Preparation method for yeast-expressed type iii recombinant human collagen with tri-helical structure and use thereof

Abstract

Provided are a preparation method for a yeast-expressed type III recombinant human collagen with a tri-helical structure and use thereof. The preparation method comprises the steps of performing fermentation using an engineered bacterium and performing purification to obtain the type III recombinant human collagen with a tri-helical structure. A method for constructing the engineered bacterium comprises using a modified plasmid to construct a collagen expression vector, first performing collagen expression level screening to obtain a high-expression strain, then introducing a P4H enzyme expression plasmid into the high-expression strain to obtain a co-expression strain, and performing fermentation and purification to obtain a mature type III recombinant human collagen with a tri-helical structure. The type III recombinant human collagen with a tri-helical structure obtained using the preparation method of the present invention comprises hydroxyproline, and hydroxyproline accounts for 40% or more of total proline.

Description

A method for preparing recombinant human collagen of type III triple helix structure expressed in yeast and its application. Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for preparing recombinant human collagen of type III triple helix structure expressed in yeast and its application. Background Technology

[0002] Collagen is the earliest discovered and most abundant extracellular matrix protein, widely present in the skin, muscles, bones, and internal organs of humans and animals. It plays a vital role in maintaining the normal physiological functions of cells, tissues, and organs, as well as in repairing damage. Due to its excellent physicochemical properties, biological efficacy, biocompatibility, and biodegradability, collagen is widely used in food, cosmetics, and nutritional supplements.

[0003] Based on their origin, collagen can be broadly classified into animal-derived collagen and recombinant collagen. Animal-derived collagen mainly comes from terrestrial and marine animals, while recombinant collagen refers to proteins obtained by cloning the human collagen gene into a selected expression vector, transforming it into expression cells, and finally purifying it. Due to the rapid development of molecular biology techniques and the potential pathogenic risks and immunogenicity of animal collagen, research and the market primarily focus on recombinant collagen.

[0004] According to the "Guidelines for Naming Recombinant Collagen Biomaterials" issued by the National Medical Products Administration on March 15, 2021, recombinant collagen can be divided into three categories: ① Recombinant human collagen, which is the full-length amino acid sequence encoded by a specific type of human collagen gene prepared by DNA recombination technology, and has a triple helix structure; ② Recombinant human collagen, which is a fragment of the full-length or partial amino acid sequence encoded by a specific type of human collagen gene prepared by DNA recombination technology, or a combination containing functional fragments of human collagen; ③ Recombinant collagen-like proteins, which are amino acid sequences or fragments of a specific gene prepared by DNA recombination technology after design and modification, or a combination of such functional amino acid sequence fragments, and the gene encoding sequence or amino acid sequence has low homology with the gene encoding sequence or amino acid sequence of human collagen.

[0005] Currently, the mainstream products on the market are recombinant human collagen or recombinant collagen-like proteins. Because these types of collagen lack post-translational modifications and triple-helix structures, they can be expressed in large quantities in microorganisms at a relatively low cost. However, these two types of collagen lack a helical structure, making their internal enzyme cleavage sites highly accessible and prone to enzymatic hydrolysis. Furthermore, the flexible single-chain structure, lacking steric hindrance, allows for numerous amino acid side chains to undergo chemical reactions leading to non-enzymatic hydrolysis. The mechanical properties of single-chain collagen are also far inferior to triple-helix collagen, making it unsuitable for applications requiring high mechanical strength. The expression level of recombinant human collagen is significantly lower than the other two types of collagen. Expression systems using E. coli, yeast, plants, baculoviruses, and mammalian cells have all been used to express recombinant human collagen. Although many expression systems are described in the literature, current expression systems for large-scale production of human collagen rely on yeast and plants. For example, FibroGen, Inc. (San Francisco, California, USA) produces recombinant collagen in yeast cells, while CollPlant Ltd. (Rehovot, Israel) uses tobacco as a collagen production plant. According to published literature, these companies primarily focus on type I and type III collagen used in the manufacture of biopharmaceutical products, including artificial corneas, implants, and wound dressings.

[0006] Numerous studies have reported cases of recombinant collagen expression. In commercial applications, recombinant humanized collagen and other single-chain collagens are the mainstream. Such collagens lack key collagen features, including triple helix conformation, modification of proline and lysine residues, and resistance to enzyme degradation.

[0007] Because triple-helix collagen (recombinant human collagen) requires specific α-chain assembly and appropriate post-translational modifications, it cannot be simply expressed using conventional industrial production systems (E. coli, yeast). Current techniques primarily involve co-expressing P4H in these systems to obtain hydroxylated collagen and triple-helix collagen. Olsen et al. reported that the expression levels of type I-III human collagen in Pichia pastoris can reach 1-1.5 g / L, and mentioned optimization through genetic methods and processes, but these were not fully disclosed. Olsen's laboratory used Pichia pastoris to co-express collagen and human P4H (P4HA1) to obtain recombinant collagen with a hydroxylation degree close to that of the natural protein. Rutschmann et al. co-expressed collagen and human P4H (P4HA1) in E. coli, and human P4H (P4HA1) is also co-expressed in most other hosts such as tobacco and insect cells.

[0008] CN114853881B utilizes proline hydroxylase derived from Bacillus anthracis to hydroxylate collagen in Escherichia coli, but non-human P4H has different substrate specificity, and the resulting recombinant collagen may pose an immune risk. CN112626074B co-expresses collagen and P4H (P4HA2) in Pichia pastoris to obtain secretible, hydroxylated recombinant collagen, but the hydroxylation rate is low, and it cannot form a triple helix conformation. This invention aims to develop a yeast expression method for triple helix type III recombinant human collagen and its preparation method, to achieve efficient expression of triple helix type III recombinant human collagen and meet market demand for triple helix type III recombinant human collagen. Summary of the Invention

[0009] The purpose of this invention is to provide a method for preparing recombinant human collagen of type III with a triple helix structure expressed in yeast and its application, thereby solving the problems existing in the prior art. The recombinant human collagen prepared using the method of this invention has a triple helix structure, and the hydroxyproline content therein can reach more than 40% of the total proline.

[0010] To achieve the above objectives, the present invention provides the following solution:

[0011] This invention provides an engineered bacterium, KM71 / 3A1FL-1#-Z, expressing a triple-helix type III recombinant human collagen. S The method for constructing -P4H includes transforming the pMChZ-3A1FL plasmid and the pPIC9K-P4H(DP)-1 plasmid into the host bacteria to construct the engineered bacteria KM71 / 3A1FL-1#-Z. S -P4H steps;

[0012] The pMChZ-3A1FL plasmid was constructed by ligating the type III collagen α1 chain DNA sequence into the pMChZ-AOX plasmid;

[0013] The pMChZ-AOX plasmid was obtained by modifying the pPIC9K plasmid as follows: replacing 4647-9266 bp of the pPIC9K plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.1; deleting BamHI before αMF and αMF in the pPIC9K plasmid through point mutation; and introducing a BamHI recognition site between 1583-1584 bp of the pPIC9K plasmid through point mutation.

[0014] The method for constructing the pPIC9K-P4H(DP)-1 plasmid includes replacing KanR in the P4H 9KDP plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.5 to obtain the pPIC9K-P4H(DP)-1 plasmid.

[0015] Furthermore, the host organism is Pichia pastoris.

[0016] Furthermore, the Pichia pastoris is X33, GS115, KM71, or KM71H.

[0017] This invention also provides a method for constructing the engineered bacterium KM71 / 3A1NproΔ-2#-P4H expressing triple-helix type III recombinant human collagen, comprising the steps of transforming pMChZ-3A1NproΔ plasmid, pMCrZ-3A1NproΔ plasmid and pPIC9K-P4H(DP)-1 plasmid into a host bacterium to construct the engineered bacterium KM71 / 3A1NproΔ-2#-P4H;

[0018] The pMChZ-3A1NproΔ plasmid is constructed by linking 3A1NproΔ into the pMChZ-AOX plasmid;

[0019] The pMCrZ-3A1NproΔ plasmid is constructed by linking the 3A1NproΔ plasmid into the pMCrZ-AOX plasmid;

[0020] The nucleotide sequence of the 3A1NproΔ is shown in SEQ ID NO.7;

[0021] The pMChZ-AOX plasmid was obtained by modifying the pPIC9K plasmid as follows: replacing 4647-9266 bp of the pPIC9K plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.1; deleting BamHI before αMF and αMF in the pPIC9K plasmid through point mutation; and introducing a BamHI recognition site between 1583-1584 bp of the pPIC9K plasmid through point mutation.

[0022] The pMCrZ-AOX plasmid was constructed by replacing the PpHIS4 on the pMChZ-AOX plasmid with the DNA sequence shown in SEQ ID NO.2.

[0023] The method for constructing the pPIC9K-P4H(DP)-1 plasmid includes replacing KanR in the P4H 9KDP plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.5 to obtain the pPIC9K-P4H(DP)-1 plasmid.

[0024] Furthermore, the host organism is Pichia pastoris.

[0025] The Pichia pastoris is X33, GS115, KM71 or KM71H.

[0026] The present invention also provides a method for constructing an engineered bacterium KM71H / 3A1THR-TEV-2#-P4H expressing a triple-helix type III recombinant human collagen, comprising the steps of transforming pMChZ-3A1THR-TEV plasmid, pMCrZ-3A1THR-TEV plasmid and pPIC9K-P4H(DP)-1 plasmid into a host bacterium to construct the engineered bacterium KM71H / 3A1THR-TEV-2#-P4H;

[0027] The pMChZ-3A1THR-TEV plasmid is constructed by linking 3A1THR-TEV into the pMChZ-AOX plasmid;

[0028] The pMCrZ-3A1THR-TEV plasmid is constructed by linking the 3A1THR-TEV into the pMCrZ-AOX plasmid;

[0029] The nucleotide sequence of the 3A1THR-TEV is shown in SEQ ID NO.9;

[0030] The pMChZ-AOX plasmid was obtained by modifying the pPIC9K plasmid as follows: replacing 4647-9266 bp of the pPIC9K plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.1; deleting BamHI before αMF and αMF in the pPIC9K plasmid through point mutation; and introducing a BamHI recognition site between 1583-1584 bp of the pPIC9K plasmid through point mutation.

[0031] The pMCrZ-AOX plasmid was constructed by replacing the PpHIS4 on the pMChZ-AOX plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.2;

[0032] The method for constructing the pPIC9K-P4H(DP)-1 plasmid includes replacing KanR in the P4H 9KDP plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.5 to obtain the pPIC9K-P4H(DP)-1 plasmid.

[0033] Furthermore, the host organism is Pichia pastoris.

[0034] Furthermore, the Pichia pastoris is X33, GS115, KM71, or KM71H.

[0035] The present invention also provides an engineered bacterium expressing type III recombinant human collagen with a triple helix structure, constructed according to the above-described construction method.

[0036] The present invention also provides the application of the above-mentioned engineered bacteria in the preparation of triple-helix type III recombinant human collagen.

[0037] The present invention also provides a method for preparing recombinant human collagen of triple helix type III expressed by yeast, comprising the step of fermenting and purifying the recombinant human collagen of triple helix type III using the above-mentioned engineered bacteria.

[0038] The present invention also provides a triple-helix type III recombinant human collagen prepared according to the above preparation method.

[0039] The present invention also provides the application of the above-mentioned triple-helix type III recombinant human collagen in the preparation of biopharmaceutical products, wherein the biopharmaceutical products are skin care products, skin repair dressings, implants, artificial skin, biomaterials and / or medical devices.

[0040] The present invention discloses the following technical effects:

[0041] This invention modifies plasmids and constructs recombinant expression vectors. First, high-expression strains are obtained by screening for collagen expression levels. Then, P4H expression plasmids are introduced into these vectors to obtain co-expression strains, which can be used to study the differences in hydroxylation of full-length collagen by Pichia pastoris from different sources. The modified plasmids of this invention, which can be used for both in vitro and in vivo multi-copy studies, facilitate the screening of high-copy strains, resulting in collagen with a triple-helix structure.

[0042] The co-expression strain provided by this invention can be purified by fermentation to obtain triple-helix type III recombinant human collagen. This co-expression strain has a high expression level of triple-helix recombinant humanized type III collagen and is easy to purify. The triple-helix type III recombinant human collagen obtained after purification contains hydroxyproline.

[0043] Compared with animal-derived type III collagen, the triple-helix type III recombinant human collagen provided by this invention has advantages such as uniform molecular weight, high purity, and no risk of viral transmission. This triple-helix type III recombinant human collagen has the characteristic triple-helix structure of collagen and can self-assemble to form characteristic collagen microfibrils. This triple-helix type III recombinant human collagen can significantly promote the adhesion, proliferation, and migration of human fibroblasts, exhibiting high biocompatibility and biological activity.

[0044] The triple-helix recombinant humanized type III collagen provided by this invention has good water solubility and stable quality. This triple-helix mature type III collagen can be applied in medical and health and medical aesthetic fields that require collagen with high molecular weight and high support, such as skin care products, skin repair dressings, implants, artificial skin, biomaterials, medical devices and other fields. Attached Figure Description

[0045] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0046] Figure 1 shows the plasmid map of pPIC9K;

[0047] Figure 2 shows the plasmid map of pMChZ-AOX;

[0048] Figure 3 shows the plasmid map of pMCrZ-AOX;

[0049] Figure 4 shows the plasmid map of pMCeH-AOX;

[0050] Figure 5 is a flowchart of the construction process for 3A1FL-related expression strains; in the diagram, the rounded rectangles contain yeast strains, and the right-angled rectangles contain expression plasmids; Z R This indicates resistance to Zeocin, Z S Indicates sensitivity to Zeocin; the number before # indicates the number of electroporation cycles of the collagen expression plasmid, and the circled number indicates that the same plasmid was used for electroporation in the previous round;

[0051] Figure 6 shows the expression identification results of 3A1FL-related strains; where a represents KM71 / 3A1FL-1#-Z strains with 6His as the primary antibody. R Identification results; b is KM71 / 3A1FL-②#-Z with 6His as the primary antibody. R The identification results; c is KM71 / 3A1FL-②#-Z R and KM71 / 3A1FL-1#-Z R The identification result of F9; d is KM71 / 3A1FL-1#-Z S -P4H P4HA detection result; e is KM71 / 3A1FL-1#-Z S -P4H P4HB test results;

[0052] Figure 7 is a flowchart of the construction process for 3A1NproΔ related expression strains; in the diagram, the rounded rectangles contain yeast strains, and the right-angled rectangles contain expression plasmids; Z R This indicates resistance to Zeocin, Z S Indicates sensitivity to Zeocin; the number before # indicates the number of electroporation cycles of the collagen expression plasmid, and the circled number indicates that the same plasmid was used for electroporation in the previous round;

[0053] Figure 8 shows the expression identification results of 3A1NproΔ related strains; where a represents KM71 / 3A1NproΔ-1#-Z with COL3A as the primary antibody.R The identification results; b is KM71 / 3A1NproΔ-1#-Z with 6His as the primary antibody. R The identification results; c is KM71 / 3A1NproΔ-②#-Z R The identification results; d is KM71 / 3A1NproΔ-1#-Z with COL3A as the primary antibody. R The detection results of KM71 / 3A1NproΔ-2# and KM71 / 3A1NproΔ-2#-P4H; e is the detection results of P4HA1 and P4HB of KM71 / 3A1NproΔ-2#-P4H;

[0054] Figure 9 is a flowchart of the construction process for 3A1THR-TEV-related expression strains; in the diagram, the rounded rectangles contain yeast strains, and the right-angled rectangles contain expression plasmids; Z R This indicates resistance to Zeocin, Z S Indicates sensitivity to Zeocin; the number before # indicates the number of electroporation cycles for the collagen expression plasmid;

[0055] Figure 10 shows the expression identification results of 3A1THR-TEV related strains; where a represents KM71H / 3A1THR-TEV-1#-Z R The identification results; b is KM71H / 3A1THR-TEV-1#-Z S KM71H / 3A1THR-TEV-2#-Z R The identification results of KM71H / 3A1THR-TEV-2#-P4H are shown in Figure 1; c is the WB detection result of sample b; d is the detection result of KM71H / 3A1THR-TEV-2#-P4H and KM71H / 3A1THR-TEV-3#-P4H with COL3A and 6His as primary antibodies, respectively.

[0056] Figure 11 is a statistical chart of the copy number of the relevant strain 3A1THE-TEV;

[0057] Figure 12 shows the electrophoresis images of the 3A1THR-TEV purification process sample and the purified sample; where a is the electrophoresis image of the purified sample; b is the result of non-denaturing electrophoresis after reconstitution of the purified lyophilized powder.

[0058] Figure 13 shows the results of circular dichroism detection by 3A1THR-TEV;

[0059] Figure 14 shows the results of transmission electron microscopy. Detailed Implementation

[0060] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0061] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0062] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0063] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0064] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0065] The Pichia pastoris KM71, Pichia pastoris KM71H and plasmid pPIC9K used in the following examples were purchased from Invitrogen.

[0066] Example 1

[0067] 1. Construct plasmids that can be used for both in vitro and in vivo multicopy construction.

[0068] The SEQ ID NO.1 sequence was synthesized to replace 4647-9266 bp of pPIC9K (see Figure 1 for the map), that is, the KanR resistance gene, Bom and AOX13' fragment element in pPIC9K were deleted. The replacement sequence contains lox71 and lox66 sequences at both ends and Cre transcription unit inside. The Cre gene is started by the pAOX1 promoter with a mutated SacⅠ restriction site. There are prokaryotic and eukaryotic promoters before BleoR, which can be used for prokaryotic and eukaryotic screening. There is a prokaryotic promoter before AmpR, which can be used for prokaryotic screening. BamHI (938-943 bp) and αMF in pPIC9K were deleted by point mutation. The BamHI recognition site was introduced by point mutation between 1583-1584 bp to obtain plasmid pMChZ-AOX (h represents the recombination site PpHIS4, Z represents the selection marker BleoR; see Figure 2 for the map). This plasmid can express Cre recombinase when induced by methanol, catalyzing the recombination of lox71 and lox66 to recover the resistance gene. The BamHI located after AOXtt can be used to construct in vitro multicopy plasmids with the same tail enzyme.

[0069] SEQ ID NO.1:

[0070] Among them, the 5' end The image shows the 3' end of a lox71. The value shown is 66. The image shown is of AmpR. P is shown AmpR , The figure shows the P at the mutated SacⅠ site. AOX1 , The image shows the Cre recombinase gene. The image shows AOX1tt. P is shown TEF The lowercase letter is P. EM7 , For BleoR, The image shows CYC1tt.

[0071] The SEQ ID NO.2 sequence (PpRGI2) was synthesized to replace PpHIS4 on pMChZ-AOX, resulting in plasmid pMCrZ-AOX (r represents the recombination site as PpRGI2; the map is shown in Figure 3).

[0072] The SEQ ID NO.3 sequence (PpENO) was synthesized to replace PpHIS4 on pMChZ-AOX, and the SEQ ID NO.4 sequence was synthesized to replace PEM7-BleoR-CYC1 tt on pMChZ-AOX, to obtain plasmid pMCeH-AOX (e represents the recombination site as PpENO, and H represents the selection marker as HygR; the map is shown in Figure 4).

[0073] SEQ ID NO.4 (underscore is HygR, 3' end is TEFtt):

[0074] 2. Construction of recombinant expression vectors

[0075] DNA sequences expressing 3A1FL, 3A1NproΔ, and 3A1THR-TEV were synthesized, and a DNA sequence encoding a 6×HisTag tag was added to the carboxyl terminus to provide a specific affinity purification label for easy immunological detection. Exogenous DNA was ligated into expression vectors pMChZ, pMCrZ, and pMCeH to construct recombinant expression vectors pMChZ-3A1FL, pMChZ-3A1NproΔ, pMCrZ-3A1NproΔ, pMChZ-3A1THR-TEV, pMCrZ-3A1THR-TEV, and pMCeH-3A1THR-TEV, respectively.

[0076] The synthesized SEQ ID NO.5 replaces KanR in the P4H 9K DP plasmid (disclosed in Chinese patent CN114480471A), that is, P is added to the 5' end of the screening marker (in the direction of the pPIC9K plasmid). TEF and P EM7 Add CYC1tt to the 3' end to obtain the P4H expression vector pPIC9K-P4H(DP)-1.

[0077] SEQ ID NO.5:

[0078] in, P is shown TEF , P is shown EM7 , The image shows KanR. The image shows CYC1tt.

[0079] Example 2

[0080] 1. Construction and expression identification of 3A1FL-related strains

[0081] 1.1 Construction of 3A1FL-related expression plasmids

[0082] The complete amino acid sequence (1466aa) of human type III collagen (https: / / www.uniprot.org / uniprot / P02461) was reverse-translated according to the Pichia pastoris codon table. The GC content and secondary structure were optimized to obtain the corresponding nucleic acid sequence, namely the type III collagen α1 chain DNA sequence in CN114480471A.

[0083] The type III collagen α1 chain DNA sequence was amplified using primers 3A1FL F (5'-cgGAATTCGAAACGATGATGTCATTCGTTCAAAAAG-3', SEQ ID NO 10) and 3A1FL R (5'-atagtttagcggccgcttaatgatgatgatgatgatgCAAAAAGCAAACAGGTCCAACA-3', SEQ ID NO 11). An EcoRI site was introduced at the 5' end, and a 6His tag and a NotI site were introduced at the 3' end. After double digestion with EcoRI and NotI, the DNA was cloned into the corresponding site of pMChZ-AOX to obtain pMChZ-3A1FL.

[0084] 1.2 Construction and expression identification of 3A1FL-related expression strains

[0085] The relevant expression strain KM71 / 3A1FL-1#-Z was constructed according to the procedure shown in Figure 5. R KM71 / 3A1FL-1#-Z S KM71 / 3A1FL-②#-Z R and KM71 / 3A1FL-1#-Z S -P4H, the electroporation method followed the Invitrogen Pichia Expression Kit USER GUIDE. pMChZ-3A1FL was linearized using SalⅠ, and after electroporation, it was plated on YPDZ (bleomycin, 300 μg / mL) plates; pPIC9K-P4H(DP)-1 was linearized using BspEⅠ, and after electroporation, it was plated on YPDG (genycin, 500 μg / mL) plates. Large colonies of the transformed strains were selected and streaked twice on selection plates to obtain single colonies, which were then induced in shake flasks. The engineered strains were inoculated into 100 mL Erlenmeyer flasks containing 10 mL of BMGY medium and cultured at 30 °C and 220 rpm until OD500. 600 The result was 6 (16 h). The cells were centrifuged at 3000 g for 5 min at room temperature, collected, and resuspended in BMMY medium to adjust the OD value. 600The culture medium was incubated at approximately 200 μL and placed on a shaker at 230 °C and 220 rpm for 3 days. Every 24 hours, 100% methanol was added to the culture medium until the final concentration reached 1.0%. After induction, a 1 mL sample was collected in a 1.5 mL EP tube and centrifuged at 12000 g for 5 min at 4 °C. The supernatant and bacterial cells were collected separately. 80 μL of the supernatant was added to 20 μL of loading buffer and heated at 80 °C for 5 min to prepare the sample. 20 μL of the sample was then used for SDS-PAGE. For the bacterial cells, 200 μL of cell disruption buffer and 40% of the liquid volume of 0.5 mm glass beads were added; the mixture was vortexed for 1 min, placed on ice for 1 min, and repeated 6 times; the mixture was centrifuged at 3000 g for 3 min at 4 °C. 80 μL of the supernatant (total protein) was added to 20 μL of loading buffer and heated at 99 °C for 5 min to prepare the sample. 20 μL of the sample was then used for SDS-PAGE, followed by Western blotting. Collagen was detected using anti-6His antibody (D191001; Shanghai Sangon Biotech) and COL3A antibody (B-10; SANTACRUZ), respectively. The α subunit of P4H was detected using P4HA1 antibody (Wuhan Feien Biotech), and the β subunit of P4H was detected using P4HB antibody (ET7110-92; Hangzhou Huaan Biotechnology). The internal control antibody ACT_YEAST was purchased from Bioss.

[0086] Cell wall disruption solution formulation: 50mM sodium phosphate (pH 7.4), 1mM EDTA, 5% glycerol and 1mM benzyl sulfonyl fluoride (PMSF) are dissolved in anhydrous ethanol to prepare a 100× stock solution, which is added before cell wall disruption.

[0087] The WB test results of the relevant strains are shown in Figure 6. In Figure 6a, 6His antibody was used as the primary antibody, KM71 / 3A1FL-1#-Z R Full-length collagen can be detected. However, after repeated electroporation with the same plasmid, the expression level decreases, becoming almost undetectable, as shown in Figures 6b and c. The modified P4H expression plasmid can normally express the P4HA and P4HB subunits, as shown in Figures 6d and e.

[0088] 2. Construction and expression identification of 3A1NproΔ related strains

[0089] 2.1 Construction of 3A1NproΔ related expression plasmid

[0090] Using the plasmid containing the type III collagen α1 chain DNA sequence in CN114480471A as a template, primer 3A1NproΔF was used.

[0091] (cgGAATTCGAAACGATGATGTCATTCGTTCAAAAAGGTTCTTGGCTTCTTCTTGCATTGCTTCATCCTACTATTATCTTGGCTCAATACGACTCTTATGACGTG, SEQ ID NO.12) and 3A1FL R (see 1.1) amplify the 3A1NproΔ sequence, digest it with EcoRI and NotI, and clone it into the corresponding site of pMChZ-AOX to obtain pMChZ-3A1NproΔ. Cloning it into the corresponding site of pMCrZ-AOX yields pMCrZ-3A1NproΔ.

[0092] The amino acid sequence of 3A1NproΔ is shown in SEQ ID NO.6, and the nucleotide sequence is shown in SEQ ID NO.7.

[0093] 2.2 Construction and expression identification of 3A1NproΔ related expression strains

[0094] Following the procedure shown in Figure 7, using KM71 as the starting strain, the relevant expression strain KM71 / 3A1NproΔ-1#-Z was constructed. R KM71 / 3A1NproΔ-1#-Z S KM71 / 3A1NproΔ-②#-Z R Transformants KM71 / 3A1NproΔ-2# and KM71 / 3A1NproΔ-2#-P4H were electroporated according to the Invitrogen Pichia Expression Kit USER GUIDE. pMChZ-3A1NproΔ was linearized using SalⅠ and then plated on YPDZ (300 μg / mL) plates after electroporation. The selected transformant KM71 / 3A1NproΔ-1#-Z was selected. R After induction, the bacterial suspension was streaked onto YPD plates. The resulting colonies were then spotted onto both YPD and YPDZ (100 μg / mL) plates. The strain that grew normally on YPD plates but not on YPDZ was the selected strain KM71 / 3A1NproΔ-1#-Z. S pMCrZ-3A1NproΔ was linearized using SpeⅠ and then plated on YPDZ (300 μg / mL) plates after electroporation; pPIC9K-P4H(DP)-1 was linearized using BspEⅠ and then plated on YPDG (500 μg / mL) plates after electroporation.

[0095] The strain was induced in shake flasks using the same method as in 1.2. After induction, the cell wall was disrupted to extract total protein. The same total protein was then loaded onto samples for SDS-PAGE and Western blotting. The Western blotting results are shown in Figure 8. KM71 / 3A1NproΔ-1#-Z RThe full-length 3A1NproΔ plasmid was detectable, as shown in Figures 8a and 8b. Repeated electroporation with the same plasmid resulted in decreased expression levels, making the target protein almost undetectable, as shown in Figure 8c. Two electroporations using a plasmid with a different recombination site improved expression levels, as shown in Figure 8d. The modified P4H expression plasmid could normally express the P4HA and P4HB subunits, as shown in Figure 8e.

[0096] 3. Construction and expression identification of 3A1THR-TEV related strains

[0097] 3.1 Construction of 3A1THR-TEV related expression plasmids

[0098] A 6His tag was added between the signal peptide and the N-terminal telopeptide, and a QP was added before the 6His tag to improve the enzyme digestion efficiency of the signal peptide. A TEV digestion sequence was added between the N-terminal peptide and the 3-helix region; a TEV digestion sequence was added between the 3-helix region and the C-terminal telopeptide, and a 6His tag was added to the C-terminus to obtain the 3A1THR-TEV amino acid sequence, as shown in SEQ ID NO.8, with the TEV recognition site indicated by red background. This amino acid sequence was optimized according to the expression preference of Pichia pastoris to obtain the nucleic acid sequence, as shown in SEQ ID NO.9. It was cloned into the EcoRI and NotRI sites of pMChZ-AOX and pMCrZ-AOX, respectively, to obtain the expression plasmids pMChZ-3A1THR-TEV and pMCrZ-3A1THR-TEV; and cloned into the BspERI and NotRI sites of pMCeH-AOX to obtain the expression plasmid pMCeH-3A1THR-TEV.

[0099] 3.2 Construction and expression identification of 3A1THR-TEV related expression strains

[0100] Following the procedure shown in Figure 9, using KM71H as the starting strain, the 3A1THR-TEV-related expression strain KM71H / 3A1THR-TEV-1#-Z was constructed. R KM71H / 3A1THR-TEV-1#-Z S KM71H / 3A1THR-TEV-2#-Z R Transformants KM71H / 3A1THR-TEV-2#-P4H and KM71H / 3A1THR-TEV-3#-P4H were electroporated according to the Invitrogen Pichia Expression Kit User Guide. pMChZ-3A1THR-TEV was linearized using SalⅠ and then plated on YPDZ (300 μg / mL) plates after electroporation. The selected transformant KM71H / 3A1THR-TEV-1#-Z was selected. RAfter induction, the bacterial suspension was streaked onto YPD plates. The resulting colonies were then spotted onto both YPD and YPDZ (100 μg / mL) plates. The strain that grew normally on YPD plates but not on YPDZ was the selected strain KM71 / 3A1THR-TEV-1#-Z. S pMCrZ-3A1THR-TEV was linearized using SpeⅠ, electroporated, and then plated on YPDZ (300 μg / mL) plates; pPIC9K-P4H(DP)-1 was linearized using BspEⅠ, electroporated, and then plated on YPDG (500 μg / mL) plates; pMCeH-3A1THR-TEV was linearized using XbaⅠ, electroporated, and then plated on YPDH (hygromycin, 200 μg / mL) plates.

[0101] The strain was induced by shaking flasks, using the same method as in 1.2. After induction, the cell wall was disrupted to extract total protein. The same total protein was then loaded onto samples for SDS-PAGE and Western blotting. The Western blotting results are shown in Figure 10. (KM71H / 3A1THR-TEV-1#-Z) R Full-length 3A1THR-TEV was detected, as shown in Figure 10a. Two electroporations were performed using the plasmid pMCrZ-3A1THR-TEV with the recombination site modified to RGI2. The expression levels were similar to those of KM71H / 3A1THR-TEV-1#-Z. S Similar results (Figure 10b). After transformation into pPIC9K-P4H(DP)-1, P4HA and P4HB subunits can be expressed normally (Figure 10b). Using the plasmid pMCeH-3A1THR-TEV with the recombination site changed to ENO, three electroporations were performed, and the expression levels of some strains were significantly increased (Figure 10d). Using different selection markers, the selection markers can be uniformly recovered after multiple rounds of electroporation verification.

[0102] 3.3 Identification of target gene copy number in 3A1THR-TEV-related expression strains

[0103] The copy number of the target gene 3A1THR-TEV was detected by Shanghai Sangon Biotech using digital PCR on different batches of the selected strains and plasmid pMChZ-3A1THR-TEV. The PCR instrument used was a BIO-RAD T100.

[0104] The quality of a single copy of dsDNA is determined by the formula. Perform the calculation.

[0105] Based on the yeast genome size of 9.4 Mb, the estimated mass of one copy of the genome is 10.33 × 10⁻⁶. -6The formula calculates the genome copy number in 1 μL of template. The ratio of the final DNA copy number in 1 μL to the genome copy number in 1 μL of template is the target gene copy number in yeast. The pMChZ-3A1THR-TEV size is 13938 bp. Using the formula above, the mass of one copy of the plasmid is 1.53 × 10⁻⁶. -8 ng. The ratio of the 3A1THR-TEV copy number in each strain to the 3A1THR-TEV copy number in pMChZ-3A1THR-TEV was plotted, as shown in Figure 11. The results were consistent with the WB in 3.2. The 3A1THE-TEV copy number in KM71H / 3A1THE-TEV-3#-P4H was significantly higher than that in other strains.

[0106] 4. KM71H / 3A1THR-TEV-3#-P4H High-Density Fermentation

[0107] Seed culture medium YPG (10 g / L yeast extract, 20 g / L peptone, and 10 g / L glycerol); fermentation medium BSM (85% H3PO4 26.7 ml / L, CaSO4·2H2O 0.93 g / L, K2SO4 18.2 g / L, MgSO4·2H2O 14.9 g / L, KOH 4.13 g / L, glycerol 40 g / L, and PTM1 trace element stock solution 4.0 mL / L); fed-batch medium (50% w / v glycerol, with 12 mL PTM1 trace element stock solution per liter); induction medium (100% methanol, with 12 mL PTM1 trace element stock solution per liter); PTM1 trace element stock solution: sterilized by filtration through a 0.22 μm filter membrane and stored at 4℃. After the fermentation medium was sterilized at high temperature, the PTM1 trace element stock solution was added after cooling to room temperature, and the pH was adjusted to 6.0 with ammonia.

[0108] The batch culture and induction conditions for the engineered strain KM71H / 3A1THR-TEV-3#-P4H were as follows: fed-batch culture was used at a temperature of 30℃. The engineered strain was inoculated into a 1L shake flask containing 200mL of seed culture medium YPG and cultured at 220rpm and 30℃ for 18h until OD500 was reached. 600 =8. Use a 5L fermenter (Baoxing Biotechnology), fill with 2L of fermentation medium. Before inoculation, adjust the fermentation speed to 300rpm, aeration rate to 4L / min, and temperature to 30℃. Adjust the pH to 6.0 using a concentrated ammonia solution. First, add 0.9mL of PTM1, then add 200mL of the prepared seed culture to the tank (flame ring inoculation). Then, calibrate the dissolved oxygen electrode. After calibration, start fermentation. When the dissolved oxygen drops to 30% for the first time, use the dissolved oxygen cascade speed function to maintain 30%. Wait for the glycerol to be depleted, the dissolved oxygen to rebound, and the dissolved oxygen to be greater than 70% (OD). 600(Value approximately 20), cancel dissolved oxygen cascade stirring speed, increase stirring speed to 650 rpm, and use 30% continuous feeding of glycerol, adding 150 mL at a time. Stop feeding glycerol, and after dissolved oxygen rebounds to above 70%, induce culture with methanol at a constant feeding rate of 4 mL / h. After 80 h of induction, OD... 600 If the change is not significant or decreases, the container can be placed in the tank.

[0109] 5. Purification of 3A1THR-TEV and identification of the target protein after purification

[0110] After fermentation, the bacterial culture was centrifuged at 5000 rpm for 30 min to collect the bacterial cells. The bacterial cells were resuspended in the purified cell-wall-breaking liquid at a ratio of 1:10 (W:V). The homogenizer was used to cycle and break the cells four times at 1000 bar. After the cell-wall-breaking was completed, the supernatant and precipitate were separated by centrifugation at 12000 rpm and 4℃ for 30 min.

[0111] The purification and cell wall disruption solution formula consists of 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, and 100 mM glycine.

[0112] The precipitate was dissolved in a washing buffer, centrifuged at 1000 rpm for 30 min, and then reconstituted with 8M urea. After refolding in a refolding buffer overnight, the pH was adjusted to 3.0, and pepsin was added for enzymatic digestion at 4°C overnight. After ultrafiltration and desalting, the lyophilized product was lyophilized. The obtained lyophilized powder was reconstituted and subjected to non-denaturing electrophoresis (4-16% non-denaturing precast gel, catalog number: BN1002BOX, Invitrogen). The results are shown in Figure 12b. The triple helix collagen after cleavage of the C-terminal propeptide is approximately 300 kDa, and the results shown in the figure are consistent with the theory.

[0113] Wash buffer formula: 50mM Tris-HCl (pH 7.5), 2M urea and 1mM EDTA.

[0114] Refolding buffer formulation: 50 mM sodium phosphate (pH 7.5), 5 mM DTT, 1 mM EDTA and 5% glycerol.

[0115] 6. Structural characterization of 3A1THR-TEV lyophilized powder

[0116] The 3A1THR-TEV lyophilized powder was reconstituted and acid-hydrolyzed. The processed sample was analyzed by liquid chromatography-mass spectrometry (LC-MS), and the peak area of ​​the targeted data was calculated using MassLynx quantitative software. The identification results were obtained using the standard curve method. The results showed that the hydroxyproline content reached more than 40% of the total proline.

[0117] Dilute the 3A1THR-TEV lyophilized powder with a blank control solution to a concentration of 0.2 μg / μL.

[0118] Circular dichroism chromatography (CD) detection: Parameters were set as follows: Beginning wavelength 190 nm, Ending wavelength 260 nm, Step size 1 nm, Repeat 1 time, Acquisition period 1 s / point, and cuvette width 0.1 cm. A blank control solution was loaded at a volume of 300 μL and offset after measurement. The sample was then tested at a volume of 300 μL, and the data were saved after testing. The results are shown in Figure 13, with a negative peak at 195 nm and a positive peak at 221 nm, consistent with the CD characteristics of triple-helical collagen.

[0119] After negative staining, collagen samples were examined by transmission electron microscopy. The test results are shown in Figure 14, which shows the typical structural features of collagen microfibers with alternating light and dark characteristics.

[0120] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. An engineered bacterium KM71 / 3A1FL-1#-Z expressing triple-helix type III recombinant human collagen. S The method for constructing -P4H is characterized by, This involves transforming the pMChZ-3A1FL plasmid and the pPIC9K-P4H(DP)-1 plasmid into the host bacteria to construct the engineered strain KM71 / 3A1FL-1#-Z. S -P4H steps; The pMChZ-3A1FL plasmid was constructed by ligating the type III collagen α1 chain DNA sequence into the pMChZ-AOX plasmid; The pMChZ-AOX plasmid is obtained by modifying the pPIC9K plasmid as follows: replacing 4647-9266 bp of the pPIC9K plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.1; deleting BamH Ⅰ before αMF and αMF in the pPIC9K plasmid through point mutation; and introducing a BamH Ⅰ recognition site between 1583-1584 bp of the pPIC9K plasmid through point mutation. The method for constructing the pPIC9K-P4H(DP)-1 plasmid includes replacing KanR in the P4H 9K DP plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.5 to obtain the pPIC9K-P4H(DP)-1 plasmid.

2. The construction method according to claim 1, characterized in that, The host organism is Pichia pastoris.

3. A method for constructing an engineered bacterium KM71 / 3A1NproΔ-2#-P4H expressing a triple-helix type III recombinant human collagen, characterized in that, The process includes transforming pMChZ-3A1NproΔ plasmid, pMCrZ-3A1NproΔ plasmid and pPIC9K-P4H(DP)-1 plasmid into the host bacteria to construct the engineered bacteria KM71 / 3A1NproΔ-2#-P4H; The pMChZ-3A1NproΔ plasmid is constructed by linking 3A1NproΔ into the pMChZ-AOX plasmid; The pMCrZ-3A1NproΔ plasmid is constructed by linking the 3A1NproΔ plasmid into the pMCrZ-AOX plasmid; The nucleotide sequence of the 3A1NproΔ is shown in SEQ ID NO.7; The pMChZ-AOX plasmid was obtained by modifying the pPIC9K plasmid as follows: replacing 4647-9266 bp of the pPIC9K plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.1; deleting BamH Ⅰ before αMF and αMF in the pPIC9K plasmid through point mutation; and introducing a BamH Ⅰ recognition site between 1583-1584 bp of the pPIC9K plasmid through point mutation. The pMCrZ-AOX plasmid was constructed by replacing the PpHIS4 on the pMChZ-AOX plasmid with the DNA sequence of nucleotide sequence as shown in SEQ ID NO.2; The method for constructing the pPIC9K-P4H(DP)-1 plasmid includes replacing KanR in the P4H 9K DP plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.5 to obtain the pPIC9K-P4H(DP)-1 plasmid.

4. The construction method according to claim 3, characterized in that, The host organism is Pichia pastoris.

5. A method for constructing an engineered bacterium KM71H / 3A1THR-TEV-2#-P4H expressing triple-helix type III recombinant human collagen, characterized in that, The process includes transforming pMChZ-3A1THR-TEV plasmid, pMCrZ-3A1THR-TEV plasmid and pPIC9K-P4H(DP)-1 plasmid into the host bacteria to construct the engineered bacteria KM71H / 3A1THR-TEV-2#-P4H; The pMChZ-3A1THR-TEV plasmid is constructed by linking 3A1THR-TEV into the pMChZ-AOX plasmid; The pMCrZ-3A1THR-TEV plasmid is constructed by linking the 3A1THR-TEV into the pMCrZ-AOX plasmid; The nucleotide sequence of the 3A1THR-TEV is shown in SEQ ID NO.9; The pMChZ-AOX plasmid was obtained by modifying the pPIC9K plasmid as follows: replacing 4647-9266 bp of the pPIC9K plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.1; deleting BamH Ⅰ before αMF and αMF in the pPIC9K plasmid through point mutation; and introducing a BamH Ⅰ recognition site between 1583-1584 bp of the pPIC9K plasmid through point mutation. The pMCrZ-AOX plasmid was constructed by replacing the PpHIS4 on the pMChZ-AOX plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.2; The method for constructing the pPIC9K-P4H(DP)-1 plasmid includes replacing KanR in the P4H 9K DP plasmid with a DNA molecule whose nucleotide sequence is shown in SEQ ID NO.5 to obtain the pPIC9K-P4H(DP)-1 plasmid.

6. An engineered bacterium expressing type III recombinant human collagen expressed by the construction method according to any one of claims 1-5.

7. The application of the engineered bacteria as described in claim 6 in the preparation of triple-helix type III recombinant human collagen.

8. A method for preparing recombinant human collagen of type III triple helix structure expressed in yeast, characterized in that, The method includes the step of using the engineered bacteria described in claim 6 to ferment and purify the triple-helix type III recombinant human collagen.

9. A type III recombinant human collagen with a triple helix structure prepared by the preparation method according to claim 8.

10. The application of the triple-helix type III recombinant human collagen as described in claim 9 in the preparation of biopharmaceutical products, characterized in that, The biomedical products mentioned are skin care products, skin repair dressings, implants, artificial skin, biomaterials and / or medical devices.

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