Method for producing human structural materials by biosynthesis

Abstract

The present invention provides a method for manufacturing a human structural material by biosynthesis. The polypeptide of the present invention has a structure in the (repeating unit) n or (repeating unit) n -including the structure of the C-terminal region, and the repeating unit contains the amino acid sequence shown in SEQ ID NO: 1. The recombinant humanized type V collagen obtained in the present invention has high activity in promoting cell adhesion, does not cause an immune response even when applied to the human body, and further, the manufacturing method has inventiveness, enabling mass production of recombinant humanized type V collagen, and can be widely used in the manufacture of human structural materials. Its application fields include the manufacture of advanced medical devices such as biological coatings, human biomimetic materials, cosmetic and plastic materials, organoid culture, cardiovascular stents, coatings, tissue injection filling, ophthalmic materials, obstetrics and gynecology biological materials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, and biological materials for 3D printed artificial organs.

Description

Technical Field

[0001] This application claims priority to the invention titled "Method for Producing Human Structural Materials by Biosynthesis" with Chinese Application No.: 202210849498.3 filed on July 19, 2022, and the entire disclosure content thereof is incorporated herein by reference.

[0002] The present invention belongs to the technical field of synthetic biology and relates to a method for producing human structural materials by biosynthesis.

Background Art

[0003] Human structural materials are mainly structural proteins containing collagen. Such proteins have an adhesion function and a support function for cells and tissues and are the main components of the extracellular matrix. Collagen is a type of protein widely distributed in the connective tissues of the human body and is the most abundant protein in the human body, accounting for 25% - 35% of the total protein. Currently, at least 28 subtypes of collagen have been found to be present in different tissues and organs. Among them, type V collagen belongs to fibrous collagen and is always accompanied by the expression of type I collagen, but has a low content and unique physiological functions. For example, it can inhibit the adhesion and proliferation of epidermal, endothelial, smooth muscle, and cancer cells, and can be used in the fields of medicine, materials, and biomedicine by combining with bioactive substances such as heparin, insulin, osteonectin, vascular endothelial growth inhibitor, macrophage colony-stimulating factor, etc.

[0004] In recent years, crude type V collagen has been extracted mainly from various types of seafood, placentas, kidneys, and other tissues. However, type V collagen is a minor type of collagen with low levels of content in the body, and its extraction process is complex. At the same time, animal-derived immune responses are also a limiting factor in the application of collagen. With the growth of China's collagen industry, the use of biosynthetic pathways to obtain collagen is becoming increasingly mature, and humanized collagen in particular is at the forefront of global research. In 2021, the National Medical Products Administration named and classified biosynthetic collagen. Among these, recombinant humanized collagen is a combination of full-length or partial amino acid sequence fragments encoded by specific types of human collagen genes prepared using DNA recombination technology, or fragments containing the function of human collagen.

[0005] Type V collagen was first discovered in human placenta and skin. It contains three α-chains with different strengths and mobility, forming four distinct subtypes within tissues: α1α1α2(V), α1α2α3(V), α3α3α3(V), and mixtures of type V collagen α-chains and type XI α-chains. The three different α-chains form three string-like procollagen molecules, which are then enzymatically broken down extracellularly to form elongated collagen fibers. These elongated fibers cross-link with each other in the space around the cell, forming a high-strength mature type V collagen fiber network after cross-linking, or forming heterotrimers with other types of collagen such as type XI collagen. It is distributed in areas such as the cornea, skin, ligaments, bones, tendons, and muscles. Type V collagen shares common structural features with fibrous collagen, and its central region contains an uninterrupted sequence of over 1000 glycine-Xaa-Yaa sequences, where Xaa-Yaa are any of the amino acids or imino acid residues. Structurally speaking, the natural type V collagen in the human body has a very complex structure, making it difficult to express humanized collagen using conventional methods and thus difficult to prepare in large quantities.

[0006] Currently, type V collagen is mainly produced by processing animal-derived tissues via pepsin and extracting collagen derivatives. However, collagen extracted using these methods loses its original biological activity and cannot perform its intended function. With the advancement of modern biotechnology, people can now prepare recombinant humanized collagen in animal, plant, and microbial expression systems through gene transfer technology, overcoming the shortcomings of enzymatic degradation methods. However, to date, success in obtaining recombinant humanized type V collagen has not been achieved. Therefore, there is an urgent need for a biosynthesis method for recombinant humanized type V collagen so that it can be widely used as a structural material for the human body. [Overview of the project] [Means for solving the problem]

[0007] The inventors, considering the technical challenges in the prior art, have for the first time designed a screening process for the functional region of recombinant humanized type V collagen and a protein synthesis process. The inventors identified the amino acid sequence of the core region of type V collagen (SEQ ID NO: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv) and constructed a polypeptide containing multiple repeating units using this core region as a repeating unit. The inventors found that the constructed polypeptide possesses cell adhesion-promoting activity. The inventors also found that the core region can be extended with one or more amino acids as repeating units at its N-terminus and / or C-terminus, and that the constructed polypeptide containing multiple repeating units also possesses cell adhesion-promoting activity. Furthermore, outside the repeating units, the polypeptide may contain peptide segments of a certain length outside the repeating units (e.g., at the N-terminus and / or C-terminus of a polypeptide consisting of repeating units). These peptide segments may be consecutive amino acid segments starting from the first position of the repeating unit.

[0008] In one embodiment, the present invention is (repeating unit) n or (repeating unit) n- A polypeptide comprising a C-terminal region structure is provided, wherein the repeating unit comprises the amino acid sequence shown in SEQ ID NO: 1, or the repeating unit comprises the amino acid sequence shown in SEQ ID NO: 1 and additional amino acid residues at the N-terminus and / or C-terminus of SEQ ID NO: 1, with the number of additional amino acid residues being 1 to 50. The polypeptide of the present invention is recombinant humanized type V collagen.

[0009] In one embodiment, each repeating unit is directly bonded, the number of repeating units n is 4 to 20, and the C-terminal region is a continuous amino acid segment from the 1st position of the repeating unit.

[0010] In one embodiment, the additional amino acid residue is located at the C-terminus of SEQ ID NO: 1 and is the amino acid sequence (galglk) shown in SEQ ID NO: 7 or a consecutive amino acid segment of said amino acid sequence. For example, the additional amino acid residue is a consecutive amino acid segment starting from position 1 of the amino acid sequence shown in SEQ ID NO: 7.

[0011] In one embodiment, the repeating unit includes one of the amino acid sequences of sequence numbers 2, 3, and 8-11.

[0012] In one embodiment, the polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 4-6.

[0013] In another embodiment, the present invention provides nucleic acids comprising nucleotides of the polypeptide of the present invention.

[0014] In one embodiment, the nucleic acid further comprises nucleotides encoding a purified tag such as a His tag, GST tag, MBP tag, SUMO tag, or NusA tag.

[0015] In one embodiment, the nucleic acid further comprises a nucleotide encoding a leader sequence.

[0016] In another embodiment, the present invention provides a vector comprising nucleic acid according to the present invention.

[0017] In one embodiment, the vector contains expression control elements such as a promoter, a terminator, and / or an enhancer that are operably connected to a nucleic acid.

[0018] In another aspect, the present invention provides a host cell containing the nucleic acid or vector of the present invention. In one embodiment, the host cell is a eukaryotic cell or a prokaryotic cell. In one embodiment, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell. In one embodiment, the prokaryotic cell is an Escherichia coli cell.

[0019] In another aspect, the present invention provides for the production of the polypeptide of the present invention comprising the following.

[0020] (1) Culturing the host cell of the present invention under appropriate culture conditions;

[0021] (2) Recovering the host cell and / or the medium containing the polypeptide; and

[0022] (3) polypeptide Purifying.

[0023] In another aspect, the present invention provides a composition containing the polypeptide, nucleic acid, vector, and / or host cell of the present invention. ​​​​​​​​​​In another aspect, the present invention provides for the use of the polypeptide, nucleic acid, vector, host cell and / or composition of the present invention in the manufacture of advanced medical devices such as bio-coating materials, human biomimetic materials, cosmetic and plastic materials, organoid culture, cardiovascular stents, coatings, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration, liver tissue and vascular repair and regeneration, and biomaterials for 3D printed artificial organs, as well as in high-end cosmetic raw materials, advanced pharmaceutical adjuvants, and food additives.

[0027] The present invention discloses the specific processes of screening and synthesis of the functional regions of recombinant humanized type V collagen, which can be used in the manufacture of human structural materials. The present invention belongs to the technical field of synthetic biology. The present invention discloses the specific processes of screening and synthesis of the functional regions of recombinant humanized type V collagen, which can be used in the manufacture of human structural materials. The present invention belongs to the technical field of synthetic biology. The biosynthetic method for preparing recombinant humanized type V collagen in the present invention includes: (1) screening of functional regions and construction of strains; (2) biological fermentation, induction and expression; and (3) purification of humanized type V collagen and optional enzymatic cleavage. The amino acid sequence of the recombinant humanized collagen produced by the present invention is derived from the functional region of human native type V collagen, and includes proteins in which the functional region and similar functional regions, as well as the amino acid sequences, are mutated and modified respectively. The recombinant humanized type V collagen produced by the present invention has a high activity of promoting cell adhesion, is applied so as not to cause an immune response in the human body, and its preparation method is novel. Recombinant humanized type V collagen can be obtained in large quantities and is widely used in the preparation of human structural materials. Its application fields include the manufacture of advanced medical devices such as bio-coating materials, human biomimetic materials, cosmetic and plastic materials, organoid culture, cardiovascular stents, coatings, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration, liver tissue and vascular repair and regeneration, and biomaterials for 3D printed artificial organs, as well as high-end cosmetic raw materials, advanced pharmaceutical adjuvants, food additives, etc.

[0028] The present invention provides the following. <1> In response to the current state of research, the present invention provides a method for producing recombinant humanized type V collagen by biosynthesis, that is, a method for producing human structural materials, which specifically includes (1) screening of functional regions and construction of bacterial strains, (2) large-scale biological fermentation culture and induction of protein expression, and (3) purification of humanized type V collagen and optional enzymatic cleavage. <2> <1> According to the report, functional region screening and strain construction can be performed as follows: (1) a large-scale functional region screening is conducted to obtain the functional region of the target gene; (2) the obtained functional region of the target gene is inserted into a PET-32a expression vector to obtain a recombinant expression plasmid; and (3) the recombinant expression plasmid is introduced into competent cells of E. coli BL21 (DE3), and positive recombinant E. coli can be obtained by screening. <3> <1> According to the report, large-scale biological fermentation can be carried out by adding positive genetically modified E. coli obtained through screening to a flask containing an antibiotic reservoir and culturing it in a constant temperature shaker at 220 rpm and 37°C for 7 hours. <4> <1> According to the report, protein expression induction can be performed by (1) cooling the culture flask to 16°C, (2) adding IPTG stock solution to induce expression, and (3) placing the bacterial suspension after expression induction into a centrifuge bottle and centrifuging at 8000 rpm and 4°C for 10 minutes to collect the bacterial cells. <5> <1> According to the report, the purification and enzymatic cleavage of humanized type V collagen can be performed by (1) crudely purifying humanized type V collagen using a Ni affinity chromatography column, (2) adding a certain proportion of TEV enzyme to perform enzymatic cleavage, and (3) re-purifying the humanized type V collagen using an ion exchange column. <6> <2> According to the study, the screened functional regions are as follows: (1) C5V3G1 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgd pgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpa glpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglkgkegtkgdpgpaglpgk dgppglrgfpgdrglpgpvgalglk, (2) C5V3G2 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkd gppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgd rglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal,(3) C5V3G3 amino acid sequence: gkegtkgdpg paglpgkdgppglrgfpgdrglpgpvgkegtkgdpg paglpgkdgppglrgfpgdrglpgpvgkegtkgdpg paglpgkdgppglrgfpgdrglpgpvgkegtkgdpg paglpgkdgppglrgfpgdr glpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv. <7> The amino acid sequence of recombinant humanized type V collagen produced in this invention is derived from the functional region of natural human type V collagen, and includes proteins in which the said functional region, similar functional regions, and amino acids have been mutated and modified. <8> The recombinant humanized type V collagen produced by this invention has applications in the following fields: biomimetic materials, human biomimetic materials, cosmetic surgery materials, organoid culture, cardiovascular stents, coatings, myocardial repair, tissue injection and filling, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, biomaterials for 3D printed artificial organs, tumor prevention, bone repair materials, skin repair materials, kidney tissue repair, pancreatic repair, and the manufacture of advanced medical devices; high-grade cosmetic raw materials and advanced pharmaceutical adjuvants, food additives, etc.

[0029] Compared to prior art, the present invention has the following advantages.

[0030] <1> This invention provides the core functional region and amino acid sequence of recombinant humanized type V collagen.

[0031] <2> This invention has successfully achieved the first biosynthesis of recombinant humanized type V collagen.

[0032] <3> The recombinant humanized type V collagen produced by this invention has good cell adhesion properties and does not trigger an immune response when applied to the human body.

[0033] <4> The manufacturing method is simple, and recombinant humanized type V collagen can be obtained on a large scale. [Brief explanation of the drawing]

[0034] [Figure 1] These are electrophoresis images of crudely purified recombinant humanized type V collagen C5V3G1 and C5V3G2. The electrophoresis images after each purification step are also shown. [Figure 2] Electrophoresis graphs of crudely purified recombinant humanized type V collagen C5V3G3 and C5V3G1, C5V3G2, and C5V3G3 after enzymatic cleavage (enzyme treatment). Electrophoresis graphs after each purification step are shown. [Figure 3] Electrophoretic graph of re-purified recombinant humanized type V collagen C5V3G1, C5V3G2, and C5V3G3. [Figure 4] This diagram shows the cell adhesion activity of recombinant humanized type V collagen C5V3G1. [Figure 5] This diagram shows the cell adhesion activity of recombinant humanized type V collagen C5V3G2. [Figure 6] This diagram shows the cell adhesion activity of recombinant humanized type V collagen C5V3G3. [Modes for carrying out the invention]

[0035] To further clarify the object, technical solution, and advantages of the present invention, the technical solution in the embodiments of the present invention will be described below clearly and completely together with the embodiments. Clearly, the embodiments described are not all embodiments, but rather some embodiments of the present invention. All other embodiments that a person skilled in the art could easily obtain by any effort without inventive step based on the embodiments described herein are all within the scope of the protection of the present invention.

[0036] As used herein, a polypeptide means a plurality of amino acid residues linked by peptide bonds. In this specification, a polypeptide comprises a plurality of repeating units derived from human type V collagen (GenBank:KAI4009058.1). A polypeptide is (a repeating unit) n or (repeating unit) n -The structure of the C-terminal region may be included, and the repeating unit includes the amino acid sequence shown in SEQ ID NO: 1, or includes the amino acid sequence shown in SEQ ID NO: 1 and additional amino acid residues at the N-terminus and / or C-terminus of SEQ ID NO: 1, the number of such additional amino acid residues being 1 to 50. For example, the number of such additional amino acid residues is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49. The additional amino acid residues may be derived from human type V collagen. For example, the additional amino acid residues may be a continuous amino acid segment directly adjacent to the repeating unit in situ of natural human type V collagen. The additional amino acid residue may be the amino acid sequence shown in SEQ ID NO: 7 or a continuous amino acid segment of said amino acid sequence. Preferably, the additional amino acid residue is a continuous amino acid segment starting from position 1 of the amino acid sequence shown in SEQ ID NO: 7. In this specification, the additional amino acid residue may be located at the C-terminus.

[0037] In this specification, each repeating unit may be directly connected or separated by one or more amino acid residues. The number n of repeating units may be 4 to 20, for example, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19. The polypeptide may include a C-terminal region. This C-terminal region may be a continuous amino acid segment from position 1 of the repeating unit. The repeating unit may include the amino acid sequence shown in either SEQ ID NO: 2 or 3.

[0038] As used herein, the term "segment," when used in relation to amino acids, refers to a portion of a sequence smaller than a specific sequence. For example, an amino acid segment in SEQ ID NO: 7 means any subsequence of a length shorter than the length of the sequence shown in SEQ ID NO: 7. A "consecutive amino acid segment" refers to an amino acid segment consisting of directly adjacent amino acids in a sequence.

[0039] As used herein, with respect to a polypeptide or a particular amino acid sequence, “C-terminus” and “N-terminus” mean a position relative to the polypeptide or the particular amino acid sequence, specifically, a position at the carboxyl-containing or amino-containing end of the polypeptide or the particular amino acid sequence.

[0040] As used herein, when referring to a polypeptide or a specific amino acid sequence, its position is described relative to the C-terminus of the polypeptide or specific amino acid sequence. For example, when referring to "starting from position 1 of the amino acid sequence (galglk) shown in SEQ ID NO: 7," the amino acid at position 1 refers to G.

[0041] In this specification, the repeating unit of the present invention may include the following sequences, or the following sequences. gkegtkgdpg paglpgkdgppglrgfpgdrglpgpv(Sequence 1) gkegtkgdpg paglpgkdgppglrgfpgdrglpgpvgal(Sequence No. 2) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk(Sequence No. 3) gkegtkgdpg paglpgkdgppglrgfpgdrglpgpvg(Sequence 8) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvga(Sequence 9) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalg(Sequence No. 10) gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalgl(Sequence No. 11)

[0042] As used herein, “nucleic acid” refers to a group of nucleotides linked together by internucleotide links. The internucleotide links may be, for example, phosphodiester bonds. The nucleic acid herein may include polynucleotides encoding the polypeptide of the present invention. To facilitate the post-processing of the polypeptide, the nucleic acid of the present invention may further include nucleotides encoding purified tags such as His tags, GST tags, MBP tags, SUMO tags, or NusA tags, and optionally nucleotides encoding a reader sequence.

[0043] As used herein, the term “vector” refers to a nucleic acid carrier into which polynucleotides can be inserted. A vector is called an expression vector if it can express a protein encoded by the inserted polynucleotide. A vector can be introduced into a host cell by transformation, transduction, or transfection to express the genetic material factor it carries within the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, e.g., yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); phages, e.g., λ phages or M13 phages; and animal viruses; etc. A vector may contain multiple types of expression regulatory elements. These expression regulatory elements include, but are not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporting genes. A vector may also further include a replication origin site. A vector may contain the nucleic acids of the present invention to facilitate introduction into cells for expression. The vector may include expression control elements, such as promoters, terminators, and / or enhancers, that are operablely connected to the nucleic acid.

[0044] As used herein, the term “host cell” refers to a cell into which nucleic acid molecules have been introduced by molecular biology techniques. These techniques include transfection with viral vectors, transformation with plasmid vectors, and rapid introduction of naked DNA by electroporation, lipofection, and particle guns. The host cell may be a eukaryotic cell or a prokaryotic cell. For example, eukaryotic cells are yeast cells, animal cells, and / or insect cells. Prokaryotic cells may be Escherichia coli cells.

[0045] In this specification, certain mutations may exist in each part of the polypeptide of the present invention, for example, in the repeating unit or the C-terminal region. For example, one or more of these amino acid sequences may have substitutions, deletions, additions, or insertions of amino acid residues. That is, the present invention can use mutations in each part, such as repeating unit mutations or C-terminal region mutations, as long as they retain the activity that promotes cell adhesion. Specifically, the mutations can have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with respect to a particular sequence. The particular sequence may be any sequence in the present invention, for example, SEQ ID NOs: 1 to 6, but it is preferable that these mutations retain the core sequence identified in the present invention.

[0046] The polypeptide of the present invention can be prepared by any suitable method, for example, by synthesis. Preferably, the polypeptide of the present invention can be prepared by recombinant. The production method may include one or more of the following steps: (1) culturing the host cells described in claim 8 under suitable culture conditions; (2) obtaining host cells and / or culture medium containing the polypeptide; and (3) polypeptideThis is a process for purifying the polypeptide. More specifically, a recombinant expression vector is obtained by inserting the sequence encoding the polypeptide of the present invention into a suitable expression vector, such as PET-28A. The recombinant expression plasmid is introduced into competent cells of E. coli BL21 (DE3), and positive genetically modified E. coli are obtained by screening. The obtained positive genetically modified E. coli can then be subjected to large-scale biological fermentation (e.g., cultured in a constant temperature shaker at 220 rpm and 37°C for 7 hours). When culturing to an appropriate cell density, protein expression can be induced within the cells. For example, in the case of genetically modified E. coli, (1) the flask after culture is cooled to 16°C, (2) IPTG stock solution is added to induce expression, and (3) the bacterial suspension after expression induction is placed in a centrifuge bottle, and the cells are collected after centrifugation at 8000 rpm and 4°C for 10 minutes. The cells are lysed (e.g., by high-pressure homogenization) to obtain a cell lysate, and then the supernatant is obtained by centrifugation. The supernatant is purified to obtain the purified polypeptide. For example, the purification process may include one or more of the following steps: (1) crude purification of the polypeptide using a Ni affinity chromatography column; (2) enzymatic cleavage by adding a TEV enzyme in a fixed proportion; and (3) re-purification of the polypeptide using an ion exchange column.

[0047] The polypeptides, nucleic acids, vectors, and / or host cells of the present invention can be prepared as compositions or kits. The compositions or kits may include one or more of the following: biomimetic coatings, biomimetic materials, cosmetic surgery materials, organoid culture materials, cardiovascular stent materials, coatings, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, biomaterials for 3D-printed artificial organs, cosmetic raw materials, pharmaceutical adjuvants, and food additives. The compositions or kits may also include one or more of the following: biomimetic coatings, biomimetic materials, cosmetic surgery materials, organoid culture materials, cardiovascular stent materials, coatings, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and vascular repair and regeneration materials, biomaterials for 3D-printed artificial organs, cosmetic raw materials, pharmaceutical adjuvants, and food additives. These compositions or kits can be used to promote cell adhesion in vitro or in vivo. [Examples]

[0048] The present invention will be described in more detail below with reference to examples. Those skilled in the art should understand that these examples are merely illustrative and not limiting. The present invention is limited only by the claims.

[0049] Example 1: Construction and expression of recombinant humanized type V collagen <1> A large-scale functional region screening was conducted, and the following target gene fragments of recombinant humanized type V collagen were identified: (1) C5V3G1 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk (C5V3G1 amino acid sequence: Sequence ID 6; the repeating unit is Sequence ID 3: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgalglk);

[0050] (2) C5V3G2 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal (C5V3G2 amino acid sequence: Sequence ID 5; the repeating unit is Sequence ID 2: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpvgal);

[0051] (3) C5V3G3 amino acid sequence: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv (C5V3G3 amino acid sequence: Sequence ID 4; the repeating unit is Sequence ID 1: gkegtkgdpgpaglpgkdgppglrgfpgdrglpgpv).

[0052]

[0053] <2> Competent cells of E. coli BL21(DE3) were transformed with the constructed expression plasmid. Specifically, (1) Competent cells of E. coli BL21(DE3) were removed from the Cryogenic Freezer, placed on ice, 2 μl of the plasmid to be transformed when half-thawed was taken and added to the competent cells of E. coli BL21(DE3), and gently mixed 2-3 times. (2) The mixture was placed on ice and subjected to an ice bath for 30 minutes, then subjected to a heat shock in a 42°C water bath for 45-90 seconds, removed and placed on ice and subjected to an ice bath for 2 minutes. (3) The cells were transferred to a biological safety cabinet, 700 μl of liquid LB medium was added, and the cells were incubated at 37°C and 220 rpm for 60 minutes. (4) 200 μl of bacterial suspension was taken and uniformly spread onto an LB plate containing ampicillin sodium. (5) The plates were incubated in a 37°C incubator for 15-17 hours until colonies of uniform size were obtained.

[0054] <3> Five to six single colonies were picked from the transformed LB plates and transferred to flasks containing antibiotic solution. They were incubated in a constant temperature shaker at 220 rpm and 37°C for 7 hours. After incubation, the flasks were cooled to 16°C, IPTG was added to induce expression, and after a certain period, the bacterial suspension was dispensed into centrifuge bottles and centrifuged at 8000 rpm and 4°C for 10 minutes. The bacterial cells were collected, their weight was recorded, and samples were taken for electrophoresis (see the "bacterial cells" lane in Figures 1 and 2).

[0055] <4> The collected bacterial cells were resuspended in a suitable buffer solution, the bacterial suspension was cooled to below 15°C, and homogenization and high-pressure homogenization were performed twice. After completion, the bacterial suspension was collected. The homogenized bacterial suspension was dispensed into centrifuge bottles and centrifuged at 17,000 rpm at 4°C for 30 minutes. The supernatant was collected, and the supernatant and precipitate were sampled and subjected to electrophoresis (see Figures 1 and 2; "Homogenized II" is the electrophoresis diagram of the bacterial suspension after two high-pressure homogenizations, and "Supernatant" and "Precipitate" are the electrophoresis diagrams of the supernatant and precipitate, respectively).

[0056] <5> The purification and enzymatic cleavage of recombinant humanized type V collagen were specifically carried out as follows: (1) Crude purification: a. Wash the column with water; b. Equilibrate the column; c. Load the sample: Place the centrifuged supernatant into the column and perform electrophoresis by sampling the flow-through until all the liquid has drained out (see the "flow-through" lane in Figures 1 and 2); d. Washing of impurities: Add 25 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) and perform electrophoresis by sampling the flow-through until all the liquid has drained out (see the "washing" lane in Figures 1 and 2); e. Recovery of the target protein: Add 20 mL of eluate and collect the flow-through to obtain the target protein containing the His tag. The protein concentration was detected by ultraviolet-visible spectrophotometric analysis, and the protein concentration was determined using the following formula (C(mg / ml) = A280 × dilution factor × extinction coefficient), and electrophoresis was performed (see the "elution" lane in Figures 1 and 2); f. The column was washed with 1M imidazole buffer; g. The column was washed with purified water. (2) Enzymatic cleavage: The ratio of total protein amount to total TEV enzyme amount was 20:1, TEV enzyme was added, enzymatic cleavage was performed at 16°C for 2 hours, and samples were taken and electrophoresis was performed. The enzymatically cleaved protein solution was placed in a dialysis bag, dialyzed at 4°C for 2 hours, and then transferred to a new dialysate and dialyzed overnight at 4°C (see Figure 3 for the electrophoresis diagram after the liquid exchange). (3) Re-purification: a. Column equilibration: The column was equilibrated using Solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 ml / min; b. Sample loading: The sample was loaded at a flow rate of 5 mL / min, and the flow-through (denoted as FL1, see Figure 3) was collected to obtain recombinant humanized type V collagen C5V3G1, C5V3G2, and C5V3G3. Electrophoresis was performed as shown in Figure 3; c. Elution: 100% Solution B (20 mM Tris, 1 M sodium chloride, pH 8.0) was set to elution lane 3CV, the peak was collected, and electrophoresis was performed (for the Solution B eluate, see the "Solution B Elution" lane in Figure 3); d. The column was washed; e. The proteins were stored at 4°C.Figure 3 shows that the apparent molecular weights of recombinant humanized type V collagen C5V3G1, C5V3G2, and C5V3G3 are 32 kDa, 29 kDa, and 34 kDa, respectively, which are consistent with the predicted molecular weights.

[0057] Example 2: Detection of the bioactivity of recombinant humanized type V collagen For methods of detecting collagen activity, please refer to the literature: 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). Specific implementation methods can be exemplified below.

[0058] (1) The concentrations of the protein samples to be measured, such as bovine type I collagen (National Institutes for Food and Drug Control, No.: 380002; 0.5 mg / mL, positive control well) and recombinant humanized type V collagen C5V3G1, C5V3G2, and C5V3G3 provided in the present invention, were detected by ultraviolet absorption method.

[0059] Specifically, the absorption of ultraviolet light at 215 nm and 225 nm of the sample was measured, and the protein concentration was determined using the empirical formula C(μg / M) = 144 × (A215 - A225) (detection is necessary when A215 < 1.5). The principle of this method is to measure the characteristic absorption of peptide bonds in far ultraviolet light, and it is unaffected by the content of chromophore, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not develop Coomassie brilliant blue color (Reference: Walker JM. The Protein Protocols Handbook, second edition. HumanaPress. 43-45). After detecting the protein concentration, the total measured protein concentration was adjusted to 0.25 mg / mL, 0.5 mg / mL, or 1 mg / mL using PBS.

[0060] (2) 100 μL each of the protein solutions and a blank PBS control (see the D-PBS group in Figures 4-6) were added to a 96-well plate and left at room temperature for 60 minutes.

[0061] (3) Place 10 well cultured 3T3 cells into each well. 5 The eggs were individually placed in containers and incubated at 37°C for 60 minutes.

[0062] (4) Each well was washed four times with PBS.

[0063] (5) The absorbance at OD492nm was detected using an LDH detection kit (Roche, 04744926001). The cell adhesion rate can be calculated from the values ​​of the blank control. The calculation formula is as follows:

number

[0064] As shown in Figures 4, 5, and 6, the recombinant humanized type V collagen of the present invention was found to have superior bioadhesion activity compared to bovine type I collagen (BColI group, 0.5 mg / ml). Figure 4 shows the relative cell adhesion activity of C5V3G1 to bovine type I collagen, revealing that C5V3G1 has cell adhesion activity and exhibits higher cell adhesion activity than bovine type I collagen at concentrations of 0.25, 0.5, and 1 mg / ml. Figure 5 shows the relative cell adhesion activity of C5V3G2 to bovine type I collagen, revealing that C5V3G2 exhibits better cell adhesion activity at concentrations of 0.25 mg / mL and above. Figure 6 shows the relative cell adhesion activity of C5V3G3 to bovine type I collagen, revealing that C5V3G3 has cell adhesion activity and exhibits higher cell adhesion activity than bovine type I collagen at a concentration of 0.5 mg / ml.

[0065] Example 3: Detection of recombinant humanized type V collagen by mass spectrometry [Experimental Method]

number

[0066] Protein samples were reduced with DTT, alkylated with iodoacetamide, and then enzymatically digested overnight with trypsin. The enzymatically digested peptide segments were further desalted with C18 ZipTip and then spotted after being mixed with the matrix, α-cyano-4-hydroxycinnamic acid (CHCA). Finally, the samples were analyzed using a matrix-assisted laser desorption / ionization time-of-flight mass spectrometer (MALDI-TOF / TOF Ulraflextreme). TMThe analysis was performed by Brucker, Germany (the peptide fingerprinting technique can be found in Protein J. 2016;35:212-7). Data retrieval was performed using the MS / MS Ion Search screen from the local masco site. Protein identification results were obtained from primary mass spectrometry of enzymatically cleaved peptide segments. Detection parameters: Enzymatic hydrolysis with trypsin was performed, creating two missing cleavage sites. Cysteine ​​alkylation was set as a fixed modification. Methionine oxidation is a variable modification. The database used for identification was NCBprot.

[0067] [Table 1]

[0068] The detected polypeptide segment coverage was 100%, consistent with the theoretical sequence, and the detection results are highly reliable.

[0069] [Table 2]

[0070] The detected polypeptide segments had an 83% coverage rate compared to the theoretical sequence, making the detection results highly reliable.

[0071] [Table 3]

[0072] The detected polypeptide segments had a coverage rate of 96.11% compared to the theoretical sequence, making the detection results highly reliable.

[0073] While the above embodiments represent preferred embodiments of the present invention, the embodiments of the present invention are not limited to these embodiments. Any modifications, alterations, substitutions, combinations, or simplifications that do not deviate from the spirit and principles of the present invention are all equivalent substitutions and fall within the scope of protection of the present invention.

Claims

1. (Repeating unit) ｎ Includes, A polypeptide in which each repeating unit is directly bonded, the number of repeating units n is 4 to 10, and each repeating unit contains an amino acid sequence that is 90% to 99% identical to the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3 and 8 to 11, A polypeptide that has cell adhesion-promoting activity and is recombinant humanized type V collagen.

2. The polypeptide according to claim 1, comprising an amino acid sequence shown in any one of SEQ ID NOs: 4 to 6, or an amino acid sequence having 90% to 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 4 to 6.

3. comprising a nucleotide encoding the polypeptide described in claim 1, Optionally further comprising a nucleotide encoding a purified tag, wherein the purified tag is a His tag, GST tag, MBP tag, SUMO tag, or NusA tag. Optionally, a nucleic acid further comprising nucleotides that encode a leader sequence.

4. comprising the nucleic acid described in claim 3, A vector optionally comprising an expression regulator operably attached to the nucleic acid, wherein the expression regulator is optionally a promoter, terminator, and / or enhancer.

5. comprising the nucleic acid described in claim 3, They may be eukaryotic or prokaryotic cells. A host cell in which, optionally, the eukaryotic cell is a yeast cell, an animal cell, and / or an insect cell, and / or the prokaryotic cell is an Escherichia coli cell, and optionally, the Escherichia coli cell is Escherichia coli BL21.

6. A method for producing the polypeptide described in claim 1 or 2, (1) Culturing host cells containing the vector described in claim 4 under appropriate culture conditions, (2) Recovering host cells and / or culture medium containing polypeptides, (3) Purification of polypeptides, optionally: 1) crude purification of polypeptides using a Ni affinity chromatography column; 2) enzymatic cleavage by adding TEV enzyme in a certain proportion; 3) re-purification of polypeptides using an ion exchange column. Methods that include...

7. A composition comprising the polypeptide according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4, and / or the host cell according to claim 5.

8. The composition according to claim 7, which is one or more of the following: bio-coating materials, human biomimetic materials, cosmetic surgery materials, organoid culture materials, cardiovascular stent materials, coating materials, tissue injection fillers, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration materials, liver tissue materials and blood vessel repair and regeneration materials, biomaterials for 3D printed artificial organs, cosmetic raw materials, pharmaceutical adjuvants, and food additives.

9. A product or kit for promoting cell adhesion, comprising the polypeptide according to claim 1 or 2, the nucleic acid according to claim 3, the vector according to claim 4, and / or the host cell according to claim 5.

10. A composition for use in the manufacture of medical devices, cosmetic ingredients and pharmaceutical adjuvants and food additives, comprising a polypeptide according to claim 1 or 2, a nucleic acid according to claim 3, a vector according to claim 4, and / or a host cell according to claim 5, A composition in which the medical device is optionally a biomedical covering material, a biomimetic material for human body, a cosmetic surgery material, an organoid culture, a cardiovascular stent, a coating, a tissue injection filler, an ophthalmic material, an obstetric and gynecological biomaterial, a nerve repair and regeneration material, a liver tissue and blood vessel repair and regeneration material, or a biomaterial for a 3D printed artificial organ.

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