Collagen production method

Abstract

The present invention relates to a method for producing a plant or isolated plant cells for producing collagen, a nucleic acid construct used therein, and a method for producing collagen using the plant or isolated plant cells.

Description

Technical Field

[0001] The present invention relates to a method for producing a collagen-producing plant or isolated plant cells and a nucleic acid construct used therein, and a method for producing collagen using the plant or isolated plant cells. Background Art

[0002] Collagen is a family of extracellular matrix proteins found in the connective tissue of all mammals. Collagen molecules contain a structure consisting of three polypeptides called α chains forming a triple helical region. Collagen such as type I collagen is involved in various biological reactions such as early development, organ formation, cell adhesion, cell proliferation, hematopoiesis, wound healing, and tissue reconstruction in animals. Due to its structural characteristics and low immunogenicity, collagen is widely used in the fields of medicine, therapy, cosmetics, and food (Non-patent document 1: Ruggiero et al., 2000, FEBS Letter, 2000, Vol. 469, pp. 132-136. Non-patent document 2: Merle et al., FEBS Letter, 2002, Vol. 515, pp. 114-118).

[0003] However, the collagen commonly used for these purposes is extracted from animal tissues and may be contaminated by viruses or other infectious substances. Therefore, in the industries in the above-mentioned fields, it is expected to ensure that collagen is obtained from other than animal tissues. As a mass production method with excellent cost performance for safely mass-producing animal-based proteins, an attempt has been made to produce collagen in plants such as genetically modified tobacco using an expression system using genetically modified plants (Non-patent document 2: Merle et al., FEBS Letter, 2002, Vol.515, pp.114-118, Patent document 1: International Publication No. 2006 / 035442).

[0004] However, collagen is a protein with a complex structure having a triple helix structure formed by three peptide chains. In order to manufacture collagen in plants that can be used in the fields of medicine, treatment, cosmetics, food, etc., further technical improvements are needed. For example, in plant cells such as tobacco plants used for protein production, enzymes that cut off the position of the cutting site that deviates from the original proregion of humans, even if expressed in plant cells alone, are difficult to completely manufacture high-quality collagen that is consistent with the structure of human collagen. Therefore, even if collagen is expressed in plant cells alone, it is difficult to manufacture collagen with quality that can be used in the fields of medicine, treatment, cosmetics, food, etc.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: International Publication No. 2006 / 035442

[0008] Non-patent literature

[0009] Non-patent literature 1: Ruggiero et al., 2000, FEBS Letter, 2000, Vol.469, pp.132-136.

[0010] Non-patent document 2: Merle et al., FEBS Letter, 2002, Vol.515, pp.114-118. Summary of the invention

[0011] Technical problem solved by the invention

[0012] The object of the present invention is to provide a further improved method for producing collagen in plants or isolated plant cells, which collagen can be used in the medical, therapeutic, cosmetic, food and other fields.

[0013] Technical means of solving problems

[0014] The inventors of the present invention have conducted in-depth experiments and studies on methods for producing the collagen in plants or isolated plant cells. As a result, they found that by transiently expressing procollagen in plant cells and inhibiting the N-terminal cleavage of procollagen in the plant body, collagen was successfully produced efficiently in plants or isolated plant cells, thereby completing the invention of the present application.

[0015] In one embodiment of the present invention, there is provided a method for producing procollagen or collagen from a plant or an isolated plant cell, comprising the step of transiently expressing procollagen in the plant or the isolated plant cell.

[0016] In one embodiment of the present invention, there is provided collagen or procollagen produced in a plant or isolated plant cells.

[0017] In one embodiment of the present invention, a nucleic acid construct is provided for producing collagen or procollagen in a plant or an isolated plant cell.

[0018] In one embodiment of the present invention, a vector and a plant or an isolated plant cell containing the vector are provided, wherein the vector contains a nucleic acid construct for producing collagen or procollagen in the plant or the isolated plant cell.

[0019] In one embodiment of the present invention, there is provided a procollagen or collagen having a non-native sequence, wherein the non-native sequence is encoded by a nucleic acid construct for producing collagen or procollagen in a plant or an isolated plant cell.

[0020] In one embodiment of the present invention, a composition containing procollagen or collagen is provided.

[0021] More specifically, the present invention provides the following.

[0022] [Item 1] A method for producing procollagen or collagen from a plant or isolated plant cells, comprising:

[0023] A) a step of preparing a plant or an isolated plant cell comprising a nucleic acid construct encoding procollagen;

[0024] B) a step of transiently expressing procollagen in a plant or isolated plant cells;

[0025] C) A step of isolating the procollagen or collagen secreted outside the cells in step B.

[0026] [Item 2] A method for producing procollagen or collagen from a plant or isolated plant cells, comprising:

[0027] After step B, a step is performed to cleave the N-terminus or C-terminus of the procollagen secreted outside the cells.

[0028] [Item 3] The method according to Item 1 or 2, wherein

[0029] The nucleic acid construct comprises a nucleic acid sequence encoding procollagen, and the procollagen has an amino acid sequence identity of more than 80% with respect to natural procollagen.

[0030] [Item 4] The method according to Item 1 or 2, wherein

[0031] The nucleic acid construct comprises a nucleic acid sequence encoding the full length of procollagen and has a mutation that prevents the N-terminus of the procollagen from being cut off in plant cells.

[0032] [Item 5] A procollagen or collagen produced by the method according to any one of Items 1 to 4.

[0033] [Item 6] A nucleic acid construct expressing collagen, which is used in the method according to any one of Items 1 to 4.

[0034] [Item 7] An expression vector comprising the nucleic acid construct described in Item 6.

[0035] [Item 8] A plant body or an isolated plant cell comprising the nucleic acid construct described in Item 5 or the expression vector described in Item 7.

[0036] [Item 9] A procollagen or collagen protein, which is a procollagen or collagen protein encoded by the nucleic acid construct according to Item 6, wherein:

[0037] The procollagen or collagen has a non-natural amino acid sequence.

[0038] [Item 10] The procollagen or collagen according to Item 9, wherein

[0039] A heterologous peptide is fused to the N-terminal pro-region.

[0040] [Item 11] A composition as a pharmaceutical composition, a cosmetic composition or a food composition, comprising:

[0041] Procollagen or collagen produced by the method of any one of items 1 to 4, or

[0042] The procollagen or collagen described in item 9 or 10.

[0043] Effects of the Invention

[0044] The present invention has the following effects: providing a further improved method for producing collagen in plants or isolated plant cells, wherein the collagen can be used in the medical field, the therapeutic field, the cosmetic field, the food field, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] [ Figure 1a ] Figure 1a It is a schematic diagram showing the structure of the type I collagen α1 expression cassette.

[0046] [ Figure 1b ] Figure 1b It is a schematic diagram showing the structure of the type I collagen α2 expression cassette.

[0047] [ Figure 1c ] Figure 1c It is a schematic diagram showing the structure of the expression cassette of prolyl 4-hydroxylase α subunit (P4Hα) and prolyl 4-hydroxylase β subunit (P4Hβ).

[0048] [ Figure 1d ] Figure 1dis a schematic diagram showing the structure of the lysyl hydroxylase 3 (LH3) expression cassette.

[0049] [ Figure 1e ] Figure 1e It is a schematic diagram showing the structure of the bone morphogenetic protein 1 (BMP1) expression cassette.

[0050] [ Figure 1f ] Figure 1f It is a schematic diagram showing the structure of the expression cassette of the RNA silencing suppressor p19 derived from Tomato bushy stunt virus.

[0051] [ Figure 2 ] Figure 2 This is a diagram showing that full-length collagen having an N-terminal pro-region and a C-terminal pro-region was accumulated in the plant by transiently expressing a type I collagen α1 expression cassette in a Nicotiana benthamiana plant expression system. Lane 3 shows that full-length collagen expressed by the type I collagen α1 expression cassette was extracted. Lanes 4 to 18 show that full-length collagen was cleaved by corresponding proteases, wherein the full-length collagen was expressed by a type I collagen α1 expression cassette having a cleavage site mutation corresponding to various proteases.

[0052] [ Figure 3 ] Figure 3 The graph shows that fluorescence was observed by irradiating an extract of a green fluorescent protein (GFP)-fused collagen α1 extract with excitation light.

[0053] [ Figure 4 ] Figure 4 The figure shows the results of a Western blot experiment confirming the simultaneous accumulation of α1 and α2 collagens in cells by co-expressing a type I collagen α1 expression cassette and a type I collagen α2 expression cassette in a plant expression system.

[0054] [ Figure 5 ] Figure 5 This is a diagram showing the results of an experiment in which the expression of α1 and α2 collagen was confirmed by a mass spectrometer. DETAILED DESCRIPTION

[0055] Collagen is composed of three peptide chains called α chains, and each peptide synthesized from mRNA by ribosomes bound to the endoplasmic reticulum membrane of the cytoplasm is transferred into the endoplasmic reticulum while being synthesized, and a three-chain (triple helix) procollagen molecule containing three peptides is formed by SS bonding reaction at each C-terminus, etc. For the three-chain procollagen molecule secreted from the endoplasmic reticulum to the outside of the cell via the Golgi apparatus, mature three-chain collagen is obtained by cutting the N-terminus and the C-terminus (Ricard-Blum, Cold Spring Harb Perspect Biol. 2011 Jan 1;3(1):a004978).

[0056] Mature three-chain collagen is classified into various types such as type I collagen, type II collagen, type III collagen, and type XXVIII collagen according to the type, combination, overall structure, or characteristics of the α chains that make them up. For example, type I collagen is mainly composed of two α1 peptides and one α2 peptide, and type II collagen is composed of three α1 peptides. Each α chain further varies depending on the splicing method during biosynthesis. Type III collagen is composed of three α1 peptides like type II collagen, but the splicing method of the α chains that make them up is different (Ricard-Blum, Cold Spring Harb Perspect Biol. 2011 Jan 1;3(1):a004978).

[0057] Some types of mature triple-chain collagens form fiber structures by further cross-linking each other, while some types of mature triple-chain collagens form network structures. These various collagens are produced in various locations in the body and are responsible for various biological functions (Ricard-Blum, Cold Spring Harb Perspect Biol. 2011 Jan 1;3(1):a004978).

[0058] In order to mass produce such collagen for use in the medical, therapeutic and cosmetic fields, the inventors of the present invention conducted intensive experiments and studies on methods for producing collagen in plants or isolated plant cells, and found that by transiently expressing procollagen in plant cells, the degradation of expressed collagen by intrinsic enzymes of plant cells can be greatly suppressed. In addition, it was found that by introducing mutations into collagen genes, three-chain procollagen molecules or mature three-chain collagen molecules can be recovered from plants, and collagen can be produced efficiently.

[0059] More specifically, in mammals, natural collagen is secreted outside the cell as procollagen with extended regions at the N-terminus and C-terminus, and then the N-terminus and C-terminus are cut by enzymes to obtain mature collagen molecules. In contrast, when procollagen is expressed in plant cells, the N-terminus and C-terminus are cut in the plant cells, resulting in a significant reduction in water solubility. The N-terminal cut-off site cut in the plant cells is located closer to the C-terminus than the site where the N-terminus of the procollagen is cut when it matures into collagen. The mature collagen obtained from the procollagen expressed in the plant cells is shortened compared to the natural mature collagen. In addition, the cut-off position of the N-terminal cut-off site cut in the plant cells is unclear. Through the cut-off at these non-natural positions in the plant cells, the collagen produced in the plant cells can have different properties from the natural collagen.

[0060] The inventors of the present invention have found that by transiently expressing collagen in plant cells, the N-terminal cleavage of the tropocollagen in plant cells can be suppressed. In addition, amino acid mutations that can suppress the cleavage of the N-terminal or C-terminal cleavage of the tropocollagen in plant cells or in the production process have been found, and by expressing the tropocollagen with these mutations, the tropocollagen or collagen secreted to the extracellular space can be efficiently recovered. In addition, the N-terminal and C-terminal extension regions of the tropocollagen are cut by a desired enzyme, and by introducing an enzyme recognition sequence into a collagen gene, the tropocollagen can be converted into a mature collagen molecule by a desired process, and the enzyme recognition sequence is used to make the amino acid sequence of the N-terminal and C-terminal of the mature collagen molecule after cutting the same as the amino acid sequence of the natural mature collagen molecule.

[0061] (definition)

[0062] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art in the technical field to which the present invention belongs.

[0063] In this specification, when a plurality of numerical ranges are expressed, it is also intended that the range includes combinations of arbitrary lower limits and upper limits of these plurality of ranges.

[0064] The term "substantially" in this specification has the same meaning as that generally understood by those skilled in the art in the technical field to which the present invention belongs. For example, considering the situation where there are biological or chemical phenomena that cannot completely achieve the target state, the use of this expression is intended to include the target state and the state that cannot be achieved inevitably due to biological or chemical characteristics.

[0065] In this specification, when the term "about" is used in relation to a numerical value, the value means, for example, that it can vary within the range of 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01%.

[0066] In this specification, the term "comprising" has the same meaning as commonly understood by those skilled in the art in the technical field to which the present invention belongs, for example, including "comprising" and "consisting of..." Specifically, a composition "comprising" A includes not only A alone, but also other constituent elements B.

[0067] In this specification, the terms "consisting of" or "composed of" used for a composition have the same meaning as commonly understood by a person skilled in the art of the technical field to which the present invention belongs, and are used to indicate the constituent elements that exclusively constitute the composition. For example, a composition "consisting of" A exclusively contains only A. However, in one embodiment, a composition "consisting of" A includes the following embodiments: it contains impurities other than A that cannot be avoided in manufacturing due to biological and chemical properties.

[0068] In the present invention, the "identity" of an amino acid sequence or a base sequence refers to arranging the two sequences in such a way that the bases of the amino acids or base sequences of the two amino acid sequences to be compared are as consistent as possible, and the number obtained by dividing the consistent number of amino acids or bases by the total number of amino acids or bases is expressed as a percentage. In the arrangement, as required, a suitable gap is inserted into one or both of the two sequences to be compared. Such a sequence arrangement can be performed using well-known programs such as BLAST, FASTA, CLUSTAL W, etc. In the case of inserting a gap, the total number of amino acids or bases is the number obtained by counting a gap as one amino acid or base. In the case where the total number of amino acids or bases counted thus obtained is different between the two sequences to be compared, the identity (%) is calculated by dividing the consistent number of amino acids or bases by the total number of amino acids or bases of the longer sequence. However, in the case where the sequence to be compared is connected to any other sequence, only the corresponding region is taken out to compare the sequence and calculate the identity.

[0069] In the present invention, "transient" expression of procollagen or collagen means that it has the same meaning as that commonly understood by those skilled in the art of the present invention, and typically, the nucleic acid sequence encoding procollagen or collagen is expressed in the host cell without being introduced into the genome of the host cell. Such transient expression can be implemented, for example, by using a viral vector.

[0070] (Method for producing collagen in plants or isolated plant cells)

[0071] In one embodiment, the present invention provides a method for producing collagen in plants or isolated plant cells.

[0072] The collagen production method of the present invention comprises: a step of transiently expressing a nucleic acid construct containing a nucleic acid encoding procollagen in a plant body or isolated plant cells, and recovering procollagen or collagen molecules secreted outside the cells, thereby ultimately producing procollagen or collagen molecules.

[0073] Typically, the procollagen or collagen production method of the present invention comprises the following steps:

[0074] A) a step of preparing a plant or an isolated plant cell comprising a nucleic acid construct encoding procollagen;

[0075] B) a step of transiently expressing procollagen in a plant or isolated plant cells;

[0076] C) A step of isolating the procollagen or collagen secreted outside the cells in step B.

[0077] The nucleic acid construct used in step A) "preparing a plant body or isolated plant cells containing a nucleic acid construct encoding procollagen" is typically a nucleic acid construct having the following structure (a nucleic acid construct for producing collagen in plants or isolated plant cells).

[0078] The type of collagen produced in the method for producing procollagen or collagen of the present invention is not particularly limited, and any collagen can be produced by combining an appropriate nucleic acid construct encoding an α chain, for example, type I collagen, type II collagen, type III collagen, etc. For example, as described below (procollagen or collagen encoded by a nucleic acid construct), a collagen having a non-natural amino acid sequence or a collagen fused with a heterologous peptide can be produced.

[0079] Examples of plants used in the procollagen or collagen production method of the present invention include plants of the genus Nicotiana, bryophytes (P. patens, etc.), potatoes (S. tuberosum, etc.), gramineous plants (O. sativa, etc.), cruciferous plants, and lettuce, preferably plants of the genus Nicotiana. Examples of plants of the genus Nicotiana include, but are not limited to, Nicotiana benthamiana, Nicotiana tabacum, and Nicotiana excelsior.

[0080] In the step B) "the step of transiently expressing procollagen in a plant or isolated plant cells", typically, an expression vector such as a viral vector containing a nucleic acid sequence encoding procollagen is expressed in a plant or isolated plant cells without being introduced into the genome of the host cell.

[0081] By transiently expressing procollagen, N-terminal cleavage in plant cells can be inhibited. As for the transient expression of procollagen in a plant body or a plant cell obtained by separation, it can be carried out by a method known to those skilled in the art of the technical field to which the present invention belongs, and as for the expression amount and expression period of procollagen, by appropriately adjusting the vector, promoter, composition of the culture medium, culture conditions, etc. used, those skilled in the art can appropriately adjust within the scope of the gist of the present invention.

[0082] For example, when the nucleic acid construct of the present invention is used as a recombinant virus, the virus is multiplied in a plant infected with the virus or in a plant cell isolated, and a large amount of collagen is produced in the plant cell. For example, when collagen is produced using a plant, the plant is cultivated for about 3 to 15 days after virus inoculation, and collagen or procollagen can be extracted and recovered from plant tissues such as leaves.

[0083] Described operation C) " will be secreted into the extracellular procollagen or collagen protein by operation B and separates in the operation ", can utilize the known arbitrary method of those skilled in the art of the present invention.For example, can use CD Mount et al., Archives of Biochemistry and Biophysics, 240 33 (1985), UH Gregory and IR Willshire, Hoppe-Sayler's Z.Physiol.Chem., the method of putting down in writing in 356 1765 (1975), but is not limited to this.More specifically, for example, in tobacco plant body, make collagen protein expression and produce under the situation of collagen protein or procollagen, can grind in suitable buffer solution through the leaf of the tobacco plant of freezing preservation, extract, purify collagen protein or procollagen protein from buffer solution by salting out.

[0084] In one embodiment, the method for producing the procollagen or collagen of the present invention may include: after step B, a step of cutting off the N-terminus or C-terminus of the procollagen secreted outside the cell. The cutting can be implemented by treating the procollagen with a chemical treatment or an enzyme treatment or a combination of these. At least a portion of the chemical or enzyme treatment is performed at a location other than the plant cell, and can be performed in a culture step, an extraction and purification step, or after the extraction and purification step. For example, it is known that the C-terminus of the natural procollagen can be removed by extracting and purifying the fully grown procollagen in an acidic environment. In one embodiment, the N-terminus or C-terminus of the procollagen can be removed by a protease (proteinase) after the extraction and purification step. The N-terminus or C-terminus of the procollagen can be removed by a protease after the extraction and purification step, thereby inhibiting the mixing of impurities into the excision reaction system, allowing the protease to perform an excision reaction under appropriate reaction conditions, thereby improving the specificity of the excision reaction and obtaining high stability of the product. In this way, not only can stable collagen be produced as the final sales form, but also the following application method can be used: fully grown collagen is used as the raw material of bio-ink for bio-3D printers, and after the three-dimensional structure is constructed by the bio-3D printer, an enzyme that cuts the N-terminus or C-terminus (e.g. BMP1) is added to make the triple helix structure more solid. This is equivalent to the "development" process of photo film before the popularization of digital cameras.

[0085] The enzyme that cuts off the N-terminal or C-terminal extension region of the tropocollagen is not particularly limited. In one embodiment, a natural mature collagen can be generated from a natural tropocollagen by using an enzyme that cuts off the N-terminal and C-terminal of the natural tropocollagen in mammalian cells. As a non-limiting example of such an enzyme that cuts off the N-terminal and C-terminal of natural tropocollagen, Procollagen N-Endopeptidase (tropocollagen N endopeptidase), BMP1, etc. can be used. In addition, the corresponding protease can be utilized by utilizing amino acid mutations to introduce the desired protease recognition sequence into the N-terminal and C-terminal of the tropocollagen. As such an enzyme, for example, HRV3C, Furin, and Enterokinase can be used, but are not limited thereto. It should be noted that the recognition sequences and cut-off sites of HRV3C, Furin, and Enterokinase are as follows.

[0086]

[0087] In one embodiment, the method for producing tropocollagen or collagen of the present invention may include a step of post-translationally modifying tropocollagen or collagen. For example, tropocollagen or collagen can be post-translationally modified by P4Hα, P4Hβ or LH3, etc., so as to improve the stability of tropocollagen or collagen. In the method for producing tropocollagen or collagen of the present invention, the step of post-translationally modifying can be performed at any time point after the translation of tropocollagen or collagen, and can be performed in plant cells or at the stage after secretion from plant cells. For example, by expressing P4Hα and P4Hβ in the same plant cell as tropocollagen, post-translationally modifying can be performed in plant cells, such as in the endoplasmic reticulum. Preferably, at least a portion of the post-translationally modified is performed at a location other than the vacuole of the plant cell.

[0088] (Collagen produced in plants or isolated plant cells)

[0089] In one embodiment, the present invention provides a procollagen or collagen produced in a plant or an isolated plant cell. As for the collagen provided in the past in the fields of medicine, cosmetics or food, most of them are provided by decomposing collagen fibers obtained by further polymerizing collagen molecules present in biological tissues, and such collagen loses part of the structure and function of the collagen through the decomposition process. On the other hand, as for the procollagen or collagen produced in the plant or isolated plant cell of the present invention, it can be produced without such decomposition treatment, so the structure and function of the collagen can be maintained in a more complete state.

[0090] In one embodiment, the procollagen produced by the production method of the present invention includes procollagen having an N-terminus and a C-terminus. Such procollagen can be produced by transiently expressing a nucleic acid construct comprising a nucleic acid encoding a native procollagen in plant cells, and extracting and purifying the procollagen secreted outside the cells.

[0091] In one embodiment, the procollagen produced in the present invention includes procollagen having a natural amino acid sequence. In the case of the procollagen having a natural sequence, the amino acid sequence is the same as that of the natural procollagen, but the post-translational modification state and the like may be different depending on various conditions in the production process. As described above, in the method of producing collagen in the plant or isolated plant cell of the present invention, the post-translational modification state can be controlled by various methods, thereby having various advantages in physiological activity.

[0092] In one embodiment, the procollagen produced in the present invention includes: a non-natural procollagen having an amino acid mutation at the N-terminal cleavage site or the C-terminal cleavage site or both. In one embodiment, the mutation inhibits the cleavage of the procollagen in plant cells or in the production process. In one embodiment, the mutation provides a recognition sequence corresponding to a desired protease. In one embodiment, the mutation provides a sequence that is induced to cleave by chemical treatment of the production process. With respect to the amino acid mutation at the N-terminal cleavage site or the C-terminal cleavage site of the non-natural full-length procollagen, it may be a mutation that ultimately produces a natural mature collagen, or it may be a mutation that produces a mature collagen having a non-natural sequence at the N-terminus or the C-terminus or both ends.

[0093] The amino acid mutation may be an amino acid addition, deletion, substitution, or a combination thereof relative to the native amino acid sequence.

[0094] In one embodiment, the procollagen produced by the production method of the present invention includes N-terminal deleted procollagen and N-terminal deleted procollagen in which only one of the N-terminus or the C-terminus is removed. The N-terminal deleted procollagen has an N-terminal deletion different from that obtained by excision in plant cells. In one embodiment, the N-terminal or C-terminal deleted procollagen can be produced by chemically treating or enzymatically treating or a combination of these fully length or incomplete length procollagen secreted outside the cell.

[0095] In one embodiment, the procollagen produced in the present invention is a procollagen containing a peptide having a specific function fused to the N-terminus or the C-terminus or both. The fused peptide is not particularly limited and can be a peptide derived from a heterologous protein or a newly synthesized non-natural peptide. For example, peptides that promote secretion outside the cell, affinity peptides that can be used for purification of procollagen, labeled peptides that produce fluorescence, etc. can be used. In one embodiment, the fused peptide can be fused to the procollagen using a linker sequence known to a person skilled in the art in the technical field to which the present invention belongs. In one embodiment, a partial region or the entire region of the fused peptide can be removed from the procollagen by chemical or enzymatic reaction.

[0096] In one embodiment, the mature collagen produced in the present invention has a natural amino acid sequence at the N-terminus and the C-terminus. In another embodiment, the collagen produced in the present invention has a non-natural amino acid sequence at at least one of the N-terminus and the C-terminus. The non-natural amino acid sequence at the N-terminus is different from that generated by excision in plant cells.

[0097] In one embodiment, the tropocollagen or collagen produced in the present invention includes tropocollagen or collagen having any amino acid mutation other than the above. In one embodiment, the tropocollagen or collagen produced in the present invention includes tropocollagen or collagen formed by fusion of the whole region or a part of the tropocollagen or collagen other than the above with other peptides.

[0098] (Nucleic acid construct for collagen production in plants or isolated plant cells)

[0099] In one embodiment, the present invention provides a nucleic acid construct for use in collagen production in plants or isolated plant cells.

[0100] The nucleic acid construct of the present invention has a nucleic acid sequence encoding at least one α chain constituting collagen. In one embodiment, the nucleic acid construct of the present invention has a sequence expressing tropocollagen. As for the tropocollagen, it includes tropocollagen with various mutations mentioned above (collagen produced in plants or isolated plant cells) and tropocollagen fused with other peptides. The type of α chain encoded by the nucleic acid construct of the present invention is not particularly limited, and any of α1, α2 and these splicing isomers can be encoded.

[0101] The nucleic acid construct of the present invention can be DNA, RNA or any analogues thereof known to those skilled in the art in the art of the present invention. For example, the nucleic acid construct of the present invention can include non-natural bases, and a portion thereof can include hydrocarbon chains, peptide chains.

[0102] The nucleic acid construct of the present invention can be prepared by chemical synthesis methods or genetic engineering methods of conventional methods known to those skilled in the art of the present invention. For example, after obtaining a cDNA with a naturally occurring sequence by synthesis, the cDNA can be used as a template and suitable primers can be used to introduce suitable mutations and synthesize. Alternatively, the nucleic acid construct to be synthesized can be divided into some shorter regions and multiple DNA fragments can be chemically synthesized, and each fragment can be connected by a known fusion PCR method to synthesize a DNA construct. RNA constructs can be prepared by transcription reactions from DNA constructs.

[0103] As a method for introducing mutations into the sequence of a nucleic acid construct, any method known to those skilled in the art of the present invention can be used. For example, those skilled in the art can use methods such as Kunkel method, Gapped duplex method and other site-directed mutagenesis methods, overlap extension PCR (overlap extension PCR) method, QuikChange method, but are not limited thereto.

[0104] In one embodiment, the nucleic acid construct of the present invention may include a nucleic acid fragment for transient expression in plants, such as a nucleic acid fragment encoding an appropriate RNA polymerase promoter (e.g., a bacteriophage polymerase promoter such as a T7 promoter), a viral replicase, a migration protein, a coat protein, etc., required for synthesizing recombinant viral RNA by transcription reaction.

[0105] In one embodiment, the nucleic acid construct of the present invention may include a nucleic acid fragment encoding an enzyme useful for stabilizing the expression of collagen in plants. For example, a nucleic acid fragment encoding P4Hα, P4Hβ, or LH3, etc., which enhances the stability of collagen, or a combination thereof may be included. As these enzymes, enzymes of various biological origins may be used, for example, enzymes derived from nematodes, mice, humans, etc. may be used.

[0106] In one embodiment, part or all of the nucleic acid fragment encoding these enzymes, etc. can be included in a second nucleic acid construct different from a nucleic acid construct containing a nucleic acid encoding the α chain of procollagen, and the first nucleic acid construct containing a nucleic acid encoding the α chain of procollagen can be used in combination with the second nucleic acid construct.

[0107] When a first nucleic acid construct comprising a nucleic acid encoding the α chain of procollagen is used in combination with a second nucleic acid construct comprising a nucleic acid fragment encoding an enzyme, etc., these can be introduced into the same plant body or isolated plant cells, or into different plants or isolated plant cells, and the procollagen secreted outside the cell can react with the enzyme.

[0108] (Expression vector containing nucleic acid construct)

[0109] In one embodiment, the present invention provides a vector comprising a nucleic acid construct, which is used for collagen production in plants or isolated plant cells. The vector can be used to transiently express collagen in a plant host, and is typically provided as a plant virus vector. In one embodiment, the expression vector of the present invention can be provided in the form of a "recombinant virus". When the virus is an RNA virus, the nucleic acid construct of the present invention is RNA, and when the virus is a DNA virus, the nucleic acid construct is DNA. In one embodiment, the vector plasmid can be transformed into Agrobacterium tuminiferens, and the Agrobacterium tuminiferens can be used to infect plants to transiently express the target protein.

[0110] The expression vector provided as the recombinant virus of the present invention can be prepared by modifying any plant virus vector known to those skilled in the art (see, for example, "Protein Nuclease", vol. 45, p. 607-613, etc.) by conventional methods. In one embodiment, it is preferred to modify the tomato bushy stunt virus as the recombinant virus.

[0111] (Plant or isolated plant cell containing nucleic acid construct or expression vector)

[0112] In one embodiment of the present invention, a plant or plant cell isolated from a nucleic acid construct or expression vector is provided. The plant or plant cell isolated from a nucleic acid construct or expression vector may be obtained by growth, development or proliferation of the treated plant or plant cell, in addition to being obtained by introduction of the nucleic acid construct or expression vector.

[0113] (Nucleic acid construct encoding procollagen or collagen)

[0114] In one embodiment of the present invention, the procollagen or collagen encoded by the nucleic acid construct of the present invention is included. The procollagen or collagen is not limited to those produced from plant cells or isolated plant bodies. As for the procollagen or collagen, typically, it has a non-natural amino acid sequence. In one embodiment, the procollagen or collagen of the present invention has an amino acid sequence identity of more than 70%, more than 75%, more than 80%, more than 85%, more than 90% or more than 95% relative to natural procollagen or collagen. The procollagen or collagen of the present invention may contain one or more amino acid substitutions, insertions, or deletions relative to natural procollagen or collagen.

[0115] In one embodiment, the procollagen or collagen of the present invention can be fused with heterologous proteins instead of the N-terminal proregion to add various functions. For example, fluorescent collagen can be produced by fusing GFP instead of the N-terminal proregion. The procollagen or collagen can be expressed and purified by any method known to those skilled in the art of the present invention, and can be used for purposes such as analyzing chemical properties and physiological activities.

[0116] (Composition containing procollagen or collagen)

[0117] In one embodiment of the present invention, a composition is provided, comprising: procollagen or collagen produced from a plant or isolated plant cells, or procollagen or collagen having a non-natural amino acid sequence produced by other methods.

[0118] In one embodiment, the composition of the present invention is not limited in form, for example, it is provided in the form of a pharmaceutical composition, a cosmetic composition or a food composition, and in addition to the tropocollagen or collagen, it may also contain carriers, excipients, etc. allowed in the compositions of these various fields. In the composition of the present invention, the tropocollagen or collagen may be in a polymeric state having a fibrous or network structure comprising a plurality of tropocollagen or collagen molecules further through intermolecular cross-linking treatment. The amount of collagen contained in the composition of the present invention is not particularly limited and can be appropriately adjusted according to the dosage form, purpose, etc.

[0119] The pharmaceutical composition comprising procollagen or collagen of the present invention includes the case where the composition itself is a drug and the composition as a raw material or intermediate product when manufacturing a drug. The form of the pharmaceutical composition of the present invention is not particularly limited, and may be solid, powdery, granular, or liquid. In the case of the pharmaceutical composition of the present invention being administered to a subject, the dosage form is not particularly limited, and for example, it may be provided as tablets, granules, pills, lozenges, capsules, or raw materials or intermediate products thereof.

[0120] The pharmaceutical composition of the present invention comprises a regenerative medicine drug or a composition used as a raw material or intermediate product when manufacturing a regenerative medicine drug. For example, the pharmaceutical composition of the present invention is provided as a sheet-like composition, which can be provided as a composition for promoting the regeneration of in vivo or surface tissue of a subject or a scaffold material for culturing sheet-like tissue. In one embodiment, the pharmaceutical composition of the present invention can be provided as a material composition for constituting a regenerative medicine product having a 3D structure using 3D printing technology, for example, as a raw material for bio-ink.

[0121] In one embodiment, the pharmaceutical composition of the present invention comprises a composition as an implantable medical device or a raw material or intermediate product for manufacturing the medical device. These compositions can be used for the prevention or treatment of diseases or disorders, cosmetic purposes, etc. Non-limiting examples of the composition include: wound dressings, artificial skin, artificial blood vessels, catheters and other devices for removing fluids or delivering fluids to patients, artificial hearts, artificial kidneys, needles, plates and implants for plastic surgery. For example, the pharmaceutical composition of the present invention can be used as a post-mastectomy implant obtained by 3D bioprinting, a filler for cosmetic surgery.

[0122] The cosmetic composition containing procollagen or collagen of the present invention includes the case where the composition itself is a cosmetic and the composition used as a raw material or intermediate product when manufacturing cosmetics. The form of the cosmetic composition of the present invention is not particularly limited, and it can be solid, powdery, granular, or liquid. The cosmetics of the present invention can be topically applied or orally administered, for example, it can be provided as ointments, lotions, creams, microemulsions, gels, oils, solutions and other topical administration agents, tablets, fine granules, pills, lozenges, capsules and other oral administration agents or raw materials or intermediate products thereof.

[0123] The food composition containing procollagen or collagen of the present invention includes the case where the composition is a food itself and the composition used as a raw material or intermediate product when manufacturing food. The food includes health food, functional food, food for specific health care, food for patients, and feed when used for animals other than humans. The form of the food is not particularly limited, and it can be solid or liquid. There is no particular limitation on animals other than humans, for example, various animals such as mammals, birds, reptiles, amphibians, fish, insects, etc. can be used as objects. Specific types of food include, for example, soft drinks, carbonated drinks, nutritious drinks, fruit juice drinks, milk drinks and other beverages or concentrated raw materials and powders for preparation of these beverages; frozen desserts such as ice cream, sherbet, shaved ice, etc.; noodles such as soba noodles, udon noodles, vermicelli, dumpling wrappers, siu mai wrappers, Chinese noodles, instant noodles, etc.; baked snacks such as sugar, chewing gum, candy, soft candy, transparent fruit-flavored candy, caramel, chocolate, hard candy, snacks, biscuits, and other baked snacks, and snacks such as jelly, jam, and cream; processed seafood / livestock foods such as fish cakes, hamburgers, ham, and sausages; dairy products such as processed milk, fermented milk, yogurt, butter, and cheese; fats and fat-processed foods such as salad oil, tempura oil, margarine, mayonnaise, shortening, whipped cream, and salad dressing; seasonings such as sauces and dipping sauces; soups, stews, curry, bread, jam, salads, side dishes, kimchi, etc., but are not limited to these.

[0124] The present invention is further described in detail below using examples. These examples are for illustrative purposes only, and the scope of the examples does not limit the scope of the present invention.

[0125] Example

[0126] (Materials and methods)

[0127] The expression cassettes used in this study were constructed as Figure 1a The coding sequence in this study was optimized for expression in tobacco and chemically synthesized to have the desired flanking regions.

[0128] The sequence used is as follows.

[0129] [Table 1]

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137]

[0138]

[0139] 1. Construction of collagen gene expression cassette

[0140] The synthetic gene was cloned into an expression cassette containing the CaMV 35S promoter (SEQ ID NO.1), the Arabidopsis ADH-derived 5'-UTR (SEQ ID NO.3), and the HSP terminator (SEQ ID NO.4). Figure 1a ), the synthetic gene encodes collagen alpha-1 (I) chain (Col1, SEQ ID NO.8) fused to barley amylase signal peptide (Barley AMY 1.2 Signal Peptide, SEQ ID NO.5). Mutations were introduced into Col1 to construct an expression cassette of Col1 with the following modifications: modification of the N-terminal or C-terminal pro-region cleavage site (based on the introduction of the cleavage site of HRV3C, furin or enterokinase), removal of the N-terminal pro-region, replacement of the N-terminal pro-region based on GFP (SEQ ID NO.13), etc.

[0141] Specifically, the modification of the N-terminal pre-region cleavage site is carried out by replacing the C-terminal region of the N-terminal pre-region with the recognition sequence of HRV3C, furin or enterokinase, respectively. At this time, furin and enterokinase cut the C-terminal of the recognition sequence, so the original collagen after the cutting process is a collagen with a natural N-terminal terminal peptide sequence. On the other hand, HRV3C cuts the inside of the recognition sequence, so the original collagen after the cutting process is a collagen with an N-terminal terminal peptide sequence of the C-terminal side sequence GlyPro with the recognition sequence of HRV3C added. Cutting is performed at the position of the C-terminal side of the 2 amino acid residues (GlnLeu) of the natural N-terminal cleavage site in the plant cell. As a modification of the cleavage in the plant cell, the recognition sequence of enterokinase is introduced into the region spanning the N-terminal pre-region and the N-terminal terminal peptide region, thereby constructing an expression cassette of Col1, and the expression cassette of Col1 produces a collagen with an N-terminal terminal peptide sequence with 2 amino acid residues (GlnLeu) missing at the N-terminal.

[0142] In addition, with regard to modification of the C-terminal pre-region, in order to remove the recognition sequence of the unknown protease that cuts off the C-terminal pre-region in plant cells, multiple expression cassettes were constructed, which carried out amino acid substitutions in the recognition sequence of enterokinase at different positions from the C-terminal end peptide region to the C-terminal pre-region.

[0143] The synthetic gene was cloned into an expression cassette containing the CaMV 35S promoter (SEQ ID NO.1), the Arabidopsis ADH-derived 5'-UTR (SEQ ID NO.3), and the HSP terminator (SEQ ID NO.4). Figure 1b ), the synthetic gene encodes collagen alpha-2 (I) chain (Col2, SEQ ID NO.11) fused to a barley amylase signal peptide (SEQ ID NO.5). Mutations were introduced into Col2, and an expression cassette of Col2 with the following modifications was constructed: modification of the N-terminal or C-terminal pre-region cleavage site, removal of the N-terminal pre-region, fusion of green fluorescent protein to the N-terminal, addition of an HN tag (SEQ ID NO.16), a GST tag (SEQ ID NO.14), and an MBP tag (SEQ ID NO.15). The contents of each modification are the same as those of the expression cassette modification of Col1.

[0144] 3. Transformation of Agrobacterium tumefaciens

[0145] Agrobacterium tumefaciens GV3101 strain was transformed by freeze-thaw method using the plasmid derived from the pRI 201-AN plant transformation vector prepared in 2. Agrobacterium tumefaciens transformed with each plasmid was used for infiltration alone or as a mixture of two strains.

[0146] The plant Nicotiana benthamiana was cultivated from seeds in a plug tray containing a commercially available coconut shell substrate. The plant was grown in a greenhouse with a photoperiod of 16 hours of light / 8 hours of shading and a temperature schedule of 25°C throughout the day. Agrobacterium infiltration was implemented for the planting 4 weeks after inoculation. Before implementation, the terminal buds were removed by picking buds from the plant.

[0147] 2. Construction of the expression vector for the modified enzyme

[0148] The synthetic gene was cloned into an expression cassette containing the CaMV 35S promoter (SEQ ID NO.1) and the Arabidopsis ADH-derived 5'-UTR (SEQ ID NO.3) or TMV omega sequence (SEQ ID NO.2) and the HSP-derived terminator (SEQ ID NO.4). Figure 1c ), the synthetic gene encodes human P4Hα (SEQ ID NO.17), human P4Hβ (SEQ ID NO.19), nematode P4Hα (SEQ ID NO.18) and mouse P4Hβ (SEQ ID NO.20) fused to a vacuole signal (Thiol protease aleurain (Barley) vacuolar signal, SEQ ID NO.6) or a barley amylase signal peptide (SEQ ID NO.5). Except for the case of accumulation in the endoplasmic reticulum, the KDEL sequence was removed from the 3' end of the P4Hβ gene.

[0149] The synthetic gene was cloned into an expression cassette containing the CaMV 35S promoter (SEQ ID NO.1), the Arabidopsis thaliana ADH-derived 5'-UTR (SEQ ID NO.3), and the HSP-derived terminator (SEQ ID NO.4) ( Figure 1d ), wherein the synthetic gene encodes LH3 (SEQ ID NO.21) fused to a vacuole signal (SEQ ID NO.6) or a barley amylase signal peptide (SEQ ID NO.5).

[0150] The synthetic gene encoding BMP1 (SEQ ID NO.22) fused with an HN tag (SEQ ID NO.16) was cloned into an expression cassette containing a CaMV 35S promoter (SEQ ID NO.1), an Arabidopsis ADH-derived 5'-UTR (SEQ ID NO.3), and an HSP-derived terminator (SEQ ID NO.4) ( Figure 1f ), the HN tag (SEQ ID NO.16) was fused to the vacuole signal (SEQ ID NO.6) or the barley amylase signal peptide (SEQ ID NO.5). Only in the case of accumulation in the endoplasmic reticulum, a KDEL sequence was added to the 3' end of the BMP1 gene.

[0151] The synthetic gene (SEQ ID NO.23) was cloned into an expression cassette containing a CaMV 35S promoter (SEQ ID NO.1), an Arabidopsis ADH-derived 5'-UTR (SEQ ID NO.3), and an HSP-derived terminator (SEQ ID NO.4). Figure 1d ), the synthetic gene encodes the RNA silencing suppressor p19 (UniProtKB - P50628) from tomato bushy stunt virus.

[0152] The constructed expression cassettes of each gene were cloned into the multiple cloning site of the pRI 201-AN plant transformation vector in the following combinations.

[0153] (1)Col1+p19

[0154] (2)Col2+p19

[0155] (3)Col1+Col2+P4Hα+P4Hβ+LH3+p19

[0156] (4)Col1+Col2+p19

[0157] (5) P4Hα+P4Hβ+LH3+p19

[0158] (6)Col1+P4Hα+P4Hβ+p19

[0159] (7)Col1+LH3+p19

[0160] (8)Col1+BMP1+p19

[0161] 4. Obtaining Collagen from Agrobacterium tumefaciens

[0162] Agrobacterium tumefaciens transformed with each plasmid was cultured with shaking at 28°C overnight in a Select Alternative Protein Source (APS) medium containing 50 μg / ml kanamycin to proliferate until the OD 600 The concentration of the bacterial solution was adjusted to OD 600 The infiltration was carried out by adjusting the inoculation rate to 0.4 (0.8 when two strains were inoculated simultaneously). The infiltrated plants were incubated for 6 days, and only the leaves infiltrated with Agrobacterium tumefaciens were harvested. The harvested leaves were frozen at -80°C and crushed into small pieces.

[0163] Total soluble protein was extracted by adding 3 parts by volume of 50 mM Tris (pH 7.4) and 0.15 M NaCl to about 0.1 g of frozen ground leaves, grinding the leaves, and centrifuging them at 20,000 g for 5 minutes at 4°C. In addition, as another extraction condition, extraction was performed using 3 parts by volume of 0.5 M acetic acid and 0.2 M NaCl.

[0164] 5. Analysis of the Obtained Collagen

[0165] Collagen extracted under the following conditions was treated and confirmed by SDS-PAGE to determine whether it was cleaved.

[0166] Protease treatment of collagen extract: 16 hours at 4°C

[0167] HRV3C: Extraction solution 20μL + HRV3C (Millipore#71493) 1μL

[0168] Furin: 20μL extract + 4μL 6mM CaCl2 + 2μL 10-fold diluted furin (R&D #1503-SE-010)

[0169] Enterokinase: 20μL of extract + 10μL of 6mM CaCl2 + 2μL of 10-fold diluted enterokinase (NEB#P8070S)

[0170] 6. Quality Analysis

[0171] After reducing the fully soluble protein with DTT, the mass analysis sample was separated by SDS-PAGE electrophoresis using MOPS buffer. After electrophoresis, the separated protein was detected by CBB staining. As a result, the expected collagen band was confirmed near about 130 kDa of the fully soluble protein expressing collagen. The gel of the part was cut out and the mass analysis of the contained protein was performed. The mass analysis was commissioned to Japan PROTEOMICS (Co., Ltd.). After decolorizing the gel fragment, trypsin digestion was performed, and the mass was measured by nanoLC-MS / MS. The data was used for protein identification using MascotSearch (https: / / www.jproteomics.com / ).

[0172] 7. Confirmation of Collagen Expression in Tobacco Leaves Using Western Blotting

[0173] Total soluble proteins extracted from tobacco leaves (Nicotiana benthamiana) were separated by SDS-PAGE electrophoresis using Tris-glycine buffer, transferred to a PVDF membrane, and Western blotting was performed using antibodies against collagen α1 and tag sequences to confirm the expression of collagen α1 and collagen α2.

[0174] (result)

[0175] Example 1: Transient expression of secretory type I collagen α1 in a plant expression system

[0176] The expression cassette of Col1 was transiently expressed in the Nicotiana benthamiana plant expression system by the above method, and the cleavage was confirmed by SDS-PAGE ( Figure 2 ). The samples in each lane are shown below.

[0177]

[0178] As a result, it was confirmed that full-length collagen ( Figure 2 , lane 3). The above results are unexpected compared to the experimental results of F. Ruggiero et al. in 2000 showing that the N-terminal pro-region and C-terminal pro-region of secretory type I collagen α1 in a plant stable expression system were cleaved. In addition, it was shown that the N-terminal and C-terminal pro-regions of type I collagen α1 can be cleaved by treating the type I collagen α1 with a cleavage site modification in an extract with an enzyme that is specific for the cleavage site modification ( Figure 2 , lanes 4~18).

[0179] Example 2: Obtaining GFP-fused collagen α1

[0180] Fluorescence was confirmed by irradiating the extract of GFP-fused collagen α1 extract with excitation light. Of the N-terminal and C-terminal pro-regions, the C-terminal pro-region is necessary for collagen to form a triple chain, while the N-terminal pro-region is not necessary. Expression was also confirmed in a sequence that substantially removed the N-terminal pro-region. This experiment showed that collagen with new functions can be produced by fusing a heterologous peptide sequence such as GFP instead of the N-terminal pro-region ( Figure 3 ).

[0181] Example 3: Confirmation of simultaneous expression of collagen α1 and collagen α2

[0182] When the Col1 expression cassette and the Col2 expression cassette were transiently co-expressed in a plant expression system by the above method, it was confirmed by Western blotting that α1 and α2 collagen were accumulated in the cell simultaneously ( Figure 4 ). The samples in each lane are shown below.

[0183]

[0184] In addition, the expression of α1 and α2 collagen was confirmed by mass spectrometry ( Figure 5 ). The samples in each lane are Figure 4 same.

[0185] The above examples unexpectedly revealed that secretory collagen can be stably obtained by transiently expressing collagen in a plant expression system. In addition, it was confirmed that highly functional collagen can be obtained by expressing collagen fused with peptides having various functions instead of the N-terminal pro-region.

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

Claims

1. A method for producing procollagen or collagen from a plant or isolated plant cells, comprising: A) a step of preparing a plant or an isolated plant cell comprising a nucleic acid construct encoding procollagen; B) a step of transiently expressing procollagen in a plant or isolated plant cells; C) A step of isolating the procollagen or collagen secreted outside the cells in step B.

2. A method for producing procollagen or collagen from a plant or isolated plant cells, comprising: After step B, a step is performed to cleave the N-terminus or C-terminus of the procollagen secreted outside the cells.

3. The method according to claim 1 or 2, wherein: The nucleic acid construct comprises a nucleic acid sequence encoding procollagen, and the procollagen has an amino acid sequence identity of more than 80% with respect to natural procollagen.

4. The method according to claim 1 or 2, wherein: The nucleic acid construct comprises a nucleic acid sequence encoding the full length of procollagen and has a mutation that prevents the N-terminus of the procollagen from being cut off in plant cells.

5. A procollagen or collagen produced by the method according to claim 1 or 2.

6. A nucleic acid construct expressing procollagen or collagen, for use in the method of claim 1.

7. An expression vector comprising the nucleic acid construct according to claim 6.

8. A plant body or an isolated plant cell, comprising the nucleic acid construct according to claim 6 or the expression vector according to claim 7.

9. A procollagen or collagen, which is the procollagen or collagen encoded by the nucleic acid construct according to claim 6, wherein: The procollagen or collagen has a non-natural amino acid sequence.

10. The procollagen or collagen according to claim 9, wherein A heterologous peptide is fused to the N-terminal pro-region.

11. A composition as a pharmaceutical composition, a cosmetic composition or a food composition, comprising: Procollagen or collagen produced by the method of claim 1 or 2, or The procollagen or collagen according to claim 9 or 10.

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