Method for manufacturing biosynthetic human structural materials

Abstract

The present invention relates to a method for manufacturing a biosynthetic human structural material. The present invention provides a recombinant type IV humanized collagen in which the amino acid sequence contains n (n is an integer of 1 or more) repeating sequences, and the repeating sequence is the sequence of the functional region of human native type IV collagen. The present invention provides the amino acid sequence of a recombinant humanized type IV collagen whose amino acid sequence is derived from native human type IV collagen, does not cause a harmful immune reaction even when administered to the human body, can be accurately expressed in vitro, and has succeeded in biosynthesis in vitro. The recombinant type IV humanized collagen prepared in the present invention has a higher cell adhesion effect than commercially available human-derived collagen. Further, the manufacturing method provided by the present invention is simple, and recombinant type IV humanized collagen can be manufactured at low cost and in high yield.

Description

Technical Field

[0001] Priority and Related Applications This application claims the priority of a Chinese patent application with the application number 202210619454.1 and the invention title "Method for Manufacturing Biosynthetic Human Structural Materials", which was filed with the China National Intellectual Property Administration on June 1, 2022, and incorporates all of its content herein by reference.

[0002] The present invention belongs to the field of biotechnology, and specifically relates to a method for manufacturing biosynthetic human structural materials.

Background Art

[0003] The structural materials of the human body are mainly structural proteins including collagen. Such proteins have an adhesion function and a support function for cells and tissues and are the main components of the extracellular matrix.

[0004] Collagen is a protein widely distributed in human connective tissues and is also the most abundant protein in the human body, accounting for 25% - 35% of all proteins. Currently, there are at least 28 subtypes of collagen in the human body, and it has been found that they exist in different tissues and organs. Among them, type IV collagen is mainly present in the portal vein vascular region and around the central vein, distributed along the hepatic sinusoids, and is an important component forming the framework of the basement membrane. It presents a non-fibrous reticular collagen triple helix structure, plays the role of the extracellular matrix, and has the function of supporting cells. Since collagen is a natural biological resource with biocompatibility not possessed by other polymer materials, it can be used as an important human structural material and is widely used in the high-end pharmaceutical industry.

[0005] However, currently, collagen is mainly extracted from animal tissues, and unfortunately, the content of type IV collagen in animals is low, making it impossible to extract type IV collagen as a single component. Furthermore, immune responses from animals are another significant reason why the applications of collagen are limited. With the growth of China's collagen industry, the use of biosynthetic pathways for obtaining collagen is becoming increasingly mature, and humanized collagen, in particular, is already at the forefront of global advancements. In 2021, the National Medical Products Administration (NASM) named and classified biosynthetic collagens. Within this framework, recombinant humanized collagen refers to the full-length or partial amino acid sequence encoded by a specific genotype of human collagen synthesized using DNA recombination technology, or a combination containing functional fragments of human collagen.

[0006] In living organisms, type IV collagen has a non-fibrous triple helix structure with six genetically distinct α chains: α1(IV) to α6(IV). These chains can form three different molecular forms within tissues: α1α1α2(IV), α3α4α5(IV), and α5α5α6(IV). These can be selectively expressed in different membranes within specific tissues at different stages. Structurally, the structure of natural type IV collagen in the human body is extremely complex, making it very difficult to express and prepare large quantities of humanized collagen using conventional methods. Collagen synthesis and modification begin with tropocollagen and involve numerous chemical changes such as hydroxylation, glycosylation, and cross-linking, all intricately controlled by various biological enzymes. Tropocollagen includes not only collagen chains but also spherical heads and tails. Without these heads and tails, the collagen chains cannot fold into the correct triple helix, resulting in a lack of biological activity as collagen. Therefore, collagen prepared according to the original gene sequence cannot spontaneously organize and form the correct spatial structure in vitro. These difficulties significantly hinder the research, development, and production of humanized collagen.

[0007] Traditionally, collagen has been produced by treating animal-derived tissues with acid, alkali, or enzymatic hydrolysis to extract collagen derivatives. However, collagen extracted using these methods has already lost its original biological activity and cannot perform its intended function in the biomedical field. With the advancement of modern biotechnology, attempts are being made to utilize transgenic technology to prepare recombinant human collagen in animal, plant, and microbial expression systems, overcoming many of the shortcomings of conventional extraction processes. However, some research institutions have pointed out that receptors expressible in human B cells are not expressed in mice, suggesting that humanized models can provide expression not obtainable in conventional mice. Therefore, there is an urgent need for a biosynthesis method for humanized type IV collagen that overcomes this deficiency and can be widely used as a structural material in the human body. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] The present invention aims to solve the problem of not being able to properly express natural human type IV collagen in vitro and thus not being able to prepare it in large quantities, and to provide recombinant humanized type IV collagen, a method for its biosynthesis, and its use in the pharmaceutical field, thereby offering new possibilities for the production and application of humanized collagen. [Means for solving the problem]

[0009] A first aspect of the present invention provides recombinant humanized type IV collagen. Specifically, the present invention screens the sequence of the functional region of human natural type IV collagen, uses it as a repeating sequence, and ensures that the amino acid sequence of the recombinant humanized type IV collagen contains n (where n is an integer of 1 or more) repeating sequences, i.e., the sequence of the functional region of human natural type IV collagen. Recombinant humanized type IV collagen consists of the sequence of the functional region of human natural type IV collagen.

[0010] Furthermore, n may be 1, 2, 3, 4, 5, 6, 7, or 8, and when n is an integer of 2 or more, each repeat sequence is directly linked to the others. This makes it possible to obtain recombinant type IV humanized collagen having an amino acid sequence containing the sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3.

[0011] Furthermore, the amino acid sequence of recombinant type IV humanized collagen provided in the present invention may further include an amino acid sequence that can be cleaved by the TEV protease shown in SEQ ID NO. 7 in order to facilitate expression and purification in the actual manufacturing process, and optionally, the TEV protease-cleavable amino acid sequence may be directly ligated to the repeat sequence. Preferably, the TEV protease-cleavable amino acid sequence is located at the N-terminus of recombinant type IV humanized collagen.

[0012] The present invention further provides recombinant type IV humanized collagen having the above amino acid sequence as its core region.

[0013] A second aspect of the present invention provides a polynucleotide encoding recombinant type IV humanized collagen as described in the first aspect of the present invention. Furthermore, the polynucleotide sequences may include the sequences shown in SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, or their artificially designed and codon-optimized derivatives.

[0014] A third aspect of the present invention provides a series of recombinant expression vectors comprising the polynucleotides described in the second aspect of the present invention. The expression vectors selected in the present invention are stable and autonomously replicated within various prokaryotic or eukaryotic host cells, such as conventional plasmids (pET series), shuttle vectors PNV 18.1, phages, or viral vectors. The recombinant expression vectors are constructed by cloning the nucleotide sequences described in the second aspect of the present invention into the vector by molecular biological operations such as enzymatic digestion or ligation.

[0015] A fourth aspect of the present invention provides a series of recombinant host cells comprising the recombinant expression vector described in the third aspect of the present invention, which are selected from prokaryotic cells, yeast, or eukaryotic cells, and may be further selected from Escherichia coli, Rhodococcus rubber, Bacillus subtilis, and yeast. Such genetically engineered bacteria are obtained by transforming host cells with the recombinant expression vector described in the third aspect of the present invention.

[0016] A fifth aspect of the present invention provides a method for producing recombinant type IV humanized collagen as described in the first aspect of the present invention, which may include the following steps. S1: A step of inducing protein expression by fermenting culture recombinant host cells according to the fourth aspect of the present invention; in this step, the fermentation culture may be carried out in a shaker with a rotation speed of 200-240 rpm and a temperature of 35-38°C, preferably with a rotation speed of 220 rpm and a temperature of 37°C. Furthermore, the specific procedure for inducing protein expression may involve lowering the temperature to 16-30°C and adding IPTG, with any IPTG concentration being 0.3-0.7 mM, preferably 0.5 mM. S2: Step of collecting the protein; In this step, the specific procedure for collecting the protein is to centrifuge the mixture obtained in step S1 at 5000-7000 rpm for 10-15 minutes in an environment of 4°C, resuspend the precipitate with an aqueous solution containing 20-30 mM Tris, 150-250 mM sodium chloride, and 15-25 mM imidazole, homogenize under high pressure, or disrupt the cells by ultrasound, and then centrifuge at 15000-18000 rpm for 20-40 minutes in an environment of 4°C. Preferably, the specific procedure for collecting the protein involves centrifuging the mixture obtained in step S1 at 6000 rpm for 12 minutes at 4°C, resuspending the precipitate with an aqueous solution containing 25 mM Tris, 200 mM sodium chloride, and 20 mM imidazole, lowering the temperature to below 15°C, homogenizing under high pressure, or disrupting the cells with ultrasound, and then centrifuging at 17000 rpm for 30 minutes at 4°C. S3: A step of purifying the protein; preferably, the step involves purifying the protein using a Ni affinity chromatography column and / or an ion exchange chromatography column, and optionally includes enzymatic digestion of the protein. It is preferable to enzymatic digestion of the protein using a TEV protease, and more preferably the mass ratio of the protein to the TEV protease is (15-25):1.

[0017] Furthermore, prior to step S1, the manufacturing method may further include a step of screening the functional region of human natural type IV collagen, a step of constructing a recombinant expression vector, and a step of constructing a recombinant host cell. Specifically, the specific procedure for screening the functional region of human natural type IV collagen involves first excluding uncharged amino acid motifs in the helix region of human natural type IV collagen, then screening for the potential helix functional region with the most interchain hydrogen bond structures and the most stable trimer aggregation form using computer-aided protein structure prediction, then screening for the human natural type IV collagen functional region with the highest protein expression level, ease of purification, and good stability using protein expression characteristics prediction, repeatedly linking the amino acid sequences of the functional regions of human natural type IV collagen obtained through screening, and adding amino acid sequences that can be cleaved with TEV protease as needed to obtain the amino acid sequence of recombinant humanized type IV collagen described in the first aspect of the present invention, then artificially designing and optimizing the polynucleotide sequence of the gene encoding the recombinant humanized type IV collagen according to the codon priority of the target product to obtain the nucleotide sequence described in the second aspect of the present invention, cloning it into a vector, constructing the recombinant expression vector described in the third aspect of the present invention, and transforming host cells with the vector to obtain the recombinant host cells described in the fourth aspect of the present invention.

[0018] A sixth aspect of the present invention provides the use of recombinant type IV humanized collagen as described in the first aspect of the present invention in the manufacture of products including medical devices, cosmetics, health foods, or pharmaceuticals.

[0019] Specifically, the present invention is as follows:

[0020] [1] Recombinant type IV humanized collagen having an amino acid sequence comprising n (where n is an integer greater than or equal to 1) repeat sequences, The aforementioned repeat sequence is the sequence of the functional region of human natural type IV collagen, Preferably, n is 1, 2, 3, 4, 5, 6, 7, or 8, where n is an integer of 2 or more, and each repeating array is directly concatenated to the others. More preferably, recombinant type IV humanized collagen wherein the amino acid sequence of the recombinant type IV humanized collagen contains any one of the following (i) to (iii). (i) Amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 (ii) Amino acid sequences in which one or more amino acid residues are added, substituted, deleted, or modified in the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and which retain the cell adhesion effect of human natural type IV collagen. (iii) an amino acid sequence that maintains the cell adhesion effect of human natural type IV collagen, encoded by the following nucleotide sequence, wherein the nucleotide sequence hybridizes under stringent conditions with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and the stringent conditions are medium stringent, medium-high stringent, high stringent, or very high stringent conditions.

[0021] [2] A recombinant type IV humanized collagen in which the amino acid sequence further contains an amino acid sequence cleavable by the TEV protease shown in SEQ ID NO.7, Preferably, the amino acid sequence cleavable by the TEV protease and the repeating sequence are directly linked, The recombinant type IV humanized collagen according to [1], wherein preferably the amino acid sequence cleavable by the TEV protease is located at the N-terminus of the recombinant type IV humanized collagen.

[0022] [3] A polynucleotide encoding the recombinant type IV humanized collagen according to [2], Preferably, a polynucleotide containing the sequence shown in SEQ ID NO.4, SEQ ID NO.5 or SEQ ID NO.6.

[0023] [4] A recombinant expression vector containing the polynucleotide according to [3], Preferably, the recombinant expression vector contains a pET series vector, a shuttle vector, a phage or a virus vector, More preferably, the recombinant expression vector is pET-28a-Trx-His, a recombinant expression vector.

[0024] [5] A recombinant host cell containing the recombinant expression vector according to [4], Preferably, the recombinant host cell is a prokaryotic cell, yeast or eukaryotic cell, More preferably, the host cell is Escherichia coli BL21(DE3), a recombinant host cell.

[0025] [6] A method for producing the recombinant type IV humanized collagen according to [1] or [2], S1: A step of fermentatively culturing the recombinant host cell according to claim 5 and inducing protein expression, S2: A step of collecting the protein, S3: A step of purifying the protein, and includes It is preferable to purify the protein using a Ni affinity chromatography column and / or an ion exchange chromatography column. A method for producing a protein, which optionally includes enzymatic digestion of the protein, preferably using a TEV protease, and more preferably having a mass ratio of the protein to the TEV protease of (15-25):1.

[0026] [7] The manufacturing method according to [6], wherein in step S1, the fermentation culture is carried out in a shaker with a rotation speed of 200 to 240 rpm and a temperature of 35 to 38°C.

[0027] [8] The manufacturing method according to [6] or [7], wherein the specific procedure for inducing protein expression in step S1 is to lower the temperature to 16-30°C, add IPTG, and preferably the concentration of IPTG used is 0.3-0.7 mM.

[0028] [9] The manufacturing method according to any one of the following items [6] to [8], wherein the specific procedure for collecting the protein in step S2 is to centrifuge the mixture obtained in step S1 at 5000 to 7000 rpm for 10 to 15 minutes at 4°C, resuspend the precipitate with an aqueous solution containing 20 to 30 mM Tris, 150 to 250 mM sodium chloride, and 15 to 25 mM imidazole, homogenize under high pressure, or disrupt the cells by ultrasound, and then centrifuge at 15000 to 18000 rpm for 20 to 40 minutes at 4°C.

[0029] Use of recombinant type IV humanized collagen as described in

[10] , [1], or [2] in the manufacture of products including medical devices, cosmetics, health foods, or pharmaceuticals. [Effects of the Invention]

[0030] Through the implementation of the above embodiments, the present invention provides an amino acid sequence for recombinant humanized type IV collagen whose amino acid sequence is derived from natural human type IV collagen, does not cause harmful immune responses when administered to the human body, can be accurately expressed in vitro, and has been successfully biosynthesized in vitro. The recombinant humanized type IV collagen prepared by the present invention has a higher cell adhesion effect than commercially available human-derived collagen. Furthermore, the manufacturing method provided by the present invention is simple, and recombinant humanized type IV collagen can be produced at low cost and in high yield. [Brief explanation of the drawing]

[0031] [Figure 1] This shows the electrophoretic detection results during the expression and purification process of recombinant type IV humanized collagen C007. [Figure 2] These are the electrophoretic detection results during the expression and purification process of recombinant type IV humanized collagen C4P7C6. [Figure 3] These are the electrophoretic detection results during the expression and purification process of recombinant type IV humanized collagen C4P7E5. [Figure 4] This is a comparison of the stability of recombinant type IV humanized collagen C4P7C6 and recombinant type IV humanized collagen C007 during expression purification. [Figure 5] These are the results of a comparison of the cell adhesion activity of each recombinant type IV humanized collagen. [Figure 6] This is the vector map for the pET-28a-Trx-His expression vector. [Modes for carrying out the invention]

[0032] The embodiments of the present invention will be described below, but the present invention is not limited thereto.

[0033] In this invention, the term "may" includes both cases where some processing is performed and cases where no processing is performed.

[0034] In the present invention, "optional" or "optionally" means that the following events or situations may or may not occur, and this description includes both the cases in which the events occur and the cases in which they do not occur.

[0035] In the present invention, the terms “include,” “have,” “incorporate,” or “contain” are inclusive and open form and do not exclude additional unlisted elements or process steps. On the other hand, “include,” “have,” “incorporate,” or “contain” can also mean closed form which excludes additional unlisted elements or process steps.

[0036] In this invention, the numerical range expressed as "numerical value A to numerical value B", "numerical value A or greater", and "numerical value A or less" means the range that includes the limit values ​​A and B.

[0037] In this invention, any numerical value includes the standard deviation of the error of the apparatus or method used to measure that value. The numerical ranges and parameters used to define this invention are all approximations, but they reproduce the numerical values ​​in specific embodiments as accurately as possible. However, any numerical value inherently includes the standard deviation due to the above-mentioned measuring apparatus or method.

[0038] In the present invention, the terms “polypeptide” and “protein” interchangeably mean a series of at least two amino acid residues linked together by a covalent bond (e.g., a peptide bond), and may be recombinant polypeptides, natural polypeptides, or synthetic polypeptides. Polypeptides may be linear or branched, may contain modified amino acids, or may be interrupted by non-amino acid groups. The term also encompasses amino acid polymers modified by any other operation, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or conjugation with a labeling component.

[0039] In the present invention, the term "amino acid" may include all natural amino acids, unnatural amino acids, amino acid analogs, and their D and L stereoisomers.

[0040] In the present invention, amino acid deletion can mean deleting one, two, or three or more amino acids from an amino acid sequence, provided that the modified sequence retains all or part of the activity of the original amino acid sequence.

[0041] In the present invention, the addition of amino acids can mean adding one, two, or three or more amino acids to the C-terminus, N-terminus, or any position between the C-terminus and N-terminus of an amino acid sequence, provided that the modified sequence retains all or part of the activity of the original amino acid sequence.

[0042] In the present invention, amino acid substitution can mean that an amino acid is replaced by another amino acid at any position in the amino acid sequence, provided that the modified sequence retains all or part of the activity of the original amino acid sequence. Amino acid substitution may be a conservative amino acid substitution, and it refers to the formation of a peptide (conservative mutant peptide) by replacing several amino acids with amino acids having similar properties compared to the original amino acid sequence. For example, these conservative mutant peptides can be produced by the following amino acid substitutions: substitution of Ala by Val, Leu, or Ile; substitution of Arg by Lys, Gln, Asn, or His; substitution of Asn by Gln, His, Lys, or Arg; substitution of Asp by Glu or Asn; substitution of Cys by Ser or Ala; substitution of Gln by Asn or Glu; substitution of Glu by Asp or Gln; substitution of Gly by Ala; substitution of His by Asn, Lys, Gln, or Arg; Leu, Met, Ala, Val Alternatively, substitution of Ile with Phe, substitution of Leu with Ile, Met, Ala, Val, or Phe, substitution of Lys with Asn, Gln, or Arg, substitution of Met with Ile, Leu, or Phe, substitution of Phe with Leu, Val, Ile, Ala, or Tyr, substitution of Pro with Ala, substitution of Ser with Thr, substitution of Thr with Ser or Val, substitution of Trp with Phe or Tyr, substitution of Tyr with Trp, Phe, Thr, or Ser, and substitution of Val with Phe, Ala, Met, Ile, or Leu. The amino acid substitutions may be non-conservative amino acid substitutions.

[0043] In the present invention, amino acid modification may include modification of functional groups, intramolecular covalent bonding (e.g., formation of rings between side chains), methylation, acylation, ubiquitination, phosphorylation, aminohexanolysis, biotinylation, and other modifications to the native sequence.

[0044] In the present invention, "hybridize" means the ability of polynucleotides or oligonucleotides to bind to nearly complementary sequences under stringential conditions, and to avoid nonspecific binding with non-complementary partners under these conditions. Therefore, it is preferable that the sequences are 90-100% complementary. The properties of complementary sequences that can bind specifically to each other are utilized, for example, in Northern blotting or Southern blotting techniques or in primer binding in PCR or RT-PCR. According to the present invention, hybridization is performed under medium-stringent, medium-to-high stringent, high stringent, or very high stringent conditions. Such hybridization conditions are described in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. For example, specific hybridization conditions are as follows: (1) Low-stringent hybridization conditions are those in which the material is washed twice with 6×sodium chloride / sodium citrate (SSC) at approximately 45°C, then at least 50°C, with 0.2×SSC and 0.1%SDS (under low-stringent conditions, the washing temperature can be increased up to 55°C). (2) Medium-stringent hybridization conditions are those in which the material is washed twice with 6×SSC at approximately 45°C, then at 60°C, with 0.2×SSC and 0.1%SDS. (3) Conditions for high-stringent hybridization are those in which the sample is washed once or multiple times with DS; (4) Conditions for ultra-high-stringent hybridization are those in which the sample is washed once or multiple times with 0.2×SSC and 0.1%SDS at 65°C, followed by 65°C; (5) Conditions for ultra-high-stringent hybridization are those in which the sample is washed once or multiple times with 0.5M sodium phosphate and 7%SDS at 65°C, followed by 0.2×SSC and 1%SDS at 65°C.

[0045] In the present invention, a suitable vector is one known in the field of vector construction, including the selection of a promoter and other regulatory elements such as enhancer elements. The vector according to the present invention contains a sequence suitable for introduction into cells. For example, the vector may be an expression vector in which the protein coding sequence is controlled by its own cis-acting regulatory element, and is designed to facilitate gene integration or gene replacement in host cells. Those skilled in the art will understand that in the present invention, “vector” includes a DNA molecule such as a plasmid, phage, virus, or other vector containing one or more heterologous or recombinant nucleotide sequences. Suitable phage and viral vectors include, but are not limited to, λ-phage, EMBL phage, Simian virus, bovine wart virus, Epstein-Barr virus, adenovirus, herpesvirus, mouse sarcoma virus, mouse mammary cancer virus, lentivirus, and the like.

[0046] In the present invention, the host cell may be a eukaryotic cell such as a fungus or yeast, or it may be a prokaryotic cell such as an Enterobacteriaceae bacterium.

[0047] In the present invention, "functional region of human natural type IV collagen" refers to a sequence fragment in human natural type IV collagen α1 that has functions such as cell adhesion activity, which are present in the amino acid sequence of collagen.

[0048] The application fields of recombinant type IV humanized collagen provided in this invention include the manufacture of high-end medical devices such as biomimetic bandages, human biomimetic materials, plastic surgery materials, organoid cultures, cardiovascular stents, coatings, tissue injection and filling, ophthalmic materials, obstetric and gynecological biomaterials, nerve repair and regeneration, liver tissue and blood vessel repair and regeneration, and 3D printed artificial organ biomaterials; as well as high-end cosmetic ingredients, high-end health foods, and high-end pharmaceutical additives. Examples

[0049] The present invention is further illustrated by the following embodiments, but none of these embodiments or any combination thereof should be construed as limiting the scope or embodiments of the present invention. The scope of the present invention is limited by the appended claims. Those skilled in the art will be able to clearly understand the scope defined by the claims based on this specification and common sense of the art. Those skilled in the art can make any modifications or changes to the technical solutions of the present invention without departing from the spirit and scope of the present invention, and such modifications and changes are also included within the scope of the present invention.

[0050] Unless specific conditions are described in the examples, general conditions or conditions recommended by the manufacturer shall be followed. All reagents and equipment whose manufacturers are not specified are common products that can be purchased commercially. Numerous specific details are described in the following specific embodiments in order to better illustrate the present invention. Those skilled in the art will understand that the present invention can be carried out even without such details. In other embodiments, methods, means, apparatus, and processes that are well known to those skilled in the art are not described in detail in order to clarify the gist of the present invention.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Unless otherwise noted, all units used herein are International Standard Units, and numerical values ​​and numerical ranges appearing in the present invention should be understood to contain unavoidable systematic errors.

[0052] Example 1: Construction and expression of recombinant type IV humanized collagen 1. Conducting large-scale protein function area screening. 1) Sequence screening: The Gly-XY repeat sequence in the helix region of natural type IV collagen contains many charged amino acids, and these charges bind to cells through interactions; therefore, regions that do not contain these important charged motifs were excluded. 2) Using computer-aided protein structure prediction, potential helix functional regions with the highest number of interchain hydrogen bonds and the greatest stability of trimer aggregation were screened. 3) Using protein expression characteristics prediction, human type IV collagen functional regions with the highest protein expression, ease of purification, and good stability were screened. 4) To ensure that the molecular weight of recombinant humanized type IV collagen falls within a certain range and that purification and stabilization are easy, amino acid fragments from the regions obtained through screening were directly linked n times and optimized, ultimately yielding the following target fragments of recombinant humanized type IV collagen. (1) Amino acid sequence of recombinant type IV humanized collagen C007: GQKGDQGEKGQIGPIGEKGSRGDPGTPGVPGKDGQAGQPGQPGPKGDPGISGTPGAPGLPGPKGSVGGMGLPGTPGEKGVPGIPGPQGSPGLPGDKGAKGEKGQAGPPGIGIPGLRGEKGDQGIAGFPGSPGEKGEKGSIGIPGMPGSPGLKGSPGSVGYPGSPGLPGEKGDKGLPGLDGIPGVKGEAGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHATE(SEQ ID NO.1); (2) Amino acid sequence of recombinant type IV humanized collagen C4P7C6: GFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHAGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGPPGPQGQPGLPGSPGHA(SEQ ID NO.2); (3) Amino acid sequence of recombinant type IV humanized collagen C4P7E5: GLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFMGLPGTPGPTGPAGQKGEPGSDGIPGSAGEKGEPGLPGRGFPGFPGAKGDKGSKGEVGFPGLAGSPGIPGSKGEQGFM (SEQ ID NO.3).

[0053] The recombinant humanized type IV collagen of the present invention can have an amino acid sequence ENLYFQ (SEQ ID NO.7) that can be cleaved by a TEV protease added to its N-terminus during expression. Preferably, the amino acid sequence that can be cleaved by the TEV protease is directly linked to the sequence of the functional region of the human natural type IV collagen.

[0054] 2. The synthesized polynucleotide fragments were each inserted into the pET-28a-Trx-His expression vector (vector map shown in Figure 6) to obtain the corresponding recombinant expression plasmids. The polynucleotide fragments of each recombinant type IV humanized collagen are as follows:

[0055] (1) Polynucleotide sequence of recombinant type IV humanized collagen C007 with an amino acid sequence cleavable by TEV protease added to the N-terminus: (SEQ ID NO.4);

[0056]

[0057]

[0058] 3. The successfully constructed expression plasmid was used to transform E. coli competent cells BL21(DE3). The specific procedure was as follows: (1) Remove E. coli competent cells BL21(DE3) from the ultracold refrigerator and place them on ice. While partially thawed, 2 μL of the plasmid to be transformed was taken and added to the E. coli competent cells BL21(DE3), and gently mixed 2-3 times. (2) The mixture was placed in an ice bath on ice for 30 minutes, then subjected to a heat shock in a 42°C water bath for 45-90 seconds. After removal, it was placed in an ice bath on ice for 2 minutes. (3) The cells were transferred to a biosafety 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 kanamycin sulfate. (5) The plates were incubated in a 37°C incubator for 15-17 hours until colonies of uniform size grew.

[0059] 4. Five to six single colonies were picked from the transformed LB plates and transferred to a shaking flask containing antibiotic stock solution (kanamycin sulfate 100 mg / L). The mixture was incubated at 220 rpm in a 37°C constant temperature shaker for a set period until it became atomized. Next, the temperature of the shaking flask after incubation was lowered to 16-30°C, and IPTG (final concentration 0.5 mM) was added to induce expression for a set period. The bacterial suspension was then dispensed into a centrifuge bottle, centrifuged at 6000 rpm at 4°C for 12 minutes, the bacterial cells were collected, the weight of the bacteria was recorded, and samples were taken and subjected to electrophoresis (sample name is labeled "bacterial suspension").

[0060] 5. The collected bacterial cells were resuspended in equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole), the bacterial suspension was cooled to below 15°C, homogenized twice, or the cells were disrupted by ultrasound. After completion, the bacterial suspension was collected and electrophoresis was performed (sample names were labeled "homogeneous," "homogeneous," or "ultrasonic" respectively). The cell-disrupted bacterial suspension was dispensed into centrifuge bottles and centrifuged at 17000 rpm at 4°C for 30 minutes. The supernatant was collected, and the supernatant and precipitate were separated and electrophoresis was performed (sample names were labeled "supernatant" and "precipitate" respectively).

[0061] 6. Recombinant type IV humanized collagen is purified and enzymatically digested, with the specific procedure being as follows. (1) Crude Purification: a. Column Packing Material Equilibration: The column packing material was equilibrated using an equilibration solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) at a flow rate of 10 mL / min. b. Sample Injection: The supernatant after centrifugation was added to the column packing material at a flow rate of 5 mL / min until the liquid flowed completely, and the flow-through was taken and electrophoresis was performed (the sample name was labeled "Flow-through"). c. Washing of Impurities: 100 mL of washing solution (200 mM sodium chloride, 25 mM Tris, 20 mM imidazole) was added at a flow rate of 10 mL / min until the liquid flowed completely, and the flow-through of the impurity wash was taken and electrophoresis was performed (the sample name was labeled "Impurity Wash"). d. Recovery of target protein: 20 mL of eluate (200 mM sodium chloride, 25 mM Tris, 250 mM imidazole) was added at a flow rate of 10 mL / min, the flow-through solution was collected, the protein concentration was measured by ultraviolet-visible spectrophotometric analysis, and the protein concentration was calculated using the formula (C(mg / mL) = A280 × dilution factor × extinction coefficient), and electrophoresis was performed (sample name is labeled "eluted"). e. The column packing material was washed with 1 M imidazole working solution at a flow rate of 10 mL / min, and electrophoresis was performed on the flow-through solution (sample name is labeled "1M washed"). (2) Enzyme digestion: TEV enzyme was added so that the ratio of the total mass of protein to the total mass of TEV enzyme was 20:1, and enzymatic digestion was carried out at 16°C for 2 hours. Samples were taken before and after digestion and measured by electrophoresis (the sample names were labeled "before digestion" and "after digestion," respectively). The protein solution after enzymatic digestion was placed in a dialysis bag and dialyzed at 4°C for 2 hours, then transferred to a new dialysate (20 mM sodium chloride, 20 mM Tris), dialyzed overnight at 4°C, and a sample of the protein solution was taken and measured by electrophoresis (the sample name was labeled "after liquid exchange"). (3) Purification: a. Column packing equilibration: The column packing was equilibrated with Solution A (20 mM Tris, 20 mM sodium chloride) at a flow rate of 10 mL / min. b. Sample injection: The sample was injected at a flow rate of 5 mL / min, the flow-through was collected, and electrophoresis was performed (the sample name was labeled "FL"). c. Gradient elution: A 0-15% Solution B aqueous solution was set for 2 minutes, then held for 3 CV, a 15-30% Solution B aqueous solution was set for 2 minutes, then held for 3 CV, and a 30-50% Solution B aqueous solution was set for 2 minutes, then held for 3 CV. Here, Solution B contains 1 M sodium chloride and 20 mM Tris, and the above ratios are volume fractions. Peaks were collected and electrophoresis was performed (the sample name was labeled "B elution"). d. The column packing was washed. e. The content of the target protein was measured, the protein yield was calculated, and the protein was stored at 4°C. (4) Water exchange: The recovered protein solution was added to a 10 kDa ultrafiltration concentration tube, and the concentration and water exchange process, based on centrifugation at 3500 rpm for 15 minutes, was repeated three times, and electrophoresis measurements were performed (the sample name is labeled "Water Exchange").

[0062] 7. Test Results: Figures 1 to 3 show the electrophoretic detection results during the preparation process of each recombinant protein. Figure 4 shows the results of a comparison of the stability of recombinant type IV humanized collagen C4P7C6 and C007 during expression purification.

[0063] Here, parts of Figures 1 and 4 show the electrophoretic detection results of recombinant type IV humanized collagen C007, indicating good purification and enzymatic digestion effects. Figures 2 and 4 (partially) show the electrophoretic detection results of recombinant type IV humanized collagen C4P7C6, indicating good purification and enzymatic digestion effects. Figure 3 shows the electrophoretic detection results of recombinant type IV humanized collagen C4P7E5, indicating good purification and enzymatic digestion effects.

[0064] Example 2: Measurement of the bioactivity of recombinant type IV humanized collagen For methods of measuring 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). The specific method is as follows. (1) The concentrations of target protein samples containing commercially available human collagen (Sigma, C7774) (as a positive control), and recombinant type IV humanized collagen C4P7C6, C4P7E5, and C007, which are purified and have good enzymatic digestion effects as provided in the present invention, were measured by ultraviolet absorption spectroscopy. Specifically, the ultraviolet absorption of the sample at 215 nm and 225 nm was measured, and the protein concentration was calculated using the empirical formula C(μg / mL) = 144 × (A215 - A225). Detection is required when A215 < 1.5. The principle of this method is to measure the characteristic absorption of peptide bonds under far-ultraviolet light, is unaffected by chromophore content, has few interfering substances, is easy to operate, and is suitable for detecting human collagen and its analogs that do not exhibit Coomassie brilliant blue coloration. (Reference: Walker JM. The Protein Protocols Handbook, second edition. HumanaPress. 43-45.) After measuring the protein concentration, the concentration of all analytes was adjusted to 0.5 mg / ml using PBS. (2) 100 μL each of the protein solutions and a blank PBS control solution were added to a 96-well plate and allowed to stand at room temperature for 60 minutes. (3) Place 10 well cultured 3T3 cells into each well. 5 The samples were placed individually and incubated at 37°C for 60 minutes. (4) Each well was washed four times with PBS. (5) Using an LDH detection kit (Roche, 04744926001) 492nm The absorbance was detected. Cell adhesion can be calculated from the values ​​of the blank control. The formula is: Cell adhesion = (Test well - Blank well) × 100% / (Positive well - Blank well). Cell adhesion can reflect collagen activity. The higher the protein activity, the better the external environment that can help cells adhere to the wall in a shorter time.

[0065] The results are shown in Figure 5. The recombinant type IV humanized collagen of the present invention was found to have superior bioadhesion activity compared to commercially available human collagen, namely, recombinant type IV humanized collagen C4P7C6 > C007 > C4P7E5 > human collagen.

[0066] Example 3: Detection of recombinant type IV humanized collagen by mass spectrometry

[0067] [Table 1]

[0068] Protein samples were treated with DTT reduction and iodoacetamidoalkylation, followed by enzymatic digestion with trypsin overnight. The resulting peptide fragments were further desalted using a C18 ZipTip and then mixed with α-cyano-4-hydroxycinnamic acid (CHCA) from the matrix on a plate. Finally, analysis was performed using a matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer, MALDI-TOF / TOF Ulraflextreme™, Brucker, Germany (see Protein J.2016;35:212-7 for peptide mass fingerprinting techniques).

[0069] Database searches were performed using the MS / MS Ion Search webpage on the local mascot site. Protein identification results were obtained from primary mass analysis of peptide fragments generated after enzymatic degradation. Search parameters: Trypsin enzymatic degradation, with two missing cleavage sites. Cysteine ​​alkylation was considered a standard modification, and methionine oxidation was considered an optional modification. The database used for identification was NCBprot.

[0070] [Table 2] The detection results showed a polypeptide fragment coverage rate of 75.08%, indicating a highly reliable detection result.

[0071] [Table 3] The detection results showed a 100% coverage rate for polypeptide fragments, indicating a highly reliable detection result.

[0072] [Table 4] The detection results showed a 100% coverage rate for polypeptide fragments, indicating a highly reliable detection result. The present invention includes the following embodiments: <Aspect 1> Recombinant type IV humanized collagen having an amino acid sequence containing n (where n is an integer greater than or equal to 1) repeating sequences, The aforementioned repeat sequence is the sequence of the functional region of human natural type IV collagen, Preferably, n is 1, 2, 3, 4, 5, 6, 7, or 8, where n is an integer of 2 or more, and each repeating array is directly concatenated to the others. More preferably, recombinant type IV humanized collagen characterized in that the amino acid sequence of the recombinant type IV humanized collagen contains any one of the following (i) to (iii). (i) Amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3 (ii) Amino acid sequences in which one or more amino acid residues are added, substituted, deleted, or modified in the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and which retain the cell adhesion effect of human natural type IV collagen. (iii) an amino acid sequence that maintains the cell adhesion effect of human natural type IV collagen, encoded by the following nucleotide sequence, wherein the nucleotide sequence hybridizes under stringent conditions with a polynucleotide sequence encoding the sequence shown in SEQ ID NO.1, SEQ ID NO.2, and SEQ ID NO.3, and the stringent conditions are medium stringent, medium-high stringent, high stringent, or very high stringent conditions. <Aspect 2> Recombinant type IV humanized collagen, whose amino acid sequence further includes an amino acid sequence that can be cleaved by the TEV protease shown in SEQ ID NO. 7, Preferably, the amino acid sequence that can be cleaved by the TEV protease and the repeat sequence are directly linked. Preferably, the recombinant type IV humanized collagen according to embodiment 1, characterized in that the amino acid sequence that can be cleaved by the TEV protease is located at the N-terminus of the recombinant type IV humanized collagen. <Aspect 3> A polynucleotide encoding recombinant type IV humanized collagen as described in Embodiment 2, Preferably, a polynucleotide comprising the sequence shown in SEQ ID NO.4, SEQ ID NO.5, or SEQ ID NO.6. <Aspect 4> A recombinant expression vector comprising a polynucleotide as described in Embodiment 3, Preferably, the recombinant expression vector comprises a pET series vector, a shuttle vector, a phage, or a viral vector. More preferably, the recombinant expression vector is characterized in that the recombinant expression vector is pET-28a-Trx-His. <Aspect 5> Recombinant host cells comprising the recombinant expression vector described in Embodiment 4, Preferably, the recombinant host cell is a prokaryotic cell, yeast, or eukaryotic cell. More preferably, the recombinant host cell is characterized in that the recombinant host cell is Escherichia coli BL21(DE3). <Pattern 6> A method for producing recombinant type IV humanized collagen according to embodiment 1 or 2, S1: A step of fermenting and culturing recombinant host cells as described in Embodiment 5 to induce protein expression, S2: A step of collecting the aforementioned protein, S3: A step of purifying the protein, It is preferable to purify the protein using a Ni affinity chromatography column and / or an ion exchange chromatography column. A method for producing a protein, which optionally includes enzymatic digestion of the protein, preferably using a TEV protease, and more preferably having a mass ratio of the protein to the TEV protease of (15-25):1. <Aspect 7> The manufacturing method according to embodiment 6, characterized in that in step S1, the fermentation culture is carried out in a shaker with a rotation speed of 200 to 240 rpm and a temperature of 35 to 38°C. <Aspect 8> The manufacturing method according to embodiment 6 or 7, characterized in that, in step S1, the specific procedure for inducing protein expression is to lower the temperature to 16-30°C, add IPTG, and preferably the concentration of IPTG used is 0.3-0.7 mM. <Aspect 9> The manufacturing method according to any one of embodiments 6 to 8, wherein the specific procedure for collecting the protein in step S2 is to centrifuge the mixture obtained in step S1 at 5000 to 7000 rpm for 10 to 15 minutes in an environment of 4°C, resuspend the precipitate with an aqueous solution containing 20 to 30 mM Tris, 150 to 250 mM sodium chloride, and 15 to 25 mM imidazole, homogenize under high pressure, or disrupt the cells by ultrasound, and then centrifuge at 15000 to 18000 rpm for 20 to 40 minutes in an environment of 4°C. <Aspect 10> Use of recombinant type IV humanized collagen as described in Embodiment 1 or 2 in the manufacture of products including medical devices, cosmetics, health foods, or pharmaceuticals.

Claims

1. Recombinant type IV humanized collagen having an amino acid sequence containing n (where n is an integer of 2 or more) repeating sequences, The aforementioned repeat sequence is the sequence of the functional region of human natural type IV collagen, Each repeating sequence is directly concatenated to the others. Recombinant humanized collagen of type IV, characterized in that the amino acid sequence of the recombinant humanized collagen of type IV contains any one of the following (i) to (iii). (i) The amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3 (ii) An amino acid sequence having one, two, or three amino acid residues added or deleted from the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3, and which retains the cell adhesion effect of human natural type IV collagen. (iii) An amino acid sequence that retains the cell adhesion effect of human natural type IV collagen, encoded by the following nucleotide sequence, wherein the nucleotide sequence is 90-100% complementary to the polynucleotide sequence encoding the sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3, and hybridizes under stringent conditions, where the stringent conditions are medium-stringent, medium-high stringent, high-stringent, or very high-stringent.

2. The recombinant type IV humanized collagen according to claim 1, wherein the amino acid sequence further comprises an amino acid sequence that can be cleaved by the TEV protease shown in SEQ ID NO.

7.

3. A polynucleotide encoding recombinant type IV humanized collagen as described in claim 2.

4. The polynucleotide according to claim 3, comprising the sequence shown in SEQ ID NO. 5 or SEQ ID NO.

6.

5. A recombinant expression vector comprising the polynucleotide described in claim 3.

6. The recombinant expression vector according to claim 5, wherein the recombinant expression vector comprises a pET series vector, a shuttle vector, a phage, or a viral vector.

7. Recombinant host cells comprising the recombinant expression vector according to claim 5.

8. A method for producing recombinant type IV humanized collagen according to claim 1, S1: A step of fermenting and culturing recombinant host cells according to claim 7 to induce protein expression, S2: A step of collecting the protein, S3: A step of purifying the protein, Manufacturing method.

9. The manufacturing method according to claim 8, characterized in that in step S1, the fermentation culture is carried out in a shaker with a rotation speed of 200 to 240 rpm and a temperature of 35 to 38°C.

10. The manufacturing method according to claim 8 or 9, wherein the specific procedure for inducing protein expression in step S1 is to lower the temperature to 16 to 30°C and add IPTG.

11. The manufacturing method according to claim 8 or 9, wherein the specific procedure for collecting the protein in step S2 is to centrifuge the mixture obtained in step S1 at 5000 to 7000 rpm for 10 to 15 minutes in an environment of 4°C, resuspend the precipitate with an aqueous solution containing 20 to 30 mM Tris, 150 to 250 mM sodium chloride, and 15 to 25 mM imidazole, homogenize under high pressure, or disrupt the cells by ultrasound, and then centrifuge at 15000 to 18000 rpm for 20 to 40 minutes in an environment of 4°C.

12. Use of recombinant type IV humanized collagen according to claim 1 or 2 in the manufacture of products including medical devices, cosmetics, health foods, or pharmaceuticals.

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