JP6934666B2 - A method for producing a xylose-modified protein in a plant cell, a method for producing a core oligosaccharide-modified protein, and a method for producing a proteoglycan. - Google Patents

Description

The present invention relates to a method for producing a xylose-modified protein in a plant cell, a method for producing a core oligosaccharide-modified protein, and a method for producing a proteoglycan.

Several techniques have been reported that enable animal-type glycosylation of proteins in plants, but most of them synthesize mucin-type O-linked glycosylation in plants (Non-Patent Document 1: J). . Biol. Chem., (2012), Vol.287, No.43, pp.36518-36526; Non-Patent Document 2: Front. Plant Sci., (2016), Vol.7, Art.18). In this method, the protein is modified with N-acetylgalactosamine by introducing N-acetylgalactosamine transferase into the plant as a first step.

However, there is no example of synthesizing proteoglycans such as heparin and heparin sulfate by sugar chain modification of core proteins in plants.

J. Biol. Chem., (2012), Vol.287, No.43, pp.36518-36526 Front. Plant Sci., (2016), Vol.7, Art.18

Proteoglycans are currently collected mainly from animals such as salmon and pigs. However, such an acquisition method cannot deny the possibility that an animal-derived virus or the like may be mixed into the product, and is not preferable from the viewpoint of animal protection. Therefore, a technique for producing proteoglycan animal-free is desired.

The present inventors have focused on the need to link xylose to the serine residue (Ser) of the core protein as the first step in proteoglycan synthesis. Then, a vector encoding a protein xylose transferase was introduced into a plant cell and expressed, and the core protein was modified with xylose, thereby succeeding in producing a xylose-modified protein. A plurality of types of enzymes are appropriately combined with this xylose-modified protein, and each of them is introduced into a plant cell by a vector and expressed, and further sugar chain modification is performed to perform a core oligosaccharide-modified protein in the plant cell. And found that it becomes possible to produce proteoglycans, and reached the present invention.

That is, the gist of the present invention is, for example, as follows.
[1] A method for producing a xylose-modified protein in plant cells.
A first vector containing a gene encoding protein xylose transferase was introduced into plant cells,
The protein xylose transferase is expressed in the plant cell by the first vector, and xylose is ligated to the serine residue of the core protein in the plant cell by the action of the protein xylose transferase to produce a xylose-modified protein. How to include that.
[2] The method of [1], wherein the core protein is selected from cerglycine, syndecane, glypican, collagen, and agrin, and decorin.
[3] The method of [1] or [2], wherein the protein xylose transferase is selected from protein xylose transferase 1 and protein xylose transferase 2.
[4] The method of [1] to [3], wherein the first vector is transiently or constitutively expressed in a plant cell.
[5] The first vector comprises a promoter sequence that functions in plant cells and a coding sequence that encodes the amino acid sequence of protein xylosetransferase and is operably linked to the promoter sequence [1] to [ 4] method.
[6] The methods [1] to [5], wherein the first vector is in the form of a plant viral vector or a T-DNA vector.
[7] The methods [1] to [6], wherein the core protein is an endogenous protein.
[8] The methods [1] to [6], wherein the core protein is an exogenous protein.
[9] A second vector containing a gene encoding a core protein is introduced into a plant cell, and a second vector is introduced.
The method of [8], further comprising expressing the core protein with a second vector in plant cells.
[10] The method of [9], wherein the second vector comprises a promoter sequence that functions in a plant cell and a coding sequence that encodes an amino acid sequence of a core protein and is operably linked to the promoter sequence.
[11] The method of [9] or [10], wherein the second vector is in the form of a plant viral vector or T-DNA vector.
[12] The method of [1] to [11], wherein the plant cell is a non-isolated cell of a plant body.
[13] The methods of [1] to [12], wherein the plant cells are cultured cells.
[14] A method for producing a core oligosaccharide-modified protein.
In plant cells, xylose-modified proteins are produced by the methods [1] to [13].
In plant cells, β-1,4-galactose, β-1,3-galactose, and β-1,3-glucuronic acid are sequentially ligated to xylose, which is a xylose-modified protein, to produce a core oligosaccharide-modified protein. Methods that include doing.
[15] Production of core oligosaccharide-modified proteins
A third vector containing a gene encoding β-1,4-galactosetransferase capable of transferring galactose to a xylose residue and β-1,3-that can transfer galactose to a galactose residue in plant cells. A fourth vector containing a gene encoding a galactose transferase, a fifth vector containing a gene encoding a β-1,3-glucuronic transferase capable of transferring glucuronic acid to a galactose residue, and a xylose residue. A sixth vector containing a gene encoding an enzyme capable of transferring a phosphate group was introduced into the drug.
By expressing the 3rd, 4th, 5th and 6th vectors in plant cells, β-1,4-galactose transfer enzyme capable of transferring galactose to a xylose residue and galactose to a galactose residue are transferred. Express β-1,3-galactose transferase, β-1,3-glucuronic acid transferase capable of transferring glucuronic acid to galactose residues, and enzymes capable of transferring phosphate groups to xylose residues. It is carried out by ligating β-1,4-galactose, β-1,3-galactose, and β-1,3-glucuronic acid to xylose, which is a xylose-modified protein, in order by the action of a transposase [14. ]the method of.
[16] A method for producing proteoglycan, which is a method for producing proteoglycan.
In plant cells, a core oligosaccharide-modified protein is produced by the method of [14] or [15].
A method comprising connecting amino sugars and uronic acids alternately to the glucuronic acid terminal of a core oligosaccharide-modified protein to form a sugar chain and sulfate to form a glycosaminoglycan.
[17] The formation of glycosaminoglycans
A seventh vector containing a gene encoding an enzyme capable of transferring an amino sugar to a glucuronic acid residue and an eighth vector containing a gene encoding an enzyme capable of transferring uronic acid to an amino sugar residue in a plant cell. A vector, a ninth vector containing a gene encoding an enzyme capable of transferring an amino sugar to a uronic acid residue, and a gene encoding an enzyme capable of sulfated an amino sugar residue and a uronic acid residue of a sugar chain. Introduced with a tenth vector containing,
An enzyme capable of transferring an amino sugar to a glucuronic acid residue and an enzyme capable of transferring uronic acid to an amino sugar residue by expressing the seventh, eighth, ninth, and tenth vectors in plant cells. Glucuronic acid, a core oligosaccharide-modified protein, is produced by expressing an enzyme capable of transferring an amino sugar to a uronic acid residue and an enzyme capable of sulfated an amino sugar residue and a uronic acid residue of a sugar chain and allowing each enzyme to act. The method of [16], which is carried out by alternately connecting amino sugars and uronic acids to the ends in order to form a sugar chain and sulfating the sugar chain.

According to the present invention, a xylose-modified protein can be synthesized in a plant cell, and a core oligosaccharide-modified protein and a proteoglycan can be produced based on the xylose-modified protein. This enables animal-free proteoglycan production technology.

FIG. 1 is a schematic diagram for explaining the structure of proteoglycan. FIG. 2 is a photograph showing the results of Western blot analysis in the examples. M: molecular weight marker, W: untreated tobacco plant, XYLT2: XTLY2 alone expression, SRGN: SRGN alone expression, XYLT2 + SRGN: SRGN and XTLY2 co-expressed. FIG. 3 is a photograph showing the results of SDS-PAGE analysis in the examples. M: molecular weight marker, Ser: SRGN alone expression, XylT + Ser: SRGN and XTLY2 co-expressed, Std. HRP (1 μg): Horseradish peroxidase (1 μg), a standard protein. FIG. 4 is a spectrum showing the result of the MALDI TOF-MS analysis result in the example. SRGN: SRGN alone expression, SRGN + XYLT2: co-expression of SRGN and XTLY2. FIG. 5 is a photograph showing the results of Western blot analysis in the examples. M: Molecular weight marker, W: Untreated tobacco plant, S: SRGN alone expression, 1: SRGN and XTLY2 co-expressed, 2: SRGN, XTLY2, B4GALT7 co-expressed, 3: SRGN, XTLY2, B4GALT7 and B3GALT6 co-expressed Expression 4: co-expressed SRGN, XTLY2, B4GALT7, B3GALT6 and B3GAT3, 3F: co-expressed SRGN, XTLY2, B4GALT7, B3GALT6 and FAM20B, 4F: co-expressed SRGN, XTLY2, B4GALT7, B3GALT6, B3GALT6 : FLAG labeled standard protein.

Hereinafter, the present invention will be described in detail according to specific embodiments. However, the present invention is not bound by the following embodiments, and can be implemented in any embodiment as long as the gist of the present invention is not deviated.

[1. Overview]
Proteoglycan is a bioactive glycoprotein having various functions as a biological component. In addition to being present on the extracellular matrix and cell surface in tissues throughout the body such as the brain and skin, it is also present as the main component of cartilage. Proteoglycans contribute to the maintenance of various tissues of the body by forming an extracellular matrix together with collagen and hyaluronic acid. It also functions as a transmitter and is involved in various cell functions such as cell differentiation, proliferation, and motility.

The proteoglycan has a structure in which a core oligosaccharide is linked to a serine residue (Ser) of a core protein serving as a carrier, and a glycosaminoglycan is further linked to the end of the core oligosaccharide (Fig. 1). The core oligosaccharide has a structure in which xylose, galactose, galactose, and glucuronic acid are linked in this order from the core protein binding side, and glycosaminoglycan is linked to the glucuronic acid terminal. Glycosaminoglycan is a highly sulfated polysaccharide except for hyaluronic acid, and has a repeating structure in which amino sugars (galactosamine, glucosamine, etc.) and uronic acid (glucuronic acid, iduronic acid, etc.) are alternately linked. Has.

Proteoglycans are roughly classified as follows according to the types of amino sugars and uronic acids in the glycosaminoglycan portion, and the presence or absence of sulfate. The core oligosaccharide structure of all proteoglycans is the same.

-Heparan sulfate: It has D-glucosamine as the amino sugar of the glycosaminoglycan moiety, α-L-iduronic acid or β-D-glucuronic acid as uronic acid, and a part of uronic acid is N-sulfated (iduronic acid). ) Or O-sulfated (glucuronic acid).

-Heparin: A type of heparan sulfate, which has a particularly high degree of sulfation.

-Chondroitin sulfate: N-acetyl-D-galactosamine as the amino sugar of the glycosaminoglycan moiety, D-glucuronic acid as the uronic acid, and a part of N-acetyl-D-galactosamine is sulfated (4th position or 6). Place) and / or epilated.

-Dermatan sulfate: N-acetyl-D-galactosamine as the amino sugar of the glycosaminoglycan moiety and L-iduronic acid as the uronic acid, and a part of N-acetyl-D-galactosamine is sulfated (4th position or 6). Place) and / or epilated.

In the synthesis of proteoglycan, a core oligosaccharide is constructed by sequentially adding xylose, galactose, galactose, and glucuronic acid to a serine residue (Ser) of a core protein serving as a carrier, and then an amino sugar (galactosamine, Glucosamine, etc.) and uronic acid (glucuronic acid, isulonic acid, etc.) are alternately linked.

Therefore, in order to sugar-modify the core protein and synthesize proteoglycan in plant cells, it is essential to modify the serine residue (Ser) of the core protein with xylose as the first step.

In the present invention, a vector encoding a core protein and a vector encoding a protein xylose transferase are introduced and expressed in plant cells to bind xylose to a serine residue (Ser) of the core protein. Produces xylose-modified protein. Further, by linking β-1,4-galactose, β-1,3-galactose, and β-1,3-glucuronic acid to the xylose residue of this xylose-modified protein in order, the core oligosaccharide-modified protein To manufacture. Furthermore, amino sugars and uronic acids are alternately linked to the core oligosaccharides of this core oligosaccharide-modified protein in order to form sugar chains and sulfated to form glycosaminoglycans in plant cells. Manufactures proteoglycans at.

In addition, the present invention is not limited to the final production of proteoglycan, and has various uses for producing a xylose-modified protein by binding xylose to a serine residue (Ser) of an arbitrary core protein in a plant cell, and thus. By further linking β-1,4-galactose, β-1,3-galactose, and β-1,3-glucuronic acid to the xylose residue of the obtained xylose-modified protein in this order, the cells were intracellularly linked. It can be used for various purposes to produce core oligosaccharide-modified proteins. In this case, the core protein is not limited to the extrinsic protein introduced into the plant cell from the outside, and may be an endogenous protein endogenous to the plant cell.

Hereinafter, as a representative viewpoint of the present invention, a method for producing a xylose-modified protein, a method for producing a core oligosaccharide-modified protein, and a method for producing a proteoglycan will be focused on, and each method will be specifically described.

[2. Method for producing xylose-modified protein]
According to one aspect of the present invention, there is provided a method for producing a xylose-modified protein in plant cells. The method includes the following steps.
(A1) A first vector containing a gene encoding a protein xylose transferase is introduced into a plant cell.
(A2) The protein xylose transferase is expressed by the first vector in plant cells, and xylose is ligated to the serine residue of the core protein by the action of the protein xylose transferase to produce a xylose-modified protein.

Furthermore, when an exogenous protein is used as the core protein, the method may further include the following steps.
(B1) A second vector containing a gene encoding a core protein containing a serine residue is introduced into a plant cell.
(B2) The core protein is expressed by the second vector in plant cells. The core protein thus expressed is the target of xylose modification in (a2).

In the latter case, the introduction of the first vector in step (a1) is usually carried out at the same time as or before or after the introduction of the second vector in step (b1), and the core protein serine in step (a2). Xylose modification of the residue will occur following the expression of the core protein in step (b2).

<2-1. Plant cells ＞
Plant cells are not restricted, and a first vector can be introduced to express the protein xylose transposase, and if the core protein uses an exogenous protein, a second vector can be introduced to the core. Any plant cell can be used as long as the protein can be expressed. The morphology of the plant cell is also arbitrary, and it may be a non-isolated cell existing in the plant body, or a cultured cell such as a tissue culture isolated from the plant body. In the case of cultured cells, it may be a differentiated cell, a dedifferentiated cell, or a redifferentiated cell.

The species of plant from which the plant cells are derived is also unrestricted. Examples of plant species include, but are not limited to, plant species belonging to Solanaceae, Legumes, Brassicaceae, Gramineae, Nelumbonaceae, Nelumbonaceae, Rosaceae, and the like.

Specific examples of plant species include tobacco, white sorghum, alfalfa, sorghum, green bean, canola, sorghum, cotton, corn, clover, hass, sorghum, lupinus, millet, oat wheat, peas, peanuts, rice, lime tree, sweet clover, Sunflower, sweet pea, soybean, sorghum, rye wheat, kuzuimo, hassho bean, soramame, wheat, wisteria, fruit plant, konukagusa, onion, goldfish, Dutch honeybee, sorghum, asparagus, roasted bean, crow bean, spinach, abrana Tsubaki, Asa, Sorghum, Chrysanthemum, Kenoposi, Kikunigana, Kankitsu, Coffee tree, Juzudama, Cucumber, Pumpkin, Gyogishiba, Camogaya, Chosen Asagao, Urimi fly, Digitalis, Yamanoimo, Abra palm, Oshiba Hiyos, sweet potato, lettuce, sorghum, lily, flax, ryegrass, tomato, mayorana, apple, mango, sorghum, sorghum, African sorghum, sorghum, sorghum, peas, green beans, sorghum, strawberry tuna Examples include kinpouge, radish, sorghum, bean, strawberry, sugar cane, salmenana, senecio, setaria, white sorghum, eggplant, sorghum, bean, cacao, jajic sorghum, sorghum, grapes and the like.

Among them, from the viewpoint of adaptation of recombinant technology and ease of production expansion, tobacco, broad bean, alfalfa, barley, green bean, canola, sorghum, cotton, corn, clover, hass, lens bean, lupinus, millet, oat, pea, etc. Pepper, rice, lime tree, sweet clover, sunflower, sweet pea, soybean, sorghum, triticale, kuzuimo, hasho bean, broad bean, wheat, wisteria, fruit plant and the like are preferable.

<2-2. Core protein>
The core protein is not particularly limited, and any type of protein is arbitrary as long as it contains at least one serine residue and can undergo xylose modification to the serine residue by protein xylose transferase. For example, it may be an endogenous protein endogenous to a plant cell, or it may be an exogenous protein introduced from the outside into a plant cell. In the case of an exogenous protein that is introduced into a plant cell from the outside, it is optional as long as it can be introduced into the plant cell by a second vector and expressed, but in addition to the specific examples of the core protein of proteoglycan listed below. , Cindecane, glypican, collagen, agrin, decorin and the like. The origin of the core protein is also not particularly limited. Examples include humans, mice, rabbits, cows and the like. However, for the purpose of producing a modified protein used in humans, a human-derived core protein is preferable. The molecular weight of the core protein is not particularly limited, but is usually 5 kDa or more, particularly preferably 10 kDa or more, and usually 500 kDa or less, particularly preferably 300 kDa or less.

In particular, when xylose modification is performed as one step of producing proteoglycan, the core protein of proteoglycan is preferable as the core protein. Specific examples include cerglycine, glypican, syndecane, aglycan, versican, neurocan, brevican, decorin, biglican, perlecan, collagen and the like. Among them, cerglycine, glypican, syndecane, collagen and the like are preferable, and cerglycine, glypican, collagen and the like are particularly preferable because the molecular weight is small and the recombination technology can be easily applied.

The nucleotide sequence of the human cerglycine gene (SEQ ID NO: 1) and the corresponding amino acid sequence of the human cerglycin protein (SEQ ID NO: 2) can be found at the National Center for Biotechnology Information (NCBI). They are registered as registration numbers (Accession Number) NM_002727 and NP_002718, respectively. The nucleotide sequence of the human collagen type XVIII gene (SEQ ID NO: 3) and the corresponding amino acid sequence of the human collagen type XVIII protein (SEQ ID NO: 4) are registered in NCBI as registration numbers NM_030582 and NP_085059, respectively.

It should be noted that a protein having one or more mutations with respect to the above-mentioned various core proteins and maintaining at least one serine residue also has the original amino acid sequence identity as long as it has a certain level of amino acid sequence identity. Since it is highly probable that it maintains the same action and activity as protein, it can be used as a core protein. Specifically, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% with respect to the various core proteins described above. , A protein that is at least 99% identical and maintains at least one serine residue can also be preferably utilized as the core protein.

The sequence identity between the two amino acid sequences is determined by the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277) (preferably version 5.00 or (Since then), it is determined using the Needleman-Wunsch algorithm by the Needle program (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453).

<2-3. Protein xylose transferase>
The protein xylose transferase is also not particularly limited as long as it is an enzyme capable of catalyzing the addition reaction of xylose to the serine residue of the core protein in the presence of the core protein and uridine 5'-diphosphate (UDP) -xylose. , The type is arbitrary. Specific examples include human protein xylose transferase 1, human protein xylose transferase 2, and the like. Of these, protein xylose transferase 2 is preferable because of its small molecular weight and easy adaptation to recombinant technology. The origin of the protein xylose transferase is not particularly limited, but a human-derived protein xylose transferase is preferable for the purpose of producing a modified protein used in humans.

The nucleotide sequence of the human protein xylose transferase 1 gene (SEQ ID NO: 5) and the corresponding amino acid sequence of the human protein xylose transferase 1 (SEQ ID NO: 6) are registered in NCBI as registration numbers NM_022166 and NP_017449, respectively. There is. The nucleotide sequence of the human protein xylose transferase 2 gene (SEQ ID NO: 7) and the corresponding amino acid sequence of the human protein xylose transferase 2 (SEQ ID NO: 8) are registered in NCBI as registration numbers NM_022167 and NP_071450, respectively. There is.

A protein having one or more mutations with respect to the above-mentioned various protein xylose transferases also maintains the same action and activity as the original protein xylose transferase as long as it has a certain level of amino acid sequence identity. Since it is highly probable that it is present, it can be used as a protein xylose transferase. Specifically, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least for the various proteins mentioned above. Proteins that are 99% identical can also be preferably used as protein xylose transferase.

<2-4. First and second vectors>
In the present invention, in order to express protein xylose transferase in plant cells, a first vector having a gene encoding protein xylose transferase is used.

When an exogenous protein is used as the core protein, a second vector having a gene encoding the exogenous protein is used in addition to the above-mentioned first vector in order to express the core protein in plant cells. ..

The composition of the first and second vectors is arbitrary, but usually, a promoter sequence that functions in plant cells and an enzyme to be expressed (in the case of the first vector, a protein xylose transferase, a second vector). Includes a coding sequence that encodes the amino acid sequence of the core protein) and is operably linked to the promoter sequence. In addition to these promoter sequences and coding sequences, it may have any other sequences such as terminator sequences and transcriptional regulators.

The type of promoter sequence is not limited as long as it functions in the plant cell of interest and is capable of initiating transcription of the coding sequence. Examples include promoters derived from plant viruses, such as the 35S promoter of cauliflower mosaic virus (CaMV) (Odell et al., (1985), Nature, 313: 810-812), the Cassaba mosaic virus promoter, the sesame mosaic virus promoter, Derived from Badnavirus promoter, Strawberry vane binding virus (SVBV) promoter, Mirabilis mosaic virus promoter (MMV), and other promoters such as Rubisco promoter, Actin promoter, Ubiquitin promoter, Agrobacterium Nos promoter and the like can be mentioned. Of these, the 35S promoter of cauliflower mosaic virus (CaMV) and the like are preferable.

As the coding sequence, an appropriate sequence may be selected according to the base sequence of the gene to be expressed (protein xylose transferase gene in the case of the first vector, core protein gene in the case of the second vector).

The terminator array is an optional element as described above. However, from the viewpoint of reliably stopping the transcription of the coding sequence of the foreign gene and expressing the desired functional protein, the first and second vectors preferably have a terminator sequence. The type of terminator sequence is not limited as long as it is a sequence capable of stopping transcription of the coding sequence of a foreign gene. Examples include CaMV 35S terminators, heat shock protein (hsp) terminators, Agrobacterium-derived nos terminators and the like.

In addition, the promoter sequence and the terminator sequence, which is an arbitrary sequence, are operably linked to the coding sequence so that the core protein gene or the protein xylose transfer enzyme gene, which is an expression target gene, can be expressed. That is, these promoter sequences, coding sequences, and terminator sequences, which are arbitrary sequences, constitute an expression cassette that can be autonomously expressed in plant cells.

The first and second vectors may have additional sequences as long as they do not substantially interfere with their function. Examples of other sequences include, but are not limited to, translation amplification sequences, transcriptional regulators, 5'untranslated regions, and the like. Examples of the translationally amplified sequence include, but are not limited to, the 5'-UTR omega sequence of the tobacco mosaic virus (TMV) genome. Examples of the 5'untranslated region include, but are not limited to, the Arabidopsis thaliana-derived alcohol dehydrogenase 5'untranslated region and the like.

The forms of the first and second vectors are also arbitrary. It may be in a linear form or a circular form. However, it is preferably in the form of a ring, for example, a plasmid. Examples of specific embodiments of the vector include a plant virus vector, a T-DNA vector, and the like.

In the plant virus vector method, for example, in the case of a plant RNA viral vector, the cDNA of the plant virus genome into which the target gene is inserted is transcribed in vitro, and the obtained RNA is inoculated into a plant as a vector to infect the plant, and the virus itself has the ability to propagate. It is a method of expressing a gene to be expressed in a plant by utilizing the ability to transfer to the whole body. Examples of plant virus vectors include cauliflower mosaic virus (CaMV), cucumber mosaic virus (CMV) vector (US Patent Application No. 2007/0143877), tobacco mosaic virus (TMV) vector (Takamatsu et al., EMBO J). ., (1987), 6 (2): 307-11), potato X virus (PVX) vector (Baulcombe et al., Plant J., (1995), 7: 1045-1053) and the like. Similarly, it is also possible to use a plant DNA viral vector.

The T-DNA vector (T-DNA binary plasmid vector) method is a method using the T-DNA sequence of Agrobacterium, and can be used for both transient expression and homeostatic expression. Specifically, Agrobacterium is a general term for Rhizobium, which is a soil bacterium belonging to Gram-negative bacteria, and has pathogenicity to plants, and is associated with root cancer disease as an example. Agrobacterium tumefacients can be mentioned. Agrobacterium has a giant plasmid called Ti plasmid. Ti plasmid has a region called T-DNA (transfer DNA) sequence, and has the property that the DNA fragment corresponding to the region is transferred to plant cells and integrated into the plant genome by random introduction. The T-DNA sequence is a right border sequence (hereinafter, appropriately referred to as "RB sequence" or simply "RB") and a left border sequence (hereinafter, appropriately referred to as "LB sequence", or simply "LB sequence"). LB ”) is provided at both ends, and a phytohormone biosynthesis gene and an opine biosynthesis gene are provided between the RB sequence and the LB sequence. The RB and LB sequences are involved in integration into the plant genome, as well as infecting plant cells and expressing genes intervening between the RB and LB sequences in plant cells. It is known that there is. However, since the Ti plasmid contains a sequence unnecessary for plant transformation, it is huge and has poor operability. Therefore, at present, it is the mainstream to introduce foreign genes by transforming plants with two types of plasmids modified from Ti plasmid, that is, T-DNA binary system in which a binary plasmid and a helper plasmid are combined.

The binary plasmid is a sequence obtained by removing a region unnecessary for plant transformation (for example, a region such as the above-mentioned plant hormone biosynthesis gene or opine biosynthesis gene) from the T-DNA region, and Agrobacterium and other microorganisms (for example, for example). It is a shuttle vector that can be replicated with both Agrobacterium and other microorganisms by incorporating a replication initiation point capable of functioning with both E. coli (such as Escherichia coli) and a selectable gene marker. The binary plasmid usually has the RB and LB sequences derived from the T-DNA sequence (and its neighboring regions) rather than the entire T-DNA sequence, and optionally the removed phytohormone biosynthesis gene and It has a nos promoter and a nos terminator that regulated the expression of genes such as an opine biosynthesis gene between the RB sequence and the LB sequence. The foreign gene to be introduced into the plant is placed between the RB sequence and the LB sequence, and optionally under the control of the nos promoter and nos terminator. Generally, the "T-DNA vector" usually refers to this binary plasmid. Instead of the Agrobacterium-derived nos promoter and / or terminator, any of the above-mentioned other promoters and / or terminators, such as the 35S promoter and / or terminator of cauliflower mosaic virus (CaMV), may be used. ..

When the first and second vectors are T-DNA vectors (T-DNA binary plasmid vectors), such T-DNA vectors are introduced into Agrobacterium transformed with Agrobacterium-derived T-DNA vectors. Use by doing. Agrobacterium into which a binary plasmid vector has been introduced can be used for both constitutive expression methods and transient expression methods. For example, in the transient expression method, the purpose is to introduce an Agrobacterium culture solution into a plant tissue by a physical method (a method such as injection with a syringe or permeation by decompression) and infect the plant. Transient expression of the gene in plants (Grimsley et al., Proc. Natl. Acad. Sci. USA, (1986), 83 (10): 3282-6). In constitutive expression, it is possible to obtain a redifferentiated individual that constitutively expresses the target gene by culturing a plant tissue fragment infected with Agrobacterium.

The first and second vectors may be accumulated in the cytoplasm of a plant cell and expressed transiently, or may be integrated into the plant genome and expressed constitutively. For example, when the first and second vectors are T-DNA vectors (T-DNA binary plasmid vectors), the sequences required for genomic integration, such as Agrobacterium Ti plasmid, can be incorporated into the plant genome. The vir region is inserted into a binary vector or a helper plasmid having a vir region is used in combination.

The first and second vectors may be used in combination with other helper plasmids, if desired. Examples of other helper plasmids include, for example, the helper plasmid having the above-mentioned vir region.

The first and second vectors can be easily prepared by appropriately combining various gene recombination techniques well known to those skilled in the art.

<2-5. Introduction of vector into plants / gene expression>
According to the present invention, in the step (a1), the above-mentioned first vector having a gene encoding a protein xylose transfer enzyme is introduced into a plant cell, and in the step (a2), the protein xylose transfer is performed in the plant cell. By expressing the enzyme, the protein xylose transferase acts on the serine residue of the core protein and UDP-xylose present in the plant cells, binds xylose to the serine residue, and produces a xylose-modified protein. Can be done.

When an exogenous protein is used as the core protein, in addition to the above-mentioned first vector, the above-mentioned second vector having a gene encoding the exogenous protein is introduced into plant cells in the step (b1). By doing so, in the step (b2), the core protein is expressed in the plant cells, and xylose is bound to the serine residue of the core protein by the action of the protein xylose transferase to make the exogenous protein the core protein. It is possible to produce a xylose-modified protein.

In this case, as described above, the introduction of the first vector in the step (a1) is usually carried out at the same time as or before or after the introduction of the second vector in the step (b1), and in the step (a2). Xylose modification of the serine residue of the core protein will occur following the expression of the core protein in step (b2).

The method for introducing the first and second vectors into plant cells is not limited, and any method depending on the composition of the vector can be used.

For example, when the first and second vectors are constructed as T-DNA (binary plasmid) vectors as described above and transiently expressed, a solution containing each of these vectors is prepared, and various solutions are prepared. It is preferable to introduce it into the plant tissue by a physical method such as injection or permeation.

For the preparation of the solution containing the first and second vectors, for example, a microorganism such as Agrobacterium into which the first and second vectors have been introduced is cultured, and the obtained culture (for example, an overnight culture or the like) is prepared. ) Can be used. Overnight cultures typically have an optical density (OD600) at a wavelength of 600 nm reaching 3 to 3.5 units. This is suspended so that the OD600 before the Agrobacterium infiltration treatment is 0.2 to 0.8. When each of these cell solutions is used alone, prepare a suspension having an OD600 = 0.2 to 0.6 value. When a plurality of bacterial cell solutions are mixed and used, each bacterial cell is mixed in equal amounts to adjust the bacterial cell mass of the final suspension to an OD600 = 0.3 to 1.2 value. ..

When an Agrobacterium solution containing each of the first and second vectors is introduced into a target plant by injection, the solution is forcibly injected into the target plant using, for example, a syringe or the like. When a solution containing each of the first and second vectors is introduced into a target plant by permeation, the solution is brought into contact with the target plant, and the pressure is reduced by using, for example, a vacuum desiccator, and the solution is used as the target plant. It is done by forcibly infiltrating into.

By introducing the first and second vectors into the whole or part of the tissue of the target plant using the above method, the introduced first and second vectors are highly efficiently incorporated into the cytoplasm of the plant cell. Introduced, it becomes possible to transiently express the core protein and protein xylose transferase possessed by each.

As described above, the first and second vectors may be constructed as individual vectors, but of course it is also possible to construct the first and second vectors as a single vector. In this case, a coding sequence encoding the amino acid sequences of the enzymes to be expressed (protein xylose transtransferase and core protein), a regulatory sequence that regulates the expression of the coding sequence (that is, a promoter sequence, and an optionally provided terminator sequence). And other sequences such as transcription factors) are placed in a single vector. Of these, sequence elements other than the coding sequence (promoter sequence, terminator sequence, transcriptional regulator, etc.) may be provided individually for each coding sequence, or may be shared by a plurality of coding sequences.

[3. Method for producing core oligosaccharide-modified protein]
According to one aspect of the present invention, there is provided a method for producing a core oligosaccharide modified protein in plant cells. The method includes the following steps.
(I) A step of producing a xylose-modified protein in a plant cell by the above-mentioned method for producing a xylose-modified protein.
(II) A step of producing a core oligosaccharide-modified protein by sequentially linking galactose, galactose, and glucuronic acid to xylose, which is a xylose-modified protein, in plant cells.

The conditions and procedures for producing the xylose-modified protein in the step (I) are described in [2. Method for Producing Xylose-Modified Protein].

The production of the core oligosaccharide-modified protein in the step (II) is preferably carried out by the following step.
(C1) A third vector containing a gene encoding a galactose transferase capable of transferring galactose to a xylose residue and a gene encoding a galactose transferase capable of transferring galactose to a galactose residue are contained in a plant cell. It contains a fourth vector, a fifth vector containing a gene encoding a glucuronic acid transferase capable of transferring glucuronic acid to a galactose residue, and a gene encoding an enzyme capable of transferring a phosphate group to a xylose residue. The step of introducing the sixth vector.
(C2) A galactose transfer enzyme capable of transferring galactose to a xylose residue and a galactose transfer capable of transferring galactose to a galactose residue by expressing the third, fourth, fifth and sixth vectors in plant cells. An enzyme, a glucuronic acid transferase capable of transferring glucuronic acid to a galactose residue, and an enzyme capable of transferring a phosphate group to a xylose residue are expressed, and the action of each transfer enzyme acts on the xylose residue of a xylose-modified protein. The step of linking galactose, galactose, and glucuronic acid in order.

The galactose-transferring enzyme capable of transferring galactose to a xylose residue is not particularly limited, and any enzyme capable of catalyzing the addition reaction of galactose to a xylose residue in the presence of a xylose-modified protein and UDP-galactose can be used. It is optional. Specific examples include β-1,4-galactose transferase.

The galactose-transferring enzyme capable of transferring galactose to a galactose residue is not particularly limited, and any enzyme capable of catalyzing the addition reaction of galactose to a galactose residue in the presence of a galactose-modified protein and UDP-galactose can be used. It is optional. Specific examples include β-1,3-galactose transferase and the like.

The glucuronic acid transferase capable of transferring glucuronic acid to a galactose residue is not particularly limited as long as it is an enzyme capable of catalyzing the addition reaction of glucuronic acid to a galactose residue in the presence of a galactose-modified protein and UDP-glucuronic acid. , The type is arbitrary. Specific examples include β-1,3-glucuronic acid transferase and the like.

The enzyme capable of transferring a phosphate group to a xylose residue is not particularly limited, and can catalyze the addition reaction of phosphate to a xylose residue in the presence of a xylose-modified protein or a galactose-xylose-modified protein and adenosine triphosphate. If it is an enzyme, its type is arbitrary. Specific examples include glycosaminoglycan xylose kinase and the like.

The origin of these transferases is not particularly limited, but human-derived transferases are preferable for the purpose of producing modified proteins used in humans.

The nucleotide sequence of the human β-1,4-galactose transferase 1 gene (SEQ ID NO: 9) and the corresponding amino acid sequence of the human β-1,4-galactose transferase 1 (SEQ ID NO: 10) can be found in NCBI. They are registered as registration numbers NM_007255 and NP_009186, respectively. The nucleotide sequence of the human β-1,3-galactose transferase gene (SEQ ID NO: 11) and the corresponding amino acid sequence of the human β-1,3-galactose transferase (SEQ ID NO: 12) are registered in NCBI, respectively. It is registered as numbers NM_080605 and NP_542172. The nucleotide sequence of the human β-1,3-glucuronosyltransferase gene (SEQ ID NO: 13) and the corresponding amino acid sequence of the human β-1,3-glucuronosyltransferase (SEQ ID NO: 14) can be found in NCBI. They are registered as registration numbers NM_012200 and NP_036332, respectively. The nucleotide sequence of the human glycosaminoglycan xylose kinase gene (SEQ ID NO: 15) and the corresponding amino acid sequence of the human glycosaminoglycan xylose kinase (SEQ ID NO: 16) are registered in NCBI with the registration number NM_014864, respectively. And NP_055679.

For details on the constitution, morphology, production method and the like of the third, fourth, fifth and sixth vectors containing the genes encoding these transferases, the constitution and morphology described for the first and second vectors described above. , Manufacturing method and other details can be applied.

Using these third, fourth, fifth and sixth vectors, galactose transferase, galactose transferase, glucuronic acid transferase, and glycosaminoglycan xylose phosphorylating enzyme are introduced into plant cells and expressed. By acting on the xylose residue of the xylose-modified protein and the UDP-galactose, UDP-glucuronic acid and adenosine triphosphate present in the plant cells, β-1,4 were sequentially applied to the xylose residue of the xylose-modified protein. -Galactose, β-1,3-galactose, and β-1,3-glucuronic acid can be linked to produce a core oligosaccharide modified protein.

As described above, the third, fourth, fifth and sixth vectors may be constructed as individual vectors, but any of the third, fourth, fifth and sixth vectors may be constructed. Of course, it is also possible to construct two or more types as a single vector. In this case, the coding sequence encoding the amino acid sequence of the enzyme to be expressed (galactose transfer enzyme, galactose transfer enzyme, glucuronic acid transfer enzyme, and glycosaminoglycan xylose phosphorylating enzyme) and the expression of the coding sequence are regulated. The regulatory sequences to be used (ie, the promoter sequence and other sequences such as optionally provided terminator sequences and transcriptional regulators) are placed in a single vector. Of these, sequence elements other than the coding sequence (promoter sequence, terminator sequence, transcriptional regulator, etc.) may be provided individually for each coding sequence, or may be shared by a plurality of coding sequences.

[4. How to make proteoglycans]
According to one aspect of the present invention, there is provided a method for producing proteoglycans in plant cells. The method includes the following steps.
(I) A step of producing a xylose-modified protein in a plant cell by the above-mentioned method for producing a xylose-modified protein.
(II) A step of producing a core oligosaccharide-modified protein by sequentially linking galactose, galactose, and glucuronic acid to xylose, which is a xylose-modified protein, in plant cells.
(III) In plant cells, amino sugars and uronic acids are alternately linked to the glucuronic acid terminals of core oligosaccharide-modified proteins in order to form sugar chains and sulfated to form glycosaminoglycans to produce proteoglycans. Process to do.

The conditions and procedures for the production of the xylose-modified protein in the step (I) and the production of the core oligosaccharide-modified protein in the step (II) are described in [2. Method for Producing Xylose-Modified Protein] and [3. Method for producing core oligosaccharide-modified protein].

The production of proteoglycan in the step (III) is preferably carried out by the following step.
(D1) A seventh vector containing a gene encoding an enzyme capable of transferring an amino sugar to a glucuronic acid residue and a gene encoding an enzyme capable of transferring uronic acid to an amino sugar residue are contained in a plant cell. It encodes an eighth vector, a ninth vector containing a gene encoding an enzyme capable of transferring an amino sugar to a uronic acid residue, and an enzyme capable of sulfated an amino sugar residue and a uronic acid residue of a sugar chain. A step of introducing a tenth vector containing a gene to be used.
(D2) By expressing the 7th, 8th, 9th, and 10th vectors in plant cells, an enzyme capable of transferring an amino sugar to a glucuronic acid residue and uronic acid to a amino sugar residue are transferred. A core oligosaccharide modified protein by expressing an enzyme capable of transferring an amino sugar to a uronic acid residue, an enzyme capable of transferring an amino sugar residue and a uronic acid residue of a sugar chain, and allowing each enzyme to act. A step of forming a sugar chain by alternately connecting amino sugars and uronic acids to the glucuronic acid terminal of the above, and forming a glycosaminoglycan by sulfating the sugar chain.

The gene encoding the enzyme capable of transferring the amino sugar to the glucuronic acid residue is not particularly limited, and can catalyze the addition reaction of the amino sugar to the glucuronic acid residue in the presence of the core oligosaccharide modified protein and UDP-aminosaccharide. If it is an enzyme, its type is arbitrary. As described above, examples of the amino sugar forming glycosaminoglycan include galactosamine and glucosamine. Therefore, it is preferable to select a transferase capable of appropriately linking these amino sugars to the glucuronic acid residue. Examples of such transferases include Exostosin-like 3.

The gene encoding an enzyme capable of transferring uronic acid to an amino sugar residue is not particularly limited, and in the presence of a sugar chain-modified core oligosaccharide-modified protein having an amino sugar residue and UDP-uronic acid, the gene for the amino sugar residue is Any type of enzyme can be used as long as it can catalyze the addition reaction of uronic acid. As described above, examples of uronic acid forming glycosaminoglycan include glucuronic acid and iduronic acid. Therefore, it is preferable to select a transferase capable of appropriately linking these uronic acids to amino sugar residues. Examples of such transferases include Exostosin-1, Exostosin-2 and the like.

The gene encoding an enzyme capable of transferring an amino sugar to a uronic acid residue is not particularly limited, and in the presence of a sugar chain-modified core oligosaccharide-modified protein having a uronic acid residue and a UDP-amino sugar, the gene for the uronic acid residue is Any type of enzyme can be used as long as it can catalyze the addition reaction of amino sugars. As described above, examples of the amino sugar forming glycosaminoglycan include galactosamine and glucosamine. Therefore, it is preferable to select a transferase capable of appropriately linking these amino sugars to the uronic acid residue. Examples of such transferases include Exostosin-1, Exostosin-2, Exostosin-like 1.

The gene encoding the enzyme capable of sulphating the amino sugar residue and uronic acid residue of the formed sugar chain is not particularly limited, and the amino sugar residue and uronic acid residue are present in the presence of the sugar chain-modified core oligosaccharide-modified protein. Any type of enzyme can be used as long as it can catalyze the sulfation reaction of the group. As described above, examples of the amino sugar forming glycosaminoglycan include galactosamine and glucosamine, and examples of uronic acid include glucuronic acid and iduronic acid. Therefore, it is preferable to select an enzyme capable of appropriately sulfateing these amino sugars and uronic acid. Examples of suitable sulfated enzymes are heparan sulfate 2-O-sulfating enzyme, heparan sulfate 6-O-sulfating enzyme, chondroitin sulfate 4-O-sulfating enzyme, chondroitin sulfate 6-O-sulfating. Examples include enzymes.

The origin of these enzymes is also not particularly limited, but for the purpose of producing a modified protein used in humans, human-derived enzymes are preferable.

The nucleotide sequence of the Exostosin-like 3 gene (SEQ ID NO: 17) and the amino acid sequence of Exostosin-like 3 (SEQ ID NO: 18), which are examples of enzymes capable of transferring N-acetylglucosamine to the core oligosaccharide-terminal glucuronic acid residue, are , NCBI are registered as registration numbers NM_001440 and NP_001431, respectively. In addition, the nucleotide sequence of the Exostosin-1 gene, which is an example of a glycosaminoglycan-extending glycosaminoglycan (an enzyme capable of transferring uronic acid or the like to an amino sugar residue, an enzyme capable of transferring an amino sugar to uronic acid or the like). The amino acid sequences of (SEQ ID NO: 19) and Exostosin-1 (SEQ ID NO: 20) are registered in NCBI as registration numbers NM_000127 and NP_000118, respectively, and the nucleotide sequence of the Exostosin-2 gene (SEQ ID NO: 21) is another example. The amino acid sequences of Exostosin-2 and Exostosin-2 (SEQ ID NO: 22) are registered in NCBI as registration numbers NM_000401 and NP_000392, respectively. In addition, the nucleotide sequence of the heparan sulfate-6-O-sulfating enzyme gene (SEQ ID NO: 23) and the amino acid sequence of the heparan sulfate-6-O-sulfating enzyme (sequence) are examples of enzymes capable of sulfating sugar chains. Number 24) is registered in NCBI as registration numbers NM_004807 and NP_004798, respectively, and is another example of the nucleotide sequence of the heparan sulfate-2-O-sulfating enzyme gene (SEQ ID NO: 25) and heparan sulfate-2. The amino acid sequence of -O-sulfating enzyme (SEQ ID NO: 26) is registered in NCBI as registration numbers NM_012262 and NP_030394, respectively, and is yet another example of the glucosamine heparan sulfate-3-O-sulfating enzyme gene. The nucleotide sequence (SEQ ID NO: 27) and the amino acid sequence of heparan sulfate glucosamine-3-O-sulfating enzyme (SEQ ID NO: 28) are registered in NCBI as registration numbers NM_006042 and NP_006033, respectively, and are still another example. The nucleotide sequence of the heparan sulfate N deacetylation / N sulfater gene (SEQ ID NO: 29) and the amino acid sequence of the heparan sulfate N deacetylation / N sulfater (SEQ ID NO: 30) are registered in NCBI, respectively, with registration number NM_001543. And NP_001534.

For details on the composition, morphology, production method, etc. of the seventh, eighth, ninth, and tenth vectors containing the genes encoding these enzymes, the configuration, morphology, and the above-mentioned first and second vectors have been described. Details such as the manufacturing method can be applied.

Using these 7th, 8th, 9th and 10th vectors, an enzyme capable of transferring an amino sugar to a glucuronic acid residue, an enzyme capable of transferring uronic acid to an amino sugar residue, and an amino to a uronic acid residue. Expressing an enzyme capable of transferring sugar and an enzyme capable of sulfating amino sugar residues and uronic acid residues of sugar chains, and allowing each enzyme to act on core oligosaccharide-modified proteins in plant cells and amino sugars and uronic acid. This makes it possible to produce a core oligosaccharide-modified protein that forms glycosaminoglycans by alternately linking amino sugars and uronic acids to the glucuronic acid terminals of the core oligosaccharide-modified protein in order to form a sugar chain and sulfate.

As described above, the 7th, 8th, 9th and 10th vectors may be constructed as individual vectors, but any of the 7th, 8th, 9th and 10th vectors may be constructed. Of course, it is also possible to construct two or more types as a single vector. In this case, the promoter to be expressed (an enzyme capable of transferring an amino sugar to a glucuronic acid residue, an enzyme capable of transferring uronic acid to an amino sugar residue, an enzyme capable of transferring an amino sugar to a uronic acid residue, and a sugar A coding sequence that encodes the amino acid sequence of each of the amino acid sequences of the amino sugar residue and the uronic acid residue of the chain, a regulatory sequence that regulates the expression of the coding sequence (that is, a promoter sequence, and an optionally provided terminator sequence). And other sequences such as transcriptional regulators) are placed in a single vector. Of these, sequence elements other than the coding sequence (promoter sequence, terminator sequence, transcriptional regulator, etc.) may be provided individually for each coding sequence, or may be shared by a plurality of coding sequences.

The method described above may have yet another step. Examples of such a step include a step in which glucuronic acid contained in glycosaminoglycan can be converted into iduronic acid. In such a step, as in each of the above steps, for example, a vector containing a gene encoding an enzyme that converts glucuronic acid to iduronic acid is introduced into a plant cell, and such a vector is expressed in the plant cell. , Can be achieved by expressing an enzyme capable of converting glucuronic acid to iduronic acid. An example of an enzyme capable of converting glucuronic acid to iduronic acid is glucuronic acid-5-epimerase. The nucleotide sequence of the glucuronic acid-5-epimerase gene (SEQ ID NO: 31) and the amino acid sequence of glucuronic acid-5-epimerase (SEQ ID NO: 32) are registered in NCBI as registration numbers NM_015554 and NP_056369, respectively. The composition of the vector is the same as that of the first to tenth vectors.

[5. Brief Summary]
Any modification may be made to the method of the present invention described above as long as the gist of the present invention is not deviated.
For example, only one type of enzyme and gene may be used as each of the above-mentioned enzymes and each gene, but a plurality of types of enzymes and genes may be used respectively. In this case, a plurality of types of enzymes may be expressed by using a plurality of types of vectors each carrying a plurality of types of genes.

Of course, it is also possible to construct any two or more of the various vectors described above as a single vector. In this case, a coding sequence that encodes the amino acid sequence of the enzyme to be expressed, a regulatory sequence that regulates the expression of the coding sequence (that is, a promoter sequence, and other sequences such as a terminator sequence and a transcriptional regulatory factor that are optionally provided). ) Is placed in a single vector. Of these, sequence elements other than the coding sequence (promoter sequence, terminator sequence, transcriptional regulator, etc.) may be provided individually for each coding sequence, or may be shared by a plurality of coding sequences.

As described above, according to the present invention, a xylose-modified protein can be synthesized in a plant cell, and a core oligosaccharide-modified protein and a proteoglycan can be produced based on the xylose-modified protein. This enables animal-free xylose-modified protein, core oligosaccharide-modified protein, and proteoglycan production techniques.

As described above, the technique for synthesizing mucin-type O-linked glycosylation in plants is described in Non-Patent Document 1 (J. Biol. Chem., (2012), Vol.287, No.43, pp.36518-36526). And non-patent document 2 (Front. Plant Sci., (2016), Vol.7, Art.18). However, its sugar chain structure is extremely simple, and it has been thought that a similar approach cannot be applied to sugar-modified proteins having a complicated sugar chain structure, such as core oligosaccharide-modified proteins and proteoglycans. .. On the other hand, in the present invention, not only the synthesis of the xylose-modified protein in which xylose is linked to the serine residue of the core protein by plant cells was successful for the first time, but also a large number of enzymes (in addition to various glycosyltransferases, phosphate transfer). Enzymes, uronic acid transferases, sulfate enzymes) are appropriately combined and introduced into plant cells by vectors to express them, thereby producing core oligosaccharide-modified proteins having a complex sugar chain structure in plant cells. For the first time, we succeeded and showed the path to the intracellular synthesis of proteoglycan, which has a more complicated sugar chain structure. The findings of the present invention in which such complicated sugar chain modification is artificially realized in plant cells cannot be assumed from the above-mentioned items described in Non-Patent Documents 1 and 2, and are extremely surprising findings.

It is also possible to synthesize various glycoproteins other than proteoglycan in plant cells by applying the method of the present invention described above. For example, ketalan sulfate, which is formed by linking a sulfated sugar chain in which amino sugars and galactose are alternately linked to a core protein, can transfer the amino sugar to a desired amino sugar residue in the core protein in a plant cell. A gene encoding an enzyme, a gene that can encode an enzyme that transfers galactose to an amino sugar residue, a gene that can encode an enzyme that transfers amino sugar to a galactose residue, and an amino sugar residue that forms a sugar chain. And a gene capable of sulphating a galactose residue can be realized by introducing and expressing each using a vector.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not bound by the following examples, and can be carried out in any form as long as the gist of the present invention is not deviated.

[1. Preparation of vector for human SRGN expression]
A vector for expressing human cerglycin (SRGN), which is one of the core proteins of proteoglycan, was prepared by the following procedure.

<1a. Isolation of human SRGN gene>
The human SRGN gene (NCBI registration number NM_002727: SEQ ID NO: 1) was isolated by the following procedure.

First, using the human liver cell cDNA library (ORIGENE Technologies) as a template, PCR is performed using a pair of primers SR-F and SR-R having the nucleotide sequences of SEQ ID NO: 33 and SEQ ID NO: 34, respectively, to carry out PCR. The gene was amplified.

Next, the amplified DNA fragment of the SRGN gene was cloned into the cloning vector pTA2 (Toyobo), and then the DNA sequence was analyzed. The obtained vector is designated as pTA2: SRGN.

<1b. Introduction of human SRGN gene into a binary vector for plant expression>
SRGN is performed by performing PCR using the above-mentioned pTA2: SRGN as a template and using a pair of primers SR-F (N) and SR-R (S) FL having the nucleotide sequences of SEQ ID NO: 35 and SEQ ID NO: 36, respectively. A DNA sequence encoding a FLAG tag sequence and a restriction enzyme NdeI recognition sequence were added to the 3'end of the gene, and a restriction enzyme SalI recognition sequence was added to the 3'end.
The synthesized DNA fragment was inserted into a plant expression binary vector pRI201-AN (Takara Bio) treated with restriction enzymes NdeI and SalI (Takara Bio) using a Gibson assembly (New England Biolabs). The obtained plasmid vector is designated as pRI: SRGN.

<1c. Preparation of Agrobacterium for human SRGN gene expression>
Transformation of Agrobacterium LBA4404 strain was performed using the above-mentioned plasmid vector pRI: SRGN.

Specifically, the plasmid vector pRI: SRGN was added to 100 μl of the Agrobacterium LBA4404 strain cell fluid in a 1.5 ml plastic tube, and frozen with liquid nitrogen. After freezing, the tube was kept warm in a water bath at 37 ° C. for 5 minutes to thaw the bacterial cell fluid. After adding 1 ml of YEP liquid medium to the thawed bacterial cell fluid, the mixture was kept warm for 2 hours while shaking slowly in a water bath at 28 ° C. After the heat insulation was completed, the bacterial cell solution was applied to a YEP agar medium containing kanamycin, and the cells were cultured at 28 ° C. for 2 days. A part of the colonies formed on the YEP agar medium was inoculated into a YEP liquid medium containing antibiotics (kanamycin, streptomycin, and rifampicin) and cultured at 28 ° C. for 1 day to obtain agrobacterium for human SRGN expression. A cell culture medium was obtained. The obtained bacterial cell culture solution was added with glycerol for storage, stored in a -80 ° C freezer, and used for subsequent experiments.

[2. Preparation of vector for human XTLY2 expression]
<2a. Isolation of human XTLY2 gene>
The human protein xylose transferase 2 (Xylosyltransferase 2: XTLY2) gene (NCBI registration number NM_022167: SEQ ID NO: 7) was isolated by the following procedure.

First, XTLY2 is carried out by performing PCR using a human liver cell cDNA library (ORIGENE Technologies) as a template and using a pair of primers XT2-F and XT2-R having the nucleotide sequences of SEQ ID NO: 37 and SEQ ID NO: 38, respectively. The gene was amplified.

Next, the amplified DNA fragment of the XTLY2 gene was cloned into the cloning vector pTA2 (Toyobo), and then the DNA sequence was analyzed. The obtained vector is designated as pTA2: XTLY2.

<2b. Introduction of human XTLY2 gene into a binary vector for plant expression>
By carrying out PCR using the above-mentioned pTA2: XTLY2 as a template and using a pair of primers XT2-F (N) and Xm-R having the nucleotide sequences of SEQ ID NO: 39 and SEQ ID NO: 40, respectively, the XTLY2 gene can be obtained. A restriction enzyme NdeI recognition sequence was added to the 5'end, and a DNA sequence encoding the c-Myc tag sequence was added to the 3'end. Furthermore, by performing PCR using a pair of primers XT2-F (N) and m-SR having the nucleotide sequences of SEQ ID NO: 39 and SEQ ID NO: 41, respectively, the restriction enzyme SalI at the 3'end of the XTLY2 gene A recognition sequence was added.

The synthesized DNA fragment was inserted into a plant expression binary vector pRI201-AN (Takara Bio) treated with restriction enzymes NdeI and SalI (Takara Bio) using a Gibson assembly (New England Biolabs). The obtained plasmid vector is designated as pRI: XTLY2.

<2c. Preparation of Agrobacterium for human XTLY2 gene expression>
Transformation of Agrobacterium LBA4404 strain was performed using the above-mentioned plasmid vector pRI: XTLY2.

Specifically, the plasmid vector pRI: XTLY2 was added to 100 μl of the Agrobacterium LBA4404 strain cell fluid in a 1.5 ml plastic tube, and frozen with liquid nitrogen. After freezing, the tube was kept warm in a water bath at 37 ° C. for 5 minutes to thaw the bacterial cell fluid. After adding 1 ml of YEP liquid medium to the thawed bacterial cell fluid, the mixture was kept warm for 2 hours while shaking slowly in a water bath at 28 ° C. After the heat retention was completed, the bacterial cell solution was applied to a YEP agar medium containing kanamycin, and the cells were cultured at 28 ° C. for 2 days. A part of the colonies formed on the YEP agar medium was inoculated into a YEP liquid medium containing antibiotics (kanamycin, streptomycin, and rifampicin) and cultured at 28 ° C. for 1 day to obtain human XTLY2 expression agrobacterium. A bacterial cell culture medium was obtained. The obtained bacterial cell culture solution was added with glycerol for storage, stored in a -80 ° C freezer, and used for subsequent experiments.

[3. Expression of human SRGN in plants and xylose modification with human XTLY2]
<3a. Transient expression of human SRGN in plants>
Each cell culture solution of SRGN and XTLY2 expression agrobacterium was thawed, seeded in a YEP liquid medium containing antibiotics (kanamycin, streptomycin, and rifampicin), and cultured with shaking at 28 ° C. overnight. Then, the cells were collected by centrifugation and suspended in 10 mM MES buffer (pH 5.7, containing 10 mM magnesium chloride, 150 μM acetosyringone) to adjust the _{cell concentration to OD 600 = 0.5.}

Vacuum infiltration into Nicotiana benthamiana was performed using the obtained cell cultures for expressing SRGN and XTLY2 whose cell concentration had been adjusted. These SRGN and XTLY2 expression cell cultures were used alone or in equal amounts of both cell cultures and used in the experiment. After infiltration, N. Nicotiana benthamiana plants were grown in a growth chamber (23 ° C., 16 hours light period, 8 hours dark period). Leaf samples were obtained from the grown plants and used for later experiments.

<3b. Expression analysis of plant-expressed human SRGN by Western blotting>
The leaf sample was ground under ice-cooling, and the ground product was suspended in 50 mM Tris-hydrochloride buffer (pH 7.5, 0.1% Tween 20, containing protease inhibitor). The suspension was centrifuged (16,000 rpm, 10 minutes, 4 ° C.) and the centrifugation supernatant was subjected to Western blot analysis. A photograph showing the obtained Western blot analysis results is shown in FIG. As is clear from FIG. 2, a difference was observed in the molecular weight of SRGN between the case where SRGN was expressed alone and the case where SRGN and XTLY2 were co-expressed, and the latter molecular weight was larger. From this, it can be seen that SRGN undergoes some modification when co-expressed with XTLY2.

<3c. Separation of plant-expressed human SRGN by SDS-PAGE and mass spectrometry by MALDI TOF-MS>
The leaf sample was ground under ice-cooling, and the ground product was suspended in 50 mM Tris-hydrochloric acid buffer (pH 7.5, 150 mM sodium chloride, 0.1% Tween 20, including protease inhibitor). The suspension was centrifuged (16,000 rpm, 10 minutes, 4 ° C.), and then SRGN was purified from the centrifugal supernatant using anti-FLAG M2 antibody magnetic beads (Sigma-Aldrich). The obtained SRGN was separated by Sodium Dodecyl Sulfate-Poly-Acrylamide Gel Electrophoresis (SDS-PAGE), and the gel was subjected to Kumazi staining. A photograph showing the obtained SDS-PAGE analysis result is shown in FIG.

A band containing SRGN was excised from the SDS-PAGE gel and subjected to trypsin treatment, and then the obtained peptide was subjected to mass spectrometry by Matrix Assisted Laser Desorption / Ionization (MALDI TOF-MS). Was done. The spectrum showing the obtained MALDI TOF-MS analysis result is shown in FIG. As shown in FIG. 4, in the case of SRGN alone expression, only unmodified peptides were detected. On the other hand, when SRGN and XTLY2 were co-expressed, 9 types of peptides having different mass numbers of about 132 (mass numbers when xylose was added) were detected. Since SRGN has eight serine residues that are presumed to undergo xylose modification, it is suggested that the serine residues were heterogeneously modified with xylose.

From the above results, it was clarified that the human XTLY2 gene introduced into plant cells functions and can bind xylose to a protein in plant cells.

[4. Further modification of xylose-modified human SRGN]
Human β-1,4-galactosyltransferase 7: B4GALT7 (NCBI registration number NM_007255), human β-1,3-galactose transferase codon-optimized for Nicotiana benthamiana. Enzyme (beta-1,3-galactosyltransferase 6: B3GALT6) (NCBI registration number NM_080605) and human β-1,3-glucuronosyltransferase (Galactosylgalactosylxylosylprotein 3-beta-glucuronosyltransferase 3: B3GAT3) (NCBI registration number NM_012200). Each gene was obtained as an artificially synthesized gene. The human glycosaminoglycan xylosylkinase (FAM20B) (NCBI registration number NM_014864) has the nucleotide sequences of SEQ ID NO: 42 and SEQ ID NO: 43, respectively, using the human liver cell cDNA library (ORIGENE Technologies) as a template. The FAM20B gene was amplified by performing PCR with a pair of primers FAB-F and FAB-R. Using the amplified FAM20B gene as a template, each primer was amplified by PCR using a pair of primers FAB-FG and FAB-RG having the nucleotide sequences of SEQ ID NO: 44 and SEQ ID NO: 45, respectively.

Using each of the above genes as a template, for the B4GALT7 gene, a pair of primers B4G7-F and B4G7-R having the nucleotide sequences of SEQ ID NO: 46 and SEQ ID NO: 47, respectively, was used, and for the B3GALT6 gene, SEQ ID NO: 48 and the sequence. Using the pair of primers B3G6-F and B3G6-R having the base sequence of No. 49, respectively, for the B3GAT3 gene, the pair of primers B3G3-F and B3G3-R having the base sequences of SEQ ID NO: 50 and SEQ ID NO: 51, respectively. Was amplified by PCR, respectively.

The synthesized DNA fragment was inserted into a plant expression binary vector pRI201-AN (Takara Bio) treated with restriction enzymes NdeI and SalI (Takara Bio) using a Gibson assembly (New England Biolabs). The obtained plasmid vectors are designated as pRI: B4GALT7, pRI: B3GALT6, pRI: B3GAT3, and pRI: FAM20B, respectively.

In the same manner as in the previous section, transformation of the Agrobacterium LBA4404 strain was carried out using each plasmid vector to obtain Agrobacterium for expression of each gene. A transient expression test was carried out in Nicotiana benthamiana using each Agrobacterium, and the sugar chain elongation effect on cerglycine was confirmed.

As an example, FIG. 5 shows the human protein xylose transferase 2, human β-1,4-galactose transferase, human β-1,3-galactose transferase, and human β-1,3-glucuronosyl transferase for cerglycine. The result of analyzing the sugar chain elongation effect is shown. As is clear from FIG. 5, when SRGN is expressed alone (“S” in the figure), when SRGN and XTLY2 are co-expressed (“1” in the figure), and SRGN, XTLY2 and B4GALT7 are co-expressed. Comparing with the case of expression (“2” in the figure), a difference was observed in the molecular weight of SRGN, and the molecular weight was larger in the latter part. From this, compared with the case where SRGN and XTLY2 are co-expressed (“1” in the figure), when B4GALT7 is further co-expressed (“2” in the figure), some modification is further received. I understand. Here, since B4GALT7 is a β-1,4-galactose transferase, it is considered that galactose was further added to the xylose residue of SRGN modified with xylose by SRGN and XTLY2. ..

On the other hand, when SRGN, XTLY2, B4GALT7 and B3GALT6 are co-expressed (“3” in the figure) and SRGN, XTLY2, B4GALT7, B3GALT6 and B3GAT3 are co-expressed (“4” in the figure), Since the migration rate of cerglycine did not change to "2" in the figure, it was considered that the sugar chain was not elongated. However, when co-expressed with FAM20B, a difference in the molecular weight of SRGN was observed, and the molecular weight was larger later (“3F” and “4F” in the figure). Since it has been reported that B3GALT6 transfers galactose to a xylose-galactose structure in which a xylose residue is phosphorylated (Biochem J., (2009), 421: 157-162), cerglycine was converted by FAM20B. It is considered that phosphorylation occurred on the xylose residue of the xylose-galactose structure to be modified, resulting in sugar chain elongation by B3GALT6 and B3GAT3. From this, it was clarified that core oligosaccharides can be synthesized by co-expressing FAM20B in addition to glycosyltransferase.

Thus, co-expression of B3GALT6, B3GAT3 and FAM20B in addition to SRGN, XTLY2 and B4GALT7 results in an increase in the molecular weight of SRGN. Therefore, it can be seen that xylose, galactose, galactose, and glucuronic acid are linked in this order to the serine residue of SRGN, which is a core protein, to form a core oligosaccharide-modified protein that is a source of proteoglycan. Furthermore, proteoglycans can be produced by forming glycosaminoglycans by alternately linking amino sugars and uronic acids to this proteoglycan by a well-known method.

According to the present invention, xylose-modified protein, core oligosaccharide-modified protein, and proteoglycan can be produced animal-free by plant cells. Therefore, the present invention has high usefulness in protein production fields such as pharmaceuticals and agriculture.

Claims (16)

A method for producing a xylose-modified protein in plant cells.
A first vector containing a gene encoding protein xylose transferase was introduced into plant cells,
The protein xylose transferase is expressed in the plant cell by the first vector, and xylose is ligated to the serine residue of the core protein in the plant cell by the action of the protein xylose transferase to produce a xylose-modified protein. Including that
Protein xylose transferase
(1) Human protein xylose transferase 1, which has the amino acid sequence shown in SEQ ID NO: 6.
(2) Human protein xylose transferase 2 having the amino acid sequence shown in SEQ ID NO: 8 and
(3) An enzyme having at least 90% or more sequence identity with the amino acid sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 8 and retaining the activity of linking xylose to a serine residue of a core protein in a plant cell.
The method to choose from. The method of claim 1, wherein the core protein is selected from cerglycine, syndecane, glypican, collagen, and agrin, and decorin. The method of claim 1 or 2 , wherein the first vector is transiently or constitutively expressed in plant cells. Any of claims 1 to 3 , wherein the first vector comprises a promoter sequence that functions in plant cells and a coding sequence that encodes the amino acid sequence of the protein xylose transferase and is operably linked to the promoter sequence. The method described in paragraph 1. The method according to any one of claims 1 to 4 , wherein the first vector is in the form of a plant viral vector or a T-DNA vector. The method according to any one of claims 1 to 5 , wherein the core protein is an endogenous protein. The method according to any one of claims 1 to 5 , wherein the core protein is an exogenous protein. A second vector containing the gene encoding the core protein was introduced into the plant cell,
The method of claim 7 , further comprising expressing the core protein with a second vector in plant cells. The method of claim 8 , wherein the second vector comprises a promoter sequence that functions in plant cells and a coding sequence that encodes the amino acid sequence of the core protein and is operably linked to the promoter sequence. The method according to claim 8 or 9 , wherein the second vector is in the form of a plant viral vector or a T-DNA vector. The method according to any one of claims 1 to 10 , wherein the plant cell is a non-isolated cell of a plant body. The method according to any one of claims 1 to 11 , wherein the plant cell is a cultured cell. A method for producing core oligosaccharide-modified proteins.
A xylose-modified protein is produced in a plant cell by the method according to any one of claims 1 to 12.
In plant cells, β-1,4-galactose, β-1,3-galactose, and β-1,3-glucuronic acid are sequentially ligated to xylose, which is a xylose-modified protein, to produce a core oligosaccharide-modified protein. Methods that include doing. Production of core oligosaccharide modified proteins,
A third vector containing a gene encoding β-1,4-galactosetransferase capable of transferring galactose to a xylose residue and β-1,3-that can transfer galactose to a galactose residue in plant cells. A fourth vector containing a gene encoding a galactose transferase, a fifth vector containing a gene encoding a β-1,3-glucuronic transferase capable of transferring glucuronic acid to a galactose residue, and a xylose residue. A sixth vector containing a gene encoding an enzyme capable of transferring a phosphate group was introduced into the drug.
By expressing the third, fourth, fifth and sixth vectors in plant cells, β-1,4-galactose transferase capable of transferring galactose to xylose residues, and galactose to galactose residues are transferred. Express β-1,3-galactose transferase, β-1,3-glucuronosyl transferase capable of transferring glucuronic acid to galactose residues, and enzymes capable of transferring phosphate groups to xylose residues. Claimed, which is carried out by ligating β-1,4-galactose, β-1,3-galactose, and β-1,3-glucuronic acid to xylose, which is a xylose-modified protein, in order by the action of a transposase. 13. The method according to 13. A method of producing proteoglycans
A core oligosaccharide-modified protein is produced in a plant cell by the method according to claim 13 or 14.
A method comprising connecting amino sugars and uronic acids alternately to the glucuronic acid terminal of a core oligosaccharide-modified protein to form a sugar chain and sulfate to form a glycosaminoglycan. The formation of glycosaminoglycans,
A seventh vector containing a gene encoding an enzyme capable of transferring an amino sugar to a glucuronic acid residue and an eighth vector containing a gene encoding an enzyme capable of transferring uronic acid to an amino sugar residue in a plant cell. A vector, a ninth vector containing a gene encoding an enzyme capable of transferring an amino sugar to a uronic acid residue, and a gene encoding an enzyme capable of sulfated an amino sugar residue and a uronic acid residue of a sugar chain. Introduced with a tenth vector containing,
An enzyme capable of transferring an amino sugar to a glucuronic acid residue by expressing the seventh, eighth, ninth, and tenth vectors in a plant cell, an enzyme capable of transferring uronic acid to an amino sugar residue, Glucuronic acid, a core oligosaccharide-modified protein, is produced by expressing an enzyme capable of transferring an amino sugar to a uronic acid residue and an enzyme capable of sulfated an amino sugar residue and a uronic acid residue of a sugar chain and allowing each enzyme to act. The method according to claim 15 , wherein a sugar chain is formed by alternately connecting amino sugar and uronic acid to the terminal in order, and the sugar chain is sulfated.

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