WO2009119649A1

WO2009119649A1 - Kit for producing o-glycosylated protein in plant and use thereof

Info

Publication number: WO2009119649A1
Application number: PCT/JP2009/055927
Authority: WO
Inventors: 和仁藤山; 正之森; 浩二土肥
Original assignee: 独立行政法人科学技術振興機構; 国立大学法人大阪大学
Priority date: 2008-03-27
Filing date: 2009-03-25
Publication date: 2009-10-01
Also published as: JP2011115043A

Abstract

A kit for producing O-glycosylated protein in a plant contains at least one member selected from polypeptide N-acetylgalactosamine transferase and a polynucleotide encoding the same.

Description

Kit for producing O-type sugar chain binding protein in plants and use thereof

The present invention relates to a kit for producing an O-type sugar chain binding protein in a plant and its use.

Most proteins expressed in eukaryotic cells have some kind of post-translational modification.

Above all, it has been clarified that modification with N-type sugar chains plays an important physiological and biological role. For example, in plants, modification with N-type sugar chains has been reported to be involved in environmental stress tolerance. For example, Patent Document 1 discloses a method for producing a human-type N-type sugar chain binding protein using plant cells.

On the other hand, regarding modification with O-type sugar chains, related enzyme genes and the like have been clarified in mammals, yeasts and the like.
International Publication Number WO / 2000/034490 Pamphlet (released on June 15, 2000)

By the way, there are various advantages in producing useful proteins that can be used in medicines and the like in plants. For example, there are advantages such as relatively easy genetic manipulation for plants and avoidance of animal pathogenic contamination during culturing.

However, plants do not have a modification mechanism for O-type sugar chains. Therefore, plants could not be used for the production of proteins in which modification with an O-type sugar chain has a major role in function and the like.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a kit and a technique using the kit for producing a useful protein modified with an O-type sugar chain using a plant. There is to do.

In order to solve the above problems, the present inventors have conducted intensive studies.

If the O-type sugar chain modification of a protein is important not only for mammals but also for plants, the plant should have an O-type sugar chain modification mechanism. However, plants do not have this. From this, it was possible to reasonably assume that O-type sugar chains do not bind to proteins in plants. Moreover, it was naturally expected that even if a gene related to O-type sugar chain modification was introduced into a plant that does not have an O-type sugar chain modification mechanism, the gene would not function normally.

However, the present inventors have found that when a polynucleotide encoding the polypeptide N-acetylgalactosamine transferase is introduced into a plant and expressed, a protein modified with an O-type sugar chain can be obtained. I came to the idea.

As described above, the present invention has been made on the basis of completely new knowledge contrary to conventional expectations, and includes the following inventions.

That is, a kit for producing an O-type sugar chain binding protein in a plant according to the present invention comprises at least one of a polypeptide N-acetylgalactosamine transferase and a polynucleotide encoding the same. It is a feature.

Furthermore, it is more preferable that the kit according to the present invention further comprises at least one of a galactose transferase and a polynucleotide encoding the enzyme.

The plant according to the present invention is characterized by comprising a polynucleotide encoding a polypeptide N-acetylgalactosamine transferase and a polynucleotide encoding a polypeptide to which N-acetylgalactosamine is bound.

The composition for producing an O-type sugar chain binding protein in a plant according to the present invention comprises at least one of a polypeptide N-acetylgalactosamine transferase and a polynucleotide encoding the same. It is said.

In addition, the method for producing an O-type sugar chain binding protein according to the present invention may include a synthesis step of synthesizing a polypeptide N-acetylgalactosamine transferase in a plant or a plant extract.

In addition, the method for producing an O-type sugar chain binding protein according to the present invention may include a mixing step of mixing a polypeptide N-acetylgalactosamine transferase with a plant extract.

Other objects, features, and superior points of the present invention will be fully understood from the following description. The advantages of the present invention will become apparent from the following description with reference to the accompanying drawings.

It is a figure which shows the result of the electrophoresis in Example 2, (a) of FIG. 1 shows the result using the cell extract of TX13 strain, (b) of FIG. 1 used cell extraction of BY2 wild strain. Results are shown. It is a figure which shows the result of HPLC in Example 2. It is a figure which shows the result of MALDI-TOF-MS in Example 2, (a) of FIG. 3 shows the result of having measured the molecular weight of the PA-ized sugar chain derived from TX13 cell, (b) of FIG. The result of having measured the molecular weight of derived Gal (beta) (1-3) GalNAc-PA is shown. FIG. 6 is a diagram showing the result of electrophoresis in Example 3.

<1. Kit for producing O-type sugar chain binding protein in plant according to the present invention>
A kit for producing an O-type sugar chain binding protein in a plant according to the present invention (hereinafter referred to as “kit according to the present invention”) is a polypeptide N-acetylgalactosamine transferase and a polynucleotide encoding the same. It suffices to have at least one of them. According to the kit of the present invention, an O-type sugar chain binding protein can be produced using a plant.

The kit according to the present invention does not have a plant as a target protein to which N-acetylgalactosamine is bound (also referred to as a target protein to which an O-type sugar chain is bound. Hereinafter, referred to as “target protein”). When protein is employed, a particularly advantageous effect is exhibited. For example, in the case of producing a protein in which binding of an O-type sugar chain has a significant effect on its function, it has conventionally been necessary to use animal cells. Since a chain-binding protein can be produced, it is advantageous in that contamination of animal pathogens can be avoided during culturing.

In the present specification, the “O-type sugar chain” means a sugar chain bound to a serine residue and / or a threonine residue of a protein. The “O-type sugar chain binding protein” means a protein to which an O-type sugar chain is bound. In this specification, “A and / or B” means “A”, “B”, or “A and B”.

In the present specification, “plants” include various forms of plants. For example, whole plant, plant organ (eg leaf, petal, stem, root, seed, etc.), plant tissue (eg epidermis, phloem, soft tissue, xylem, vascular bundle, fence-like tissue, spongy tissue, etc.), Various forms of plant cells (for example, cultured cells such as suspension culture cells), protoplasts, callus and the like can be mentioned.

In addition, in the present specification, “producing an O-type sugar chain binding protein in a plant” means that an O-type sugar chain binding protein is produced using a plant. The type and form of the plant used here is not particularly limited, and an extract obtained from a plant may be used. That is, the production of an O-type sugar chain binding protein using a plant extract is also included in the category of “producing an O-type sugar chain binding protein in a plant”.

In the present specification, “polypeptide N-acetylgalactosamine transferase” means an enzyme having an activity of binding N-acetylgalactosamine to a serine residue and / or a threonine residue in a protein. Hereinafter, the polypeptide N-acetylgalactosamine transferase is referred to as “ppGalNAcT”, the polynucleotide encoding the polypeptide N-acetylgalactosamine transferase is referred to as “ppGalNAcT gene”, and serine residues and / or threonine in the protein. The activity of binding N-acetylgalactosamine to a residue is referred to as “ppGalNAcT activity”.

The types of plants that can be used in the kit according to the present invention are not particularly limited. For example, it can be applied to various plants such as various monocotyledonous plants, dicotyledonous plants and trees. For example, monocotyledonous plants include, for example, duckweed plants including duckweed plants (duckweed) and duckweed plants (duckweed, Hingemo); cattleya plants, cymbidium plants, dendrobium plants, phalaenopsis plants, vanda genera Plants, Paphiopedilum spp., Oncidium spp., Etc .; Rubiaceae plants; Rubiaceae plants; Rubiaceae plants; Relatives plants; Thorny plants; Tochika plant, Japanese cucurbitaceae plant, Gramineae plant, Orchardaceae plant, Palmiaceae plant, Sugar beet plant, Hosogusa plant, Thripsaceae plant, Mizuoiaceae plant, Rabbitaceae plant , Bacteriaceae, Lilyaceae, Higanbanaceae, Yamano potato, Ayameceae, Ganoderma, Ginger Canna plant of the family, can be exemplified Hinanoshakujou Plants like.

In addition, examples of dicotyledonous plants include Asagao genus plants (Asagao), Convolvulus genus plants (Convolvulus, Coleoptera, Clamgao), Sweet potato species (Gumbai convolvulus, Sweet potato), and Prunus genus plants (Nenshikazura, Mamedaoshi) Plants: Nadesico genus plants (carnations, etc.), Jacobe genus plants, Takanetus genus plants, Miminagusa genus, clover genus plants, Nominotsutsuri genus plants, Oyafusuma genus plants, Waigaiso genus plants, Hamahakobe genus plants, Otsumexa genus plants, Shiotsuma genus Plants, mantemae plants, genus plants, genus fusiflora, nanbanjakobe plants; anemoneaceae plants; aroma family plants, donut family plants, anthropaceae plants, anthropaceae plants, anthropaceae plants, yam family Plant, walnut plant , Birch family plant, beech family plant, bittern family plant, laceaceae plant, iridaceae plant, dendrobaceae plant, sweet potato family plant, shabby genus plant, duck family plant, Yadorigi, Uma no Suzukaku, Yakusou, Uchibuchi, Uchida, Azaza, Ayuyu, Oshirobana, Yamatobusa, Yamagobo Family plant, vine family plant, slippery family plant, creek family plant, mountain bear family plant, wig family plant, renaceae plant, pine family plant, genus genus plant, aceae family, urchinaceae Plants, Tsurujifuji Plants, Waxyaceae Plants, Kusanaki Family Plants, Poppy Family Plants, Japanese Psyllidaceae Plants, Oily Plant Plants, Apricot Family Plants, Utsubo Pseudophyceae Plants, Agrobiaceae Plants, Yukino Plant, laver plant, ma Succulent plant, Suzukake family plant, Rose family plant, Lepidopterous plant, Stomach family plant, Diplophyceae plant, Lariaceae plant, Habushi family plant, Tangerine family plant, Ginger family plant, Sendai Plant, Himehagi plant, Todagusa plant, Dendrobaceae plant, Boxwood plant, Gourdaceae plant, Gourdaceae plant, Urushiaceae plant, Mokinoki plant, Nishikigi plant, Mitsuba Gourdaceae plant, Kurodaki kazura family plant, Maple family plant, Tochinoaceae plant, Mukuroji family plant, Dipteraceae plant, Dendrobaceae plant, Drosophila family plant, Vineceae plant, Horono Mushroom plant, Shinano family plant, Aoi family plant, Papilio family plant, Monkeys family plant, Camellia family plant, Fairy family plant, Mizo Hachibana plant, Gyory family plant, Violet family plant, Iri family Plants, asteraceae plants, and Diatomaceous plant, asteraceae plant, cactaceae plant, rhododendron plant, gypsum plant, misoaceae plant, pomegranate plant, asteraceae plant, cucurbitaceae plant, papaveraceae plant, rhinoceros plant Family plant, red plant family plant, Arino family plant family plant, urchinaceae plant plant, urchinaceae plant plant, urchinaceae plant plant, citrus family plant plant, irrigated plant family plant, rhododendron plant plant, ivy plant family plant, Azalea family plant, Bamboo family plant, Cherry tree family plant, Isomatsu family plant, Oyster family plant, Oyster family plant, Erynaceae plant, Mokusei family plant, Fuji next family plant, Rindo Family plant, asteraceae plant, garaceae plant, bush plant family, purple plant family, kumazaku family plant, shiso family plant, eggplant family plant (tomato, etc.), sesame plant family, sesame family plant Plant, sesame plant, Urchinaceae plant, cigarette plant, raccoonaceae plant, foxglove plant, hamanchoaceae plant, staghorn plant, scallop plant, rape plant, watermelon plant, reptile plant Examples of the plant include the family plant of the family, the pine family, the cucurbitaceae plant, the asteraceae plant, and the asteraceae plant.

[Composition of the kit according to the present invention: a form comprising a ppGalNAcT gene]
The ppGalNAcT gene provided in the kit according to the present invention is not particularly limited as long as it encodes the above-mentioned polypeptide having activity as ppGalNAcT. For example, the ppGalNAcT gene derived from various organisms such as mammals such as humans, birds and reptiles can be included in the kit according to the present invention. More specifically, GalNAc-T2 (for example, GenBank accession number NM_004481) and GalNAc-T1 (for example, GenBank accession number NM_020474) are exemplified.

In addition, the polynucleotide that can be included in the kit according to the present invention includes a polynucleotide that hybridizes under stringent conditions with a polynucleotide comprising a complementary base sequence of the polynucleotide of the specific example described above and has ppGalNAcT activity. Examples thereof include a polynucleotide encoding a peptide. Here, hybridizing under stringent conditions means binding at 60 ° C. under 2 × SSC washing conditions. The hybridization can be carried out by a conventionally known method such as the method described in J. Sambrook et al. Molecular Cloning, A Laboratory Manual, 2nd Ed, Cold Spring Harbor Laboratory (1989). Usually, the higher the temperature and the lower the salt concentration, the higher the stringency (harder to hybridize).

The method for obtaining the ppGalNAcT gene is not particularly limited, and the ppGalNAcT gene can be isolated from the aforementioned mammals and the like by a conventionally known method. For example, the ppGalNAcT gene can be obtained by performing PCR using a primer pair prepared based on the base sequence of a known ppGalNAcT gene, using the cDNA or genomic DNA of the above-mentioned mammal or the like as a template. The ppGalNAcT gene can also be obtained by chemical synthesis by a conventionally known method.

The kit according to the present invention may be provided with an expression vector for expressing the ppGalNAcT gene. The expression vector for ppGalNAcT is not particularly limited as long as it includes a ppGalNAcT gene and a promoter.

Various conventionally known vectors can be used as the base vector for the expression vector. For example, a plasmid, phage, cosmid or the like can be used, and can be appropriately selected according to the plant cell to be introduced and the introduction method. Specific examples include pBR322, pBR325, pUC19, pUC119, pBluescript, pBluescriptSK, pBI vectors, and the like. In particular, when a vector comprising the ppGalNAcT gene is introduced into a plant by the Agrobacterium method using the kit according to the present invention, it is preferable to use a pBI binary vector. Specific examples of the pBI binary vector include pBIG, pBIN19, pBI101, pBI121, pBI221, and the like.

The promoter is not particularly limited as long as it is a promoter capable of expressing a gene in a plant body, and a known promoter can be suitably used. Examples of such promoters include cauliflower mosaic virus 35S promoter (CaMV35S), actin promoter, nopaline synthase promoter, tobacco PR1a gene promoter, tomato ribulose 1,5-diphosphate carboxylase oxidase small subunit promoter and the like. Can be mentioned. Among these, cauliflower mosaic virus 35S promoter or actin promoter can be used more preferably. When each of the above promoters is used, the resulting expression vector can strongly express an arbitrary gene when introduced into a plant cell.

The promoter is not particularly limited as long as it is linked so that a gene encoding a transcription factor can be expressed and introduced into the vector.

The expression vector may further contain another DNA segment in addition to the promoter and the ppGalNAcT gene. The other DNA segment is not particularly limited, and examples thereof include a terminator, a selection marker, an enhancer, and a base sequence for improving translation efficiency. The expression vector may further have a T-DNA region. The T-DNA region can increase the efficiency of gene transfer particularly when Agrobacterium is used to introduce the expression vector into a plant.

The terminator is not particularly limited as long as it has a function as a transcription termination site, and may be a known one. For example, specifically, the transcription termination region (Nos terminator) of the nopaline synthase gene, the transcription termination region of the cauliflower mosaic virus 35S (CaMV35S terminator) and the like can be preferably used. Of these, the Nos terminator can be more preferably used. In the above expression vector, by placing the terminator at an appropriate position, it is necessary to synthesize an unnecessarily long transcript after introduction into a plant cell, or a strong promoter reduces the copy number of the plasmid. Can be prevented from occurring.

As the selection marker, for example, a drug resistance gene can be used. Specific examples of such drug resistance genes include drug resistance genes for hygromycin, bleomycin, kanamycin, gentamicin, chloramphenicol and the like. Thereby, the transformed plant body can be easily selected by selecting the plant body growing in the medium containing the antibiotic.

Examples of the polynucleotide for increasing the translation efficiency include an omega sequence derived from tobacco mosaic virus. By placing this omega sequence in the untranslated region (5′UTR) of the promoter, the translation efficiency of the gene encoding the transcription factor can be increased. Thus, the expression vector can contain various DNA segments depending on the purpose.

As a specific method for constructing an expression vector, there is a method in which the promoter and the ppGalNAcT gene and, if necessary, the other DNA segments are introduced in a predetermined order into an appropriately selected parent vector. Illustrated. In addition, an expression cassette may be constructed by linking a ppGalNAcT gene and a promoter (such as a terminator if necessary) and then introducing it into a vector.

In the construction of the expression cassette, for example, the order of the DNA segments can be defined by setting the cleavage sites of each DNA segment as complementary protruding ends and reacting with a ligation enzyme. In addition, when a terminator is included in the expression cassette, the promoter, the ppGalNAcT gene, and the terminator may be in order from the upstream. Further, the types of reagents for constructing the expression vector, that is, the types of restriction enzymes and ligation enzymes are not particularly limited, and commercially available ones may be appropriately selected and used.

Also, the method for producing the above expression vector (production method) is not particularly limited, and a conventionally known method can be used. In general, Escherichia coli may be used as a host and propagated in the E. coli. At this time, a preferred E. coli type may be selected according to the type of vector.

In addition, the kit according to the present invention includes a vector, a promoter, a selection marker, a polynucleotide for enhancing translation efficiency, and a terminator, which serve as a parent of the above-described expression vector, in a separate container from the ppGalNAcT gene. You may have.

[Configuration of kit according to the present invention: a form including ppGalNAcT]
The ppGalNAcT provided in the kit according to the present invention is not limited as long as it is a polypeptide having ppGalNAcT activity. In the present specification, the term “polypeptide” is used interchangeably with “peptide” or “protein”.

For example, the ppGalNAcT provided in the kit according to the present invention may be one encoded by the ppGalNAcT gene described above.

Also by recombinant techniques from natural purified products, products of chemical synthesis procedures, and prokaryotic or eukaryotic hosts (including, for example, bacterial cells, yeast cells, higher plant cells, insect cells, and mammalian cells). Includes produced products. It can also be myristylated or non-myristylated depending on the host used in the recombinant production procedure. Furthermore, the polypeptides according to the invention may also comprise an initiating modified methionine residue in some cases as a result of a host-mediated process.

Further, ppGalNAcT provided in the kit according to the present invention may be dissolved in advance in a conventionally known protein storage reagent.

[Other configurations]
The kit according to the present invention may include various other reagents and the like as long as the effects of the present invention are not impaired. The O-type sugar chain is composed of N-acetylgalactosamine as a basic skeleton and other sugars and / or acids such as galactose, N-acetylglucosamine, and sialic acid. Therefore, the kit according to the present invention may contain a sugar and / or an acid transferase constituting the O-type sugar chain. Such transferases include galactose transferase, ST6GALNAC1 (alpha-N-acetyl-neuraminyl-2,3-beta-galactosyl-1,3) -N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 (GenBank Accession) No. NC — 000017), core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase, 1 (GenBank accession number NC — 000007), UDP-GlcNAc: betaGal beta-1,3-N-acetylglucosaminyltransferase 6 (core3 synthase) Session number NC — 000011), ST3 beta-galactoside alpha-2,3-sialyltransferase 1 (GenBank accession number NM — 003033) and the like can be preferably exemplified. Also, for example, Varki, Ajit; Cummings, Richard; Esko, Jeffrey; Freeze, Hudson; Hart, Gerald; Marth, Jamey., Essentials of Glycobiology., Cold Spring Harbor Laboratory Press, 1999, Chapter 8, Fig. 8- You may provide the enzyme which catalyzes each process in the synthesis | combination process of O-type sugar chain binding protein described in 3, 8-4, 8-5, 8-6.

Note that an enzyme other than ppGalNAcT, which is a sugar or acid transferase constituting the O-type sugar chain, is referred to as “further transferase” for convenience of explanation. By providing the kit according to the present invention with a further transferase, any O-type sugar chain can be bound to the target protein.

The kit according to the present invention may include a polynucleotide encoding the further transferase. Moreover, the polynucleotide etc. which hybridize with the polynucleotide which consists of a complementary base sequence of these polynucleotides on stringent conditions, and encodes the polypeptide which has each enzyme activity may be included.

The method for obtaining the polynucleotide encoding the further transferase is not particularly limited, and can be isolated from many mammals, birds, reptiles and the like by a conventionally known method. For example, a primer pair prepared based on the base sequence of a gene encoding a known galactose transferase can be used. Using this primer pair, a polynucleotide can be obtained, for example, by performing PCR using cDNA or genomic DNA of the above-mentioned mammal or the like as a template. A polynucleotide encoding a further transferase can also be obtained by chemical synthesis by a conventionally known method.

These polynucleotides may be provided separately from the ppGalNAcT gene in a separate container.

In addition, when the kit according to the present invention includes the polynucleotide of the further transferase, it may be in the form of an expression vector including the polynucleotide. This expression vector conforms to the description of the expression vector for ppGalNAcT described above.

In the kit according to the present invention, the polynucleotide encoding the further transferase may be introduced into the expression vector of ppGalNAcT described above, and the expression vector of ppGalNAcT and the expression vector of the further transferase are expressed separately. It is good also as a vector.

Further, the kit according to the present invention may be in the form of a target protein and / or a polynucleotide encoding the target protein (hereinafter referred to as “target protein gene”). The target protein gene may be introduced into an expression vector separate from the ppGalNAcT gene, or both genes may be introduced into one expression vector. This form is effective when adopting a protein that the plant does not have as the target protein. This is because according to the kit according to the embodiment, the user of the kit does not need to separately adjust the target protein and / or target protein gene, and can more easily produce the O-type sugar chain binding protein.

Further, other reagents that can be included in the kit according to the present invention are not limited to those described so far. For example, ppGalNAcT, the further transferase, the target protein, a reagent for stably holding the polynucleotide encoding them, a buffer, etc. may be included, a restriction enzyme for introducing the polynucleotide into a vector, A reagent such as ligase may be included. Moreover, the extract from a plant cell and a plant may be included, and the reagent for introduce | transducing the said polynucleotide into a plant cell may be included. In the kit according to the present invention, a plurality of different reagents may be mixed in an appropriate volume and / or form, or may be provided in separate containers.

Note that, as used herein, the term “kit” is intended to mean a package including a container (eg, bottle, plate, tube, dish, etc.) containing a specific material. As used herein in the context of a kit, “comprising” is intended to mean that the ppGalNAcT and / or ppGalNAcT gene is encapsulated in any of the individual containers that make up the kit. At this time, the ppGalNAcT and / or ppGalNAcT gene is not particularly limited, but may be dissolved in a conventionally known storage solution such as a buffer solution or may be purified. The kit according to the present invention may be provided with the above-mentioned ppGalNAcT, the above-mentioned additional transferase, the target protein, the polynucleotide encoding them, and other reagents mixed in the same container or in separate containers. Good.

In addition, the kit according to the present invention may include an instruction sheet describing a procedure for producing an O-type binding protein in a plant. It may be written or printed on paper or other media, or it may be affixed to electronic media such as magnetic tape, computer-readable discs or CD-ROMs.

[Method of using the kit according to the present invention]
Although the example of the usage method of the kit which concerns on this invention is demonstrated below, it is not limited to this, What is necessary is just to use it so that O-type sugar chain binding protein can be manufactured in a plant.

When the kit according to the present invention includes a ppGalNAcT gene, the ppGalNAcT gene may be expressed in a plant or an extract from a plant. The method for expressing the ppGalNAcT gene is not particularly limited, and a conventionally known gene expression method in plants such as the above-described expression vector may be used.

Moreover, the target protein only needs to contain a serine residue and / or a threonine residue. For example, the target protein may be a polypeptide possessed by a plant (hereinafter referred to as “endogenous protein”) or a polypeptide possessed by an organism other than a plant (hereinafter referred to as “foreign protein”). Good.

Intrinsic proteins include, for example, glutelin (Glutelin, eg, rice-derived) (Kishimoto T, Watanabe M, Mitsui T, Hori H.Glutelin basic subunits have a mammalian mucin-type O-linked disaccharide side chain.Arch Biochem Biophys 1999 Oct 15; 370 (2): 271-277).

Examples of the foreign protein include erythropoietin, synthetic peptide (see, for example, Otvos, L. Jr., Krivuika. Et al., Biochim. Biophys. Acta., 1995, 1267, 55-64), mucin (for example, Shogren, R , et al., Biochemistry, 1989, 28, 5525-5536), HSP-1, HSP-2 (heat shock proteins such as Calvete, JJ, et al., Biochem. J., 1995, 310, 615). Referring to -622), T1 fragment of glycophorin a N ( e.g. Pieper, J., et al., Nat. Struct. Biol., 1996, 3, see 228-232), DAF (decay accelerating factor, e.g., Coyne , KE, et al., J. Immunol., 1992, 149, 2906-2913), G-CSF (granulocyte colony stimulating factor such as Oh-eda, M., et al., J. Biol. Chem). ., 1990, 265, 11432-11435), PSGL-1 (P-selection glycoprotein ligand-1, example) See, for example, Maly, P., et al., Cell, 1996, 86, 643-653), ZP-3 (zona pelucida protein 3, eg Yurewicz, EC, et al., Mol. Reprod Dev., 1992, 33, 182), Peptides in MHC (major histocompatibility complex) -groove (eg Haurum, JS, et al., J. Exp. Med., 1994, 180, 739-744, Haurum , JS, et al., Eur. J. Immunol., 1995, 25, 3270-3276), InterLeukin-5 (eg, Kodama, S., et al., Eur. J. Biochem., 1993, 211). , 903-908), Lactase-phlorizin hydrolase (eg Naim, HY, and Lentze, MJ, J. Biol. Chem., 1992, 267, 25494-25504), Glycophorin A (eg Remaley, AT, J Biol. Chem., 1991, 266, 24176-24183), GF-II (insul) n-like growth factor II, for example, Daughaday, WH, et al., Proc. Natl. Acad. Sci., 1993, 90, see 5823-5827).

When N-acetylgalactosamine is bound to an endogenous protein, a ppGalNAcT gene may be introduced into a plant cell or extract and expressed together with a polynucleotide encoding the endogenous protein, or ppGalNAcT may be mixed with a plant extract. Also good.

When N-acetylgalactosamine is bound to a foreign polypeptide, the polynucleotide encoding the foreign polypeptide may be introduced into a plant cell or extract that expresses the ppGalNAcT gene, and this may be expressed. PpGalNAcT and the foreign protein may be mixed in the product.

The method for introducing a ppGalNAcT gene and a target protein gene into a plant is not particularly limited, and a conventionally known plant transformation method may be used. For example, an Agrobacterium method, a particle gun method, a polyethylene glycol method, an electroporation method, or the like is used. When the Agrobacterium method is used, the above expression vector is introduced into an appropriate Agrobacterium (for example, Agrobacterium tumefaciens), and this strain is introduced into the leaf disk method (by Hirofumi Uchimiya, plant gene manipulation). Manual, 1990, 27-31pp, Kodansha Scientific, Tokyo, etc.) can be used to infect sterile cultured leaf pieces to obtain transformed plants.

PpGalNAcT gene and target protein gene may be introduced into separate expression vectors, or both genes may be introduced into one expression vector.

When the ppGalNAcT gene and the target protein gene are introduced into separate expression vectors, transformation using each expression vector may be performed at one time or in two stages. “Transformation in two stages” means that one of the expression vectors is introduced and the other expression vector is introduced into a transformed plant in which the introduction of the expression vector is confirmed. Thus, by performing transformation in two steps, a plant that most stably expresses the expression vector introduced first can be selected in advance. If the other expression vector is introduced into the plant selected here, it becomes possible to obtain a plant in which both expression vectors are stably and highly expressed with high probability. Therefore, it is possible to obtain a plant capable of producing an O-type binding protein with high labor and time, and as a result, it becomes possible to mass-produce useful proteins efficiently, inexpensively and safely. .

In addition, when the kit according to the present invention is in a form including ppGalNAcT, the method of use thereof is not particularly limited. For example, ppGalNAcT may be mixed with a plant extract.

The plant extract is not particularly limited as long as it is extracted from the plant, for example, it may be extracted using a conventionally known solvent, the plant is ground and filtered, It may be obtained by squeezing.

<2. Plant according to the present invention>
The plant according to the present invention only needs to have a ppGalNAcT gene and a polynucleotide (target protein gene) encoding a polypeptide (target protein) to which N-acetylgalactosamine is bound.

For example, a ppGalNAcT gene and a target protein gene may be introduced into a genome present in a plant chromosome, mitochondria or the like.

The method for introducing a gene into a plant, the form and type of the plant into which the gene is introduced, and the like may be the same as those described for the kit according to the present invention. In addition, when employ | adopting what a plant has as a target protein, only a ppGalNAcT gene may be introduce | transduced into a plant, and in order to make a target protein express more highly, both genes may be introduce | transduced into a plant. .

<3. Composition>
A composition for producing an O-type sugar chain binding protein according to the present invention (hereinafter referred to as “the composition according to the present invention”) encodes a polypeptide N-acetylgalactosaminyltransferase (ppGalNAcT) and the same At least one of the polynucleotides (ppGalNAcT gene) may be included.

In the composition according to the present invention, when ppGalNAcT is included, the concentration thereof is not particularly limited. In this case, ppGalNAcT may be dissolved in a conventionally known protein buffer. Such a buffer solution is not limited as long as ppGalNAcT can be stored. For example, a buffer solution used in measuring enzyme activity in the following document can be used. Wandall HH, et al., J Biol Chem., 1997 Sep 19; 272 (38): 23503-23514, Zhang Y, et al., J Biol Chem., 2003, Jan 3; 278 (1): 573-584 Bennett EP, et al, J Biol Chem., 1998, Nov 13; 273 (46): 30472-30481. Also, for example, Iwasaki H, et al., J Biol Chem., 2003, Feb 21; 278 (8): 5613-5621 includes 25 mM Tris-HCl (pH 7.4), 5 mM MnCl ₂ , 0.1 A mixture of% Triton X-100 can be used as a storage buffer. Moreover, you may include enzyme stabilizers, such as glycerol, in these buffers.

Further, when the composition according to the present invention includes the ppGalNAcT gene, the concentration thereof is not particularly limited. Further, the ppGalNAcT gene only needs to be dissolved in a conventionally known nucleic acid storage buffer.

The composition according to the present invention may contain the above-described further transferase and / or a polynucleotide encoding the same, a target protein and / or a target protein gene.

<4. Production method 1 of O-type sugar chain binding protein according to the present invention>
The method for producing an O-type sugar chain binding protein according to the present invention may include a synthesis step of synthesizing a polypeptide N-acetylgalactosamine transferase in a plant or a plant extract.

The specific method of the synthesis step is not particularly limited as long as ppGalNAcT can be synthesized in a plant or a plant extract.

For example, the ppGalNAcT gene may be expressed in plants. In this case, the synthesis step may be a step of performing an introduction step of introducing a ppGalNAcT gene into a plant cell and then culturing a plant cell into which the ppGalNAcT gene has been introduced.

The introduction step is not limited as long as it is a step of introducing a ppGalNAcT gene into a plant cell. For example, a conventionally known plant transformation method described above may be used.

In the above culturing step, plant cells into which the ppGalNAcT gene has been introduced may be maintained in the state of cultured cells, differentiated into tissues such as leaves, roots, and stems, or regenerated into complete plants. May be. Alternatively, ppGalNAcT may be synthesized in a plant extract. For example, ppGalNAcT gene may be expressed by a cell-free expression system after mixing the ppGalNAcT gene in a plant extract.

After the above synthesis step, the O-type sugar chain binding protein may be purified from the plant or from the plant extract. For purification of the O-type sugar chain binding protein, a conventionally known purification method for N-type sugar chain protein may be applied. For example, a lectin that specifically recognizes an O-type sugar chain binding protein may be used.

<5. Production method 2 of O-type sugar chain binding protein according to the present invention>
The method for producing an O-type sugar chain binding protein according to the present invention may include a mixing step of mixing a polypeptide N-acetylgalactosaminyltransferase with a plant extract.

The specific method of the mixing step is not particularly limited as long as ppGalNAcT is mixed with the plant extract. For example, ppGalNAcT may be simply dropped on a plant extract, or may be stirred. O-type sugar chains are produced using UDP-GalNAc contained in plant extracts as a substrate. Further, when UDP-GalNAc is not contained in the plant extract, it may be added separately, or even if it is contained, it may be further added.

After the mixing step, the O-type sugar chain binding protein may be purified from the plant extract. The purification of the O-type sugar chain binding protein may be performed using a conventionally known purification method for the type sugar chain binding protein.

Examples will be shown below, and the embodiments of the present invention will be described in more detail. Of course, the present invention is not limited to the following examples, and it goes without saying that various aspects are possible in detail. Further, the present invention is not limited to the above-described embodiments, and various modifications can be made within the scope shown in the claims, and the present invention is also applied to the embodiments obtained by appropriately combining the disclosed technical means. It is included in the technical scope of the invention. Moreover, all the literatures described in this specification are used as reference in this specification.

<Example 1. Production of transformed plant cells>
From human tissues, the ppGalNAcT gene and the galactose transferase gene were cloned.

The ppGalNAcT gene was obtained by the following method. A liver-derived human cDNA library is used as a template (ORIGENE | TECHNOLOGIES | INC. -3 ′) (SEQ ID NO: 2) was amplified by PCR to obtain a DNA fragment containing the ppGalNAcT gene. This was digested with BamHI and SacI and then introduced into the BamHI / SacI site of Ti plasmid pBI121 to construct plasmid pBI-GalNAc-T1.

The galactose transferase gene was obtained by the following method. Liver-derived human cDNA library (ORIGENE TECHNOLOGIES INC. Multiple Choice (TM) Human cDNA) as a template, primer GT-F (5'-GGCGGATCCAAGGAGATATAACAATGGCCTCTAAATCCTGGCTGAATTTTTTA-3 ') (SEQ ID NO: 3) and GT-TT (TC'TTGATCTC -3 ′) (SEQ ID NO: 4) was amplified by PCR to obtain a DNA fragment containing the galactose transferase gene. This was cleaved with BamHI and SacI and then introduced into the BamHI / SacI site of pBI121 to construct plasmid pBI-C1GALT1.

Next, together with each vector, tobacco BY2 cultured cells (Dohi et al., Archives of Virology, 151, 1075-1084, RIKEN; RIKEN BioResource Center; experimental plant development room; plant culture cell catalog; RPC No. 00001) was co-transformed by the Agrobacterium method. MRNA was prepared from the resulting transformed cells, and the expression of N-acetylgalactosamine transferase gene and galactose transferase gene was confirmed by Northern analysis. Then, transformed cells that express both transferase genes were selected. Of the 300 plant cells used in this experiment, two transformed cells expressed both transferase genes together. Among them, a transformed cell having a high mRNA amount (hereinafter referred to as “TX13 strain”) was further selected and cultured in suspension using Murashige-Skoog medium.

<Example 2. Confirmation of O-type glycan binding protein>
The TX13 strain cultured in suspension in Example 1 was suspended in 1.5 ml of 175 mM Tris-HCl buffer (pH 7.5). The cells in the obtained suspension were homogenized using a mortar and pestle and then subjected to centrifugation (8,000 rpm, 15 minutes, 4 ° C.). Next, the supernatant (cell extract) was collected. Similarly, a cell extract was prepared from non-transformed BY2 cells (hereinafter referred to as “BY2 wild strain”).

Next, cell extracts obtained from the TX13 strain and BY2 wild strain were each passed through a lectin column to adsorb glycoproteins to the lectin. The non-adsorbed fraction was discarded. The lectin column used was a column made by Bio-Rad (product number 737-1006, inner diameter 1 cm length 5 cm) with 0.5 ml of Jacalin lectin. Jackalin recognizes the Galβ (1-3) GalNAc structure.

Next, the lectin column was washed with 20 ml of 175 mM Tris-HCl buffer (pH 7.5) to collect the eluate. Further, the adsorbed protein was eluted with 175 mM Tris-HCl buffer (pH 7.5) containing 10 mM methylgalactopyranoside, and 1 ml (fraction 1), 1 ml (fraction 2), 1 ml (fraction 3), It recovered as 1 ml (fraction 4) and 6 ml (fraction 5).

Next, fractions 1 to 3 derived from each cell extract were subjected to 12.5% SDS-polyacrylamide gel electrophoresis. The results are shown in FIG. FIG. 1 is a diagram showing the results of electrophoresis in this example. FIG. 1 (a) shows the results using a cell extract of TX13 strain, and FIG. 1 (b) uses the cell extract of BY2 wild strain. Shows the results. Moreover, the numbers shown above each lane in FIGS. 1A and 1B indicate the numbers of fractions, and “2 & 3” indicates that the mixture of

fractions

2 and 3 was subjected to electrophoresis. .

As shown by the triangle mark in FIG. 1 (a), the lectin-binding protein that was not confirmed from the cell extract of the BY2 wild strain was confirmed in the cell extract of the TX13 strain.

Next,

fractions

1 and 2 derived from each cell extract were dialyzed against ultrapure water and then freeze-dried. The obtained product was dissolved in 0.2 M sodium citrate buffer (pH 4.5), α-N-acetylgalactosaminidase (manufactured by Seikagaku Corporation) was added, and the mixture was kept at 37 ° C. for 3 days. . In the examples of the present specification, “ultra pure water” refers to water purified using a Milli-Q system (Millipore).

Next, the solution after the incubation was purified using Carbograph (GL-Science). First, the solution was passed through Carbograph, then washed with 5 ml of 50 mM ammonium acetoacetate (pH 7.0), and sugar chains were eluted with 50 mM ammonium acetoacetate (pH 7.0) containing 60% acetonitrile. The resulting solution was then lyophilized.

A suitable amount of a PA-forming reagent (2-aminopyridine (manufactured by Wako), 552 mg mixed with 200 μl of acetic acid (manufactured by Wako)) was added to the lyophilized sample and incubated at 90 ° C. for 1 hour. Next, after the sample was cooled at room temperature, an equal amount of a reducing reagent (a mixture of 200 μl of acetic acid with 39 mg of dimethylaminoborane (Wako)) was added and incubated at 80 ° C. for 40 minutes. Next, an equal amount of distilled water was added to stop the reaction.

To the resulting solution, an equal amount of ultrapure water saturated phenol solution: chloroform solution (1: 1) was added to extract the unreacted PA reagent. This extraction operation was repeated again. Next, the chloroform solution was mixed, and the obtained aqueous layer portion was extracted.

Next, the extract was allowed to stand at room temperature under vacuum to be concentrated to one-tenth of the liquid volume and subjected to analysis by liquid chromatography (HPLC).

In addition, as a control, the same operation was performed on a commercially available Galβ (1-3) GalNAc (manufactured by Toronto Research Chemicals) and subjected to HPLC. That is, an appropriate amount of the above PA reagent was added to 1 mg of Galβ (1-3) GalNAc and incubated at 90 ° C. for 1 hour.

Next, after cooling the sample at room temperature, an equal amount of the above-mentioned reducing reagent was added and incubated at 80 ° C. for 40 minutes. Next, an equal amount of distilled water was added to stop the reaction. Thereafter, an equal amount of ultrapure water saturated phenol solution: chloroform solution (1: 1) was added to extract an unreacted PA reagent. This extraction operation was repeated again, and then the chloroform solution was mixed to extract the obtained aqueous layer portion. The extract was allowed to stand at room temperature under vacuum to be concentrated to one-tenth volume and subjected to HPLC.

The analysis by HPLC was performed as follows. Acetonitrile was added to the sample so as to be 80% of the total amount and subjected to HPLC (SF-HPLC). As an HPLC column, an amide column (manufactured by Showa denko, model name: Asahipak NH2P-50; 4.6 × 250 nm) was used. As the HPLC apparatus, a HITACHI HPLC system equipped with a HITACHI FL Detector L-7480 was used. Detailed conditions such as developing solvent and gradient are as follows.
Developing solvent: 80% acetonitrile Gradient: Isocratic flow rate: 0.7 ml / min Detection: Fluorescence (Ex; 310 nm, Em; 380 nm)
Column temperature: 30 ° C
The results are shown in FIG. FIG. 2 is a diagram showing the results of HPLC in this example.

As shown in FIG. 2, as a result of using the cell extract of TX13 strain, a peak was detected at the same position as Galβ (1-3) GalNAc-PA derived from the control. The elution fraction of this peak was separated and purified. From this, it was confirmed that the protein extracted with N-acetylgalactosamine was contained in the cell extract of the TX13 strain.

Next, the molecular weight of the purified protein (PA sugar chain, fraction 2 above) was measured by MALDI-TOF-MS. Autoflex (manufactured by Bruker Daltonics) was used as the apparatus. Data was obtained under a vacuum of 3.0 × e ⁻⁷ or less with a laser intensity of 1800-2000 mbar. As a matrix reagent, a solution obtained by mixing 10 mg of 2,5-dihydroxybenzoic acid (manufactured by Sigma) with a solution obtained by mixing distilled water: acetonitrile at 1: 1 was used. In addition, an equal amount of a matrix reagent was mixed with distilled water in which PA sugar chains were dissolved, and 2 μl of this was placed on a target and crystallized by drying at room temperature, followed by reflector mode analysis.

The results are shown in FIG. FIG. 3 is a diagram showing the results of MALDI-TOF-MS of this example. FIG. 3 (a) shows the results of measuring the molecular weight of the PA13 sugar chain derived from TX13 cells, and FIG. 3 (b). Shows the result of measuring the molecular weight of Galβ (1-3) GalNAc-PA derived from the control.

As shown in FIGS. 3 (a) and 3 (b), the molecular weight (m / z for [M + Na] ⁺ = 482) of the PA-glycan derived from TX13 cells is the same as the control-derived Galβ (1-3) GalNAc−. It was the same as the molecular weight of PA, and almost coincided with the molecular weight estimated from the structure (483.5).

[Example 3: Production of O-glycan-binding protein by tobacco plant]
A ppGalNAcT gene and a galactose transferase gene were introduced into wild-type tobacco SR1 species to obtain transformed cells of SR1. The obtained transformed cells were cultured to obtain SR1 plants. Acquisition of plant bodies from the transformed cells was carried out with reference to Masaki Iwabuchi, Reiro Shimura, “Lab Manual Functional Analysis of Plant Genes”, Maruzen Co., Ltd., p32-p46.

Specifically, the leaves of SR1 tobacco plant grown aseptically were cut into 1 × 1 cm, and Agrobacterium into which plasmid pBI-GalNAc-T1 and plasmid pBI-C1GALT1 obtained by the method described in Example 1 were introduced. The suspension was infiltrated for 1 minute. Then, MS1 (2 mg / l NAA, 0.02 mg / l BAP, 30 g / l sucrose, 0.8% agar) was added to the MS salt, leaves were placed on the medium and cultured at 26 degrees for 2 days. MS4 containing 100 mg / l kanamycin, 100 mg / l carbenicillin (MSA salt with 0.1 mg / l NAA, 0.5 mg / l BAP, 30 g / l sucrose, 0.8% agar) medium, The cells were cultured at 26 ° C. and 8000 Lux for 2 to 3 weeks. Germinated 5-10 mm shoots were added to MSR medium containing 100 mg / l kanamycin and 100 mg / l carbenicillin (MS medium containing 525 mg / l NAA and 100 mg / l BAP and using gellite instead of agar) Moved to. Two weeks later, young plants that grew to a total length of about 5 cm were acclimated and potted to obtain transformed plants.

Next, in the same manner as in Example 2, a cell extract was obtained from the plant body, passed through a lectin column, the eluate was collected, and the adsorbed protein was eluted and collected as fractions 1-5. Subsequently, fractions 1 to 5 were subjected to SDS-polyacrylamide gel electrophoresis in the same manner as in Example 2. The results are shown in FIG. FIG. 4 is a diagram showing the results of electrophoresis in this example. The number above each lane corresponds to the fraction number. As shown in FIG. 4, humanized glycoprotein was detected in fractions 2-4.

From the above results, it was revealed that the N-acetylgalactosamine transfer gene and galactose transfer gene introduced into plant cells function and can bind O-type sugar chains to plant proteins.

As described above, the kit for producing an O-type sugar chain binding protein in a plant according to the present invention comprises at least one of the polypeptide N-acetylgalactosamine transferase and the polynucleotide encoding the same. As a result, it is possible to produce useful proteins modified with O-type sugar chains using plants.

The specific embodiments or examples made in the detailed description section of the invention are merely to clarify the technical contents of the present invention, and are limited to such specific examples and are interpreted in a narrow sense. It should be understood that various modifications may be made within the spirit of the invention and the scope of the following claims.

If the kit according to the present invention is used, an O-type sugar chain can be bound to a protein in a plant, so that a useful protein can be produced in the plant. Therefore, the present invention contributes to the expansion of industrial uses of plants, and can be used in a wide range of industries such as the pharmaceutical industry and the food industry.

Claims

A kit for producing an O-type sugar chain binding protein in a plant, comprising at least one of a polypeptide N-acetylgalactosamine transferase and a polynucleotide encoding the same.
The kit according to claim 1, further comprising at least one of galactose transferase and a polynucleotide encoding the enzyme.
A transformed plant comprising a polynucleotide encoding a polypeptide N-acetylgalactosamine transferase and a polynucleotide encoding a target polypeptide to which N-acetylgalactosamine is bound.
A composition for producing an O-type sugar chain binding protein in a plant, comprising at least one of a polypeptide N-acetylgalactosamine transferase and a polynucleotide encoding the same.
A method for producing an O-type sugar chain binding protein comprising a synthesis step of synthesizing a polypeptide N-acetylgalactosamine transferase in a plant or a plant extract.
A method for producing an O-type sugar chain-binding protein, comprising a mixing step of mixing a polypeptide N-acetylgalactosamine transferase with a plant extract.