was only one, and the 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.

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

The only difference between proteins 1703 and 1703NT in their amino acid sequences is the tag sequences at the N-terminus and C-terminus. The amino acid sequences of the recombinant humanized collagen as the main part are 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 be combined with the corresponding affinity chromatography medium).

(1) Hydrophobic chromatography

Each buffer solution is prepared with deionized water:

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.

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 tube: Lisui Technology Co., Ltd., XK50/30. Hydrophobic filler: Capto Phenyl, loaded on AKTA pure 150M) until the A215 absorbance value dropped to 30 Mau and the conductivity was maintained at about 192 ms/cm. The flow rate was set to 20 mL/min for loading, and the loading volume was 30 0mL/time, after loading, set the flow rate to 30mL/min, and rebalance with buffer A until the A215 absorbance value drops to 30Mau and the conductivity remains at about 192ms/cm. Under the condition of constant flow rate, use buffer B for washing (0.45μm filter membrane filtration) until the A215 absorbance value drops to 30Mau, set buffer C for elution, open the sample collector when the A215 absorbance value rises, 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.

(2) Cationic chromatography

Each buffer solution is prepared with deionized water:

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.

Set a flow rate of 30 mL/min to balance the cationic chromatography medium (column: Lisui Technology Co., Ltd., XK50/30. Cationic filler: SP Sepharose Fast Flow, loaded on AKTA pure 150M) with buffer D (0.45 μm filter membrane) until the A215 absorbance value drops to 30 Mau and the conductivity remains at around 11 ms/cm. Take out eluent 1 and adjust the pH to 4.0. Set a flow rate of 20 mL/min to load eluent 1. After loading, set a flow rate of 30 mL/min and rebalance with buffer D until the A215 absorbance value drops to 30 Mau and the conductivity remains at around 11 ms/cm. Elute with buffer E while keeping the flow rate unchanged. When the A215 absorbance value rises, open the sample collection valve and start collecting eluent 2 until the A215 absorbance value drops to 100 Mau, then stop collecting. The eluate 2 was ultrafiltered and freeze-dried (ultrafiltration equipment: AKTA Flux, AKTA Bioengineering Technology Co., Ltd.), and the freeze-dried product was finally collected.

Dissolve the lyophilized products of 1703 and 1703NT in ultrapure water to 2 mg/mL, filter through a 0.22 μm filter membrane, inject 10 μL (Sepax Bio-C18 column, HPLC: Waters 2695 or Agilent LC 1260), and analyze the purity.

Take the lyophilized products of 1703 and 1703NT and dissolve them in ultrapure water to 1 mg/mL, filter them through a 0.22 μm filter membrane, inject 5 μL, and detect by SDS-PAGE electrophoresis.

The purification results are shown in Figure 11. The purified lyophilized products of 1703 (Figure 11a) and 1703NT (Figure 11b) have a single peak after liquid phase analysis and measurement (area normalization method), and the purity is high, 1703 can reach 94%, and 1703NT can reach 95%. The electrophoresis diagram of Figure 12 shows that the purified lyophilized products of 1703 (Figure 12a) and 1703NT (Figure 12b) both show a single band.

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 cationic chromatography.

Example 4. Characterization of properties and detection of biological activity of recombinant humanized type XVII collagen 1703 and 1703NT

(1) Molecular weight detection

The theoretically predicted molecular weight of protein 1703 is 24968.16Da, and the theoretically predicted molecular weight of protein 1703NT is 22566.54Da. The high-purity lyophilized products of 1703 and 1703NT were subjected to LC-MS analysis (capillary high-performance liquid chromatograph Thermo Fisher Scientific Ultimate 3000, electrospray-quadrupole time-of-flight mass spectrometer AB SCIEX TripleTOF 5600 Mass Spectrometer, chromatographic column ACQUITY UPLC Protein BEH C4 Column) to obtain their deconvoluted molecular weights, and the detection was commissioned to Beijing Biotech Biotechnology Co., Ltd. As shown in Figure 13, the molecular weight of 1703 is mainly 24967.82Da, which is basically consistent with the theoretical molecular weight (24967.81Da); as shown in Figure 14, the molecular weight of 1703NT is mainly 22566.12Da, which is basically consistent with the theoretical molecular weight (22566.22Da). (2) N-terminal, C-terminal sequencing, and full sequence sequencing verification

Beijing Biotech Biotechnology Co., Ltd. was commissioned to conduct N-terminal and C-terminal sequencing verification and LC-MS/MS-based protein full sequence analysis on the high-purity lyophilized products of 1703 and 1703NT.

N-terminal sequencing was performed by Shimadzu fully automatic protein peptide sequencer (PPSQ-33A) to analyze the N-terminal sequence of the sample (Edman degradation method): take an appropriate amount of 1703 and 1703NT freeze-dried samples and dissolve them, drop the sample solution on the PVDF membrane, place it in the reactor, assemble the reactor and place it in a fixed position of the instrument, set the sample name, sample number, test cycle number, and select the method file through the software PPSQ-30Analysis, and start the test after the settings are completed. The raw data and spectrum generated by PPSQ-33A are identified by the PPSQ-30DataProcessing software and the corresponding spectrum is exported. After data analysis, the protein N-terminal sequence is determined. The N-terminal sequence of the 1703NT freeze-dried sample was detected to be: NH2-Tyr-Val-Glu-Phe-Trp-Ser-His-Pro-Glu-Phe-Glu-Lys-Gly-Ser-Pro, that is, YVEFWSHPQFEKGSP, which is consistent with the theoretical N-terminal amino acid sequence (YVEFWSHPQFEKGSP). The N-terminal sequence of the 1703NT freeze-dried sample was detected as: NH2-Gly-Ser-Pro-Gly- Pro-Lys-Gly-Asp-Met-Gly, i.e., GSPGPKGDMG, is consistent with the theoretical N-terminal amino acid sequence (GSPGPKGDMG).

C-terminal sequencing: Take an appropriate amount of 1703 and 1703NT high-purity lyophilized products for trypsin and pepsin enzymatic treatment, then analyze the treated samples by liquid chromatography-mass spectrometry (LC-MS/MS) to obtain the raw file of the original mass spectrometry results, and analyze and match the data through the software Byonic to obtain the identification results. After the mass spectrometry data is retrieved from the database, the secondary mass spectrum of the C-terminal peptide of the 1703 lyophilized sample is shown in Figure 15, and its sequence is: PGTPGRPGIKGEPGAPGKIHHHHHH, which is consistent with the theoretical C-terminal amino acid sequence (PGTPGRPGIKGEPGAPGKIHHHHHH). The secondary mass spectrum of the C-terminal peptide of the 1703NT lyophilized sample is shown in Figure 16, and its sequence is: PGAPGKI, which is consistent with the theoretical C-terminal amino acid sequence (PGAPGKI).

Protein full sequence analysis based on LC-MS/MS: Further, high-purity freeze-dried samples of 1703 and 1703NT were treated with trypsin, chymotrypsin, pepsin, trypsin & Glu-C protease, trypsin & Asp-N protease, and then the treated samples were analyzed by liquid chromatography-mass spectrometry (LC-MS/MS) to obtain the raw file of the original mass spectrometry results. After analysis by the software Byonic, the data was matched to obtain the results of full sequence sequencing verification. Comprehensive analysis of the test results showed that the amino acid sequence and total coverage 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.

The biosynthesis of proteins starts at the N-terminus and ends at the C-terminus. The correctness of the amino acid sequences at the N- and C-termini directly indicates whether the amino acid sequence is correct and complete. The protein full sequence analysis based on LC-MS/MS can verify whether the expressed amino acid sequence is correct. The above test results show that the amino acid sequences expressed by 1703 and 1703NT are correct.

(3) Fourier transform infrared spectroscopy (FT-IR) analysis

The characteristic absorption peaks of collagen groups can be detected by infrared spectroscopy. In the experiment, a small amount of 1703, 1703NT, 1703M, and 1703MNT high-purity freeze-dried samples were ground into powder with KBr and pressed into tablets. At room temperature, the range of 4000 to 400 cm ^-1 was scanned (Thermo Scientific, Nicolet ^TM iS ^TM 10 FT-IR spectrometer). The method and results were analyzed with reference 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.)

From the infrared spectra of the high-purity freeze-dried samples of 1703, 1703NT, 1703M, and 1703MNT (corresponding to Figures 17, 18, 19, and 20 respectively), it can be seen that their characteristic absorption average wavenumbers are consistent with the structural characteristics of recombinant collagen: amide A (around 3299 cm ^-1 ), amide B (around 3081 cm ^-1 ), amide I (around 1650 cm ^-1 ), amide II (around 1530-1550 cm ^-1 ), and amide III (around 1240 cm ^-1 ) (see reference [1]. Chen Jingtao et al., Infrared spectroscopy of recombinant collagen and bovine type I collagen. Materials Guide, 2008 (03): pp. 119-121. [2]. Doyle, BB, EGBendit and ERBlout, 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 collagen. Food and Fermentation Industries, 2015(03): pp. 46-52.).

(4) Recombinant collagen cell adhesion activity test

References for the cell adhesion and viability detection method of recombinant collagen: 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). This method was commissioned by the Functional Nanomaterials and Biomedical Testing Laboratory of the School of Pharmacy of Changzhou University.

Specific implementation method: Normally culture NIH/3T3 cells (purchased from the cell bank of the Chinese Academy of Sciences, catalog number GNM6, culture, The method of subculturing was carried out according to the instructions of the cell. 1703, 1703NT, 1703M purified lyophilized products, control human collagen (Sigma, catalog number C7774) and bovine serum albumin (BSA, purchased from Sangon Biotech (Shanghai) Co., Ltd.) were dissolved (ultrapure water or 1M HCl solution), and the protein concentration was determined by the UV protein quantitative empirical formula: C (mg/mL) = 0.144*(A215-A225), and then diluted to 0.5 mg/mL with PBS (pH 7.4). 100 μL of various protein solutions and blank PBS solution were added to the 96-well cell culture plate, and the plates were allowed to stand at room temperature for 60 minutes; 10 ⁵ 3T3 cells in good culture status were added to each well, and incubated at 37°C, 5% CO ₂ for 60 minutes. The cells in the wells were washed 4 times with PBS. The absorbance value at OD _{492 nm} was detected using an LDH detection kit (Roche, 04744926001) (refer to the instructions for specific operations), and the data were analyzed and significant difference analysis was performed (SPSS 22 software, Ducan method, P<0.05).

The absorbance at OD _492nm 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 collagen can help cells adhere to the wall or the extracellular matrix in a short time, which is more conducive to building a better extracellular environment. As shown in Figure 21, the cell adhesion activity of 1703, 1703NT, and 1703M is significantly better than that of commercial natural human collagen, and there is no significant difference in the cell adhesion activity of 1703, 1703NT, and 1703M, indicating that the 1703M after the amino acid sequence mutation did not significantly change the cell adhesion activity of the sequence before the mutation.

(5) Scratch assay to detect cell migration activity of recombinant collagen

Cell migration activity detection and analysis method of recombinant collagen reference Bobadilla, A., et al., In vitro cell migration quantification method for scratch assays. J R Soc Interface, 2019.16(151): p.20180709. Commissioned by the Functional Nanomaterials and Biomedical Testing Laboratory of the School of Pharmacy, Changzhou University.

Specific implementation method: Take 1703, 1703NT, 1703M purified lyophilized products, control human collagen (Sigma, catalog number C7774) and bovine serum albumin (BSA, purchased from Sangon Biotech (Shanghai) Co., Ltd.) and dissolve (ultrapure water or 1M HCl solution), and determine the protein concentration using the UV protein quantitative empirical formula: C (mg/mL) = 0.144*(A215-A225), and then dilute to 0.5 mg/mL with serum-free DMEM (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 Chinese Academy of Sciences Cell Bank, catalog number GNM6, culture and passage methods refer to the cell manual). Inoculate cells in good condition into 6-well plates, inoculate 2 mL of cell suspension in each well at a density of 20,000 cells/mL, and culture for 36 hours. Use a 200 μL gun tip to prepare scratches, wash the cells 3 times with PBS, and remove the scratched cells. Add the protein solution diluted with DMEM serum-free medium to the wells, continue to culture in a 37°C, 5% CO ₂ incubator, take samples and take pictures at 0h, 24h, and 48h. Use Image J software to process the pictures of cell migration, obtain the initial scratch area and the area of the cell-free blank area, and calculate: migration rate = (1-cell-free blank area/initial scratch area) * 100%, analyze the data and perform significant difference analysis (SPSS 22 software, Ducan method, P<0.05).

In vitro cell migration experiments simulate the process of cell migration in vivo to a certain extent, and directly reflect the interaction between cells and extracellular matrix and cells under the influence of matrix. Cell migration activity is a more effective indicator for characterizing the biological activity of collagen. The higher the migration rate, the faster the speed, and the better the biological activity of collagen. The actual comparison of cell migration taken at different times as shown in Figure 22 (the two red lines are the scratch wound areas after the initial and cell migration, and the red horizontal line part is the ruler in the lower right corner of each figure in the figure, and the ruler size is 100 μm) and the calculated cell migration rate shown in Figure 23 (Image J calculates the cell-free blank area) are compared. It can be seen that the cell migration activity of 1703, 1703NT, and 1703M purified lyophilized products is significantly better than that of natural human collagen, and the cell migration activity of 1703M is also better than that of 1703 and 1703NT.

Claims

Recombinant type XVII humanized collagen, characterized in that the recombinant type XVII humanized collagen comprises an amino acid sequence that is 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% identical to positions 567-808 of SEQ ID NO.1, and maintains the biological activity of the 15th helical region of type XVII collagen.
The recombinant type XVII humanized collagen according to claim 1 is characterized in that the recombinant type XVII humanized collagen includes an amino acid sequence that has 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% identity with SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8 and maintains the biological activity of the 15th helix region of type XVII collagen; preferably, the recombinant type XVII humanized collagen includes the amino acid sequence shown in SEQ ID NO.2, SEQ ID NO.4, SEQ ID NO.6 or SEQ ID NO.8.
A polynucleotide, characterized in that the polynucleotide encodes the recombinant type XVII humanized collagen according to claim 1 or 2.
The polynucleotide according to claim 3 is characterized in that the polynucleotide includes the nucleotide sequence shown by SEQ ID NO.3, SEQ ID NO.5, SEQ ID NO.7 or SEQ ID NO.9, or a degenerate sequence thereof.
A vector, characterized in that the vector comprises the polynucleotide according to claim 3 or 4; the vector is a eukaryotic vector or a prokaryotic vector; preferably, the vector is pPIC9K.
A host cell or a recombinant engineered bacterium, characterized in that the host cell or the recombinant engineered bacterium comprises the polynucleotide described in claim 3 or 4, or the vector described in claim 5; preferably, the cell is a eukaryotic cell or a prokaryotic cell, and more preferably, the host cell is a Pichia pastoris engineered bacterium.
The host cell or recombinant engineered bacteria according to claim 6 is characterized in that the Pichia pastoris engineered bacteria is deposited in the General Microbiology Center of China National Microbiological Culture Collection Administration, with the deposit numbers being CGMCC No. 21889, CGMCC No. 21888, CGMCC No. 21890, and CGMCC No. 21884.
The composition is characterized in that the composition comprises the recombinant humanized type XVII collagen described in claims 1-2, or the polynucleotide described in claims 3-4, or the vector described in claim 5, or the host cell or recombinant engineered bacteria described in claims 6-7.
The product is characterized in that the product comprises the recombinant humanized type XVII collagen described in claims 1-2, or the polynucleotide described in claims 3-4, or the vector described in claim 5, or the host cell or recombinant engineered bacteria described in claims 6-7, or the composition described in claim 8; preferably, the product is selected from drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.
Use of the recombinant humanized type XVII collagen according to claims 1-2, the polynucleotide according to claims 3-4, the vector according to claim 5, the host cell or recombinant engineered bacteria according to claims 6-7, the composition according to claim 8 or the product according to claim 9 in the preparation of drugs, medical devices, biomaterials, tissue engineering products, cosmetics or health products.
A preparation method for improving the production level of recombinant type XVII humanized collagen with low protein degradation, characterized in that it comprises the following steps:

(1) inoculating the recombinant engineered bacteria described in claim 6 or 7 into a seed culture medium and culturing overnight to prepare a seed solution;

(2) Setting the fermentation temperature and pH value, inoculating the bacterial seed solution into the fermentation medium, adjusting the stirring speed, air flux, tank pressure, and DO value, culturing until the carbon source is exhausted and the DO recovers rapidly, starting to feed the feed medium, and stopping the feeding of the feed medium when the bacterial OD 600 value reaches a certain value;

(3) When the glycerol is exhausted and DO ≥ 70%, the induction medium is added and the methanol induction phase is entered. The speed and ventilation are adjusted. The volume, tank pressure and flow acceleration make DO ≥ 30%;

(4) After the induction fermentation is completed, the supernatant of the fermentation liquid is taken and the protein is detected;

The recombinant humanized type XVII collagen is a recombinant humanized collagen that expresses the 15th helical region of type XVII collagen; more preferably, the recombinant humanized type XVII collagen includes what is described in claim 1 or 2, or includes the amino acid sequence shown in SEQ ID NO.2.
The preparation method according to claim 11, characterized in that

The fermentation medium components in step (2) include: 11.9-47.6 g/L NH 4 H 2 PO 4 , 2.515-10.06 g/L KH 2 PO 4 , 0.295-1.18 g/L CaSO 4 ·2H 2 O , 4.55-18.2 g/L K 2 SO 4 , 3.725-14.9 g/L MgSO 4 ·7H 2 O , 20 g/L glycerol, and 0.45 mL/L PTM 1 . Preferably, the fermentation medium components include 11.9 g/L NH 4 H 2 PO 4 , 2.515 g/L KH 2 PO 4 , 0.295 g/L CaSO 4 ·2H 2 O , 4.55 g/L K 2 SO 4 , 3.725-14.9 g/L MgSO 4 ·7H 2 O 3.725g/L, glycerol 20g/L, PTM 1 0.45mL/L.
The preparation method according to claim 11, characterized in that the pH value in step (2) is set to 3.5-6, preferably the pH value is 4.0.
The preparation method according to claim 11, characterized in that in the step (2), when the bacterial OD 600 value is 50-150, preferably when the bacterial OD 600 value is 50, the feeding medium is stopped.
The preparation method according to claim 11, characterized in that the induction medium components in step (3) include: pure methanol, 50% glycerol, and PTM 1 ; wherein the volume ratio of pure methanol to 50% glycerol is 10-7:0-3, and 12 mL of PTM 1 is added per liter; preferably, the volume ratio of pure methanol to 50% glycerol is 8:2.
The preparation method according to any one of claims 11 to 15, characterized in that, after step (4), it also includes a step of purifying the fermentation product; preferably, the purification includes the steps of hydrophobic chromatography and cationic chromatography performed sequentially.
The preparation method according to claim 16 is characterized in that the hydrophobic chromatography adopts buffer A for balancing, buffer B for washing, and buffer C for elution; the cationic chromatography adopts buffer D for balancing and buffer E for elution; preferably, buffer A comprises: 20mM KH2PO4 , 2M ammonium sulfate, pH5.0; buffer B comprises: 20mM KH2PO4 , 0.6M ammonium sulfate, pH5.0; buffer C comprises: 20mM KH2PO4 , pH5.0; buffer D comprises: 20mM tartaric acid, 100mM sodium chloride, pH4.0; buffer E comprises: 20mM tartaric acid, 500mM sodium chloride, pH4.0.
A method for purifying recombinant type XVII humanized collagen, characterized in that it comprises the following steps:

(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;

The fermentation supernatant of the recombinant type XVII humanized collagen was collected, and the hydrophobic chromatography medium was equilibrated with buffer A. After the loading was completed, the medium was re-equilibrated with buffer A; then, the impurities were washed with buffer B, and the eluate was eluted with buffer C, and the eluate 1 was collected;

(2) Cationic 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;

Buffer D is used to balance the cationic chromatography medium, and eluate 1 is loaded. After loading, the medium is re-balanced with buffer D, and eluate is eluted with buffer E. Eluate 2 is collected, and eluate 2 is ultrafiltered and freeze-dried to obtain a purified protein freeze-dried product;

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