JP2003235561A - Method for converting glycoprotein having plant type sugar chain into glycoprotein having animal type sugar chain - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for simply and rapidly producing a glycoprotein which is stable in vivo, retains an essential physiological activity and has an animal type sugar chain structure. <P>SOLUTION: The method is to convert a glycoprotein having a plant type sugar chain into the glycoprotein having the animal type sugar chain. The method comprises a step of making an enzyme capable of carrying out a transfer reaction of a nonreducing terminal acetylglucosamine residue into a galactose residue act on the glycoprotein having the plant type sugar chain. The glycoprotein having the plant type sugar chain is produced by a plant cell and can be a foreign glycoprotein. The glycoprotein having the animal type sugar chain comprises a core sugar chain and an external sugar chain. The core sugar chain consists essentially of a plurality of mannoses and acetylglycosamines. The external sugar chain contains terminal sugar chain moiety comprising nonreducing terminal galactose. Fucose or xylose is preferably not contained in the glycoprotein having the animal type sugar chain. <P>COPYRIGHT: (C)2003,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for converting a glycoprotein having a plant type sugar chain into a glycoprotein having an animal type sugar chain.

[0002]

2. Description of the Related Art Plant culture cells can be used as one of hosts for producing heterologous proteins. For example,
Attempts have been made to produce the following useful human proteins using cultured tobacco cells: GM-CSF (James E)
A, Wang C, Wang Z, Reeves R,
Shin JH, Magnuson NS, Lee J
M, Production and characte
Rization of Biologically
active human GM-CSF secre
tedby genetically modify
d plant cells. Protein Exp
r Purif. 19, 131-138 (200
0)), IL-2 and IL-4 (Magnuson)
NS, Linzmaier PM, Reeves R,
An G, HayGlass K, Lee JM, Se
CREATION OF BILOGICALLY a
ctive humaninterleukin-2
and interleukin-4 fromge
netly modified tobacc
o cellsin suspension cult
ure. Protein ExprPurif. 13,
45-52 (1998)), immunoglobulin (Magn)
uson NS, Linzmaier PM, Gao
JW, Reeves R, An G, Lee JM, E
enhanced recovery of a sec
reted mammalian protein f
rom suspension culture of
genetically modified toba
cco cells. Protein Expr Pu
rif. 7, 220-228 (1996)), erythropoietin (Matsumoto S, Ishii).
A, Ikura K, Ueda M, Sasaki
R, Expression of human ery
thropoietin in cultured t
obacco cells, Biosci. Biote
chnol. Biochem. 57, 1249-125
2 (1993)), α1-antitrypsin (Tera)
Shima M, Murai Y, Kawamura
M, Nakanishi S, Stoltz T, Ch
en L, Drohan W, Rodriguez R
L, Katoh S, Production of fu
nctional human alpha 1-an
titrypsin by plant cell c
culture. Appl Microbiol Bio
technology. 52, 516-523 (1999)).
Such.

On the other hand, plant cultured cells have been reported to secrete many proteins or glycoproteins extracellularly (Sturm A, Heterogene).
ity of the complex N-link
ed oligosaccharides at sp
specific glycosylation site
s of two secreted carrot
Glycoproteins Eur J Bioch
em. 199, 169-179 (1991); Okus
Hima Y, Koizumi N, Kusano
T, Sano H, Secreted protein
s of tobacco cultured BY2
cells: identification of
a new member of pathogen
sis-related proteins. Pl
ant Mol Biol. 42, 479-488 (2
000); and Okushima Y, Koizum
i N, Kusano T, Sano H, Glyco
sylation and it's adquire
processing is critical f
or protein security in to
bacco BY2 cells. J. Plant P
hysiolo. 154, 623-627 (199
9)). For example, in tobacco BY2 cell culture cells, two peroxidases were purified from the culture, allowing cloning of their genes (Narita).
H, Asaka Y, Ikura K, Matsumo
to S, Sasaki R, Isolation, c
harmonization and expre
session of national peroxid
ase isozymes released int
o the medium of culture
tobacco cells. Eur J Bioch
em. 228, 855-862 (1995)). When polyvinylpyrrolidone (PVP) was added to the culture medium of plant cells, the concentration of proteins secreted into the medium increased (Magnuson NS, Linzmaier P).
M, Gao JW, ReevesR, An G, Lee
JM, Enhanced recovery of
assured mammalian prote
in from suspension culture
e of genetically modified
tobacco cells, Protein Ex
pr Purif7, 220-228 (1996)).
Okushima Y et al. (1999, supra) reported that cultured cells of the BY2 strain of tobacco secrete hundreds of proteins extracellularly. Since these proteins reacted with a lectin (concanavalin A) that recognizes a high-mannose type sugar chain, it was shown that the glycoprotein was also secreted in a large amount outside the cell (Okushima Y).
Et al., 1999, supra).

Proteins produced in plant cells as described above (especially immunoglobulin, interleukin, G
M-CSF) has its own unique signal peptide recognized by the secretory mechanism in plant cells, and the mature protein was secreted into the culture medium (James).
EA et al., Supra; Magnuson NS et al., 199.
8, supra; Magnuson NS et al., 1996, supra). Many of these proteins are glycoproteins containing sugar chains, and it has been suggested that the sugar chains are involved in blood half-life, sensitivity to proteolytic enzymes, and stability in any of these glycoproteins. There is. However, in reality, the sugar chain structure of the recombinant protein produced in plant cells is different from that of the antibody (Cabanes-Machet)
eau M, Fitchette-Laine AC,
Loutier-BourhisC, Lange
C, Vine ND, MaJK, Lerouge P,
Faye L. N-Glycosylation of
a mouse IgG expressed in
transgenic tobacco plant
s. Glycobiology (1999) 9, 365
-372; Baker H, Bardor M, Mol.
thoff JW, Gomord V, Elbers
I, Stevens LH, Jordi W, Lomm
en A, Face L, Lerouge P, Bos
ch D. Galactose-extended g
lycans of antibiotics prod
UCEDBY TRANSGENIC PLANTS.
Proc Natl AcadSci USA (200
1) 98, 2899-2904) has not been investigated, and the recombinant protein is presumed to have a plant type sugar chain structure. Raskin et al.
A method for secreting proteins such as human placental alkaline phosphatase has been described (Borisjuk N.
V, Borisjuk LG, Logendra S,
Petersen F, Gleba Y, Raskin
I. Production of recombina
nt proteins in plant root
exudates. Nat Biotechnol
1999; 17: 466-469; Gleba D, B.
orisjuk NV, Borisjuk LG, Kn
er R, Poulev A, Skarzhinska
ya M, Dushenkov S, Longendr
a S, Gleba YY, Raskin I.D. Use
of plants for phytor
education and molecular
farming. Proc Natl Acad Sc
i USA 1999; 96: 5973-5977).

Analysis of the sugar chain structure of the secretory antibody molecule sIgA produced in tobacco plants revealed that this secretory antibody molecule has a plant sugar chain (Cabanes-
Macheteau M, Fitchette-Lai
ne AC, Loutier-Bourhis
C, Range C, Vine ND, Ma JK, L
error P, Face L, N-Glycosy
relation of mouse IgG expr
essed in transgenic tobac
co plants. Glycobiology. 9,
365-372. (1999)). It was also shown that the heterologous antibody molecule also produced in the tobacco plant is susceptible to degradation by proteolytic enzymes and is unstable (S
sevens LH, Stoopen GM, Elbe
rs IJ, Molthoff JW, Baker H
A, Lommen A, Bosch D, Jordi
W, Effect of climate condi
tions and plant developme
ntal stage on the stabili
ty of antibodies express
d in transgenic tobacco.
Plant Physiol. 124,173-18
2. (2000)). By the Western method using an anti-plant type sugar chain antibody, it was confirmed that a plant type sugar chain was added to this antibody. It has been reported that β1,4-linked galactose residues present in sugar chains of antibody molecules produced in humans, mice, etc. contribute to stabilization of antibody proteins, but are produced in plant cells. This sugar residue is absent in the isolated antibody molecule. For this reason, it is considered that the antibody produced in the tobacco plant became susceptible to the degradation by the proteolytic enzyme.

When erythropoietin was produced in cultured tobacco cells, in vitro biological activity was observed but in vivo activity could not be detected (Mats).
umoto S, Ikura K, Ueda M, Sa
saki R Characterization o
f a human glycoprotein (e
rythropoietin) produced i
n culturedtobacco cells.
Plant Mol Biol. 27, 1163-1
172 (1995)). It was also concluded that this is also due to the fact that sugar chains are greatly involved in biological activity in erythropoietin, and that sugar chain structures differ greatly when produced in plant cells.

Thus, including the above, the sugar chains of glycoproteins are i) protected from proteolytic degradation, such as during in vivo blood clearance, ii) involved in protein folding and physiological activity, iii. )
It is considered to play various roles such as contribution to cell-cell adhesion, iv) mediation of cell surface recognition by viruses and pathogens.

FIG. 1 shows the structures of typical sugar chains (N-linked sugar chains) present in animal cells and plant cells. As shown in FIG. 1, animal-type and plant-type sugar chains consist of a core sugar chain consisting essentially of a plurality of mannoses and acetylglucosamine, and an external sugar chain that is bound to the core sugar chain via mannose. . The outer sugar chains of animal-type sugar chains (bottom of FIG. 1) do not have β1,4 that are not found in plant-type sugar chains (top of FIG. 1)
There are bound galactose residues and sialic acid.
On the other hand, the core sugar chain of a plant type sugar chain has β1,2-bonded xyrose residues and α1,3-bonded residue fucoses that are not found in animal cells.

Furthermore, it has been suggested that plant type sugar chains may serve as allergens in mammals including humans.
That is, it has been reported that a β1,2-xyrose residue or an α1,3-fucose residue, which is a plant-specific sugar chain structure that is not found in mammalian glycoprotein sugar chains, serves as an allergen ( Fotisch K, Altmann
F, Haustain D, Vieths S, In
volvementof carbohydrate
epitopes in the IgErespon
se of celery-allergic pat
ients. Int Arch Allergy I
mmunol. 120, 30-42 (1999); Wi
lson IB, Harthill JE, Mulli
nNP, Ashford DA, Altmann F,
Core alpha1,3-fucose is a
key part of the epitope
recognized by antibodies
reacting again plant N-1
inked oligosaccharides an
dis is present in a wide va
riety of plant extracts. Gl
ycobiology. 8, 651-661 (199
8); and van Ree R, Cabanes-M.
acheteau M, Akkerdaas J, Mi
lazzo JP, Loutier-Bourhi
s C, Rayon C, Villalba M, Ko
ppelman S, Aalberse R, Rodr
iguez R, Face L, Lerouge P,
Beta (1,2) -xylose andalpha
(1,3) -fucose residues hav
e a strong contribution i
n IgE binding top plant gly
callergens. J Biol Chem. Two
000 Apr 14; 275 (15): 11451-.
11458). Therefore, it is desired that the medical protein has a sugar chain structure having no β1,2-xyloose residue or α1,3-fucose residue.

[0010]

DISCLOSURE OF THE INVENTION The present invention solves the above-mentioned conventional problems and provides a glycoprotein having a sugar chain structure that is stable in vivo, retains its original physiological activity, and does not become an allergen. The purpose is to provide a method for rapid production.

[0011]

Means for Solving the Problems FIG. 2 shows a part of the synthetic pathway of sugar chains in animal cells and plant cells. The present inventors have found that plant-type sugar chains are animal-type sugar chains except that there are β1,2-linked xylose residues and α1,3-bonded fucose residues. The present invention shows that an animal-type sugar chain can be obtained by adding galactose and sialic acid capable of binding β1,4 to the non-reducing end of a plant-type sugar chain in vitro, thus completing the present invention. It was

The present invention relates to a method for converting a glycoprotein having a plant-type sugar chain into a glycoprotein having an animal-type sugar chain, which comprises adding a non-reducing terminal acetylglucosamine residue to a glycoprotein having a plant-type sugar chain. The step of reacting with an enzyme capable of performing a transfer reaction of a galactose residue to a group is included.

[0013] Preferably, the glycoprotein having a plant type sugar chain may be a glycoprotein produced by a plant cell.

[0014] Preferably, the glycoprotein produced by the plant cell may be a heterologous glycoprotein.

Preferably, the above heterologous protein can be obtained by a method including a step of transforming a plant cell with a gene encoding the heterologous protein, and a step of culturing the obtained transformant.

[0016] Preferably, the above method may further include a step of collecting a culture solution of the above transformant.

Preferably, the glycoprotein having an animal type sugar chain comprises a core sugar chain and an outer sugar chain, and the core sugar chain essentially consists of a plurality of mannoses and acetylglucosamine, and the outer sugar chain is May contain a terminal sugar chain moiety containing a non-reducing terminal galactose.

[0018] Preferably, the glycoprotein having an animal type sugar chain does not contain fucose or xylose.

The present invention also relates to a glycoprotein having an animal type sugar chain obtained by the above method.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be described in detail below.

In the present invention, unless otherwise specified, protein separation and analysis methods and immunological methods known in the art can be employed. These techniques can be performed using commercially available kits, antibodies, labeling substances and the like.

The method of the present invention relates to a method for converting a glycoprotein having a plant-type sugar chain into a glycoprotein having an animal-type sugar chain. The step of reacting with an enzyme capable of carrying out a transfer reaction of a galactose residue to a glucosamine residue is included. Typically, the glycoprotein having a plant type sugar chain is produced by cultured plant cells.

In the present specification, the "animal type sugar chain" means
It refers to a sugar chain having a galactose residue bonded to an N-acetylglucosamine residue. The galactose residue in the animal type sugar chain may be at the end of the sugar chain, or a sialic acid residue may be bound further outside the galactose residue. In the glycoprotein of the present invention, at least one of xylose and fucose is bound in one or more portions consisting of a core sugar chain portion, a branched sugar chain portion, and a terminal sugar chain portion of an animal type sugar chain. It is more preferable that at least one of xylose and fucose is not bound to any part of the animal type sugar chain,
Most preferably, xylose and fuco-in the animal type sugar chain.
It is even more preferred that it does not contain any of these.

The plant cell can be any plant cell. The plant cells may be in the form of cultured cells, cultured tissues, cultured organs, or plant bodies. Preferably,
It is a cultured cell, a cultured tissue, or a cultured organ, more preferably a cultured cell. The plant species that can be used in the production method of the present invention can be any plant species capable of gene transfer. Examples of plant species that can be used in the production method of the present invention include plants of Solanaceae, Gramineae, Brassicaceae, Rosaceae, Leguminaceae, Cucurbitaceae, Lamiaceae, Liliaceae, Acalyptaceae, and Seriaceae. .

As an example of a plant of the Solanaceae family, Nicoti
ana, Solanum, Dataura, Lycope
Examples include plants belonging to rsion or Petunia, and examples thereof include tobacco, eggplant, potato, tomato, capsicum, petunia, and the like.

Examples of plants of the family Gramineae include Oryza,
Hordenum, Scale, Scccharu
m, Echinochloa, or Zea. Examples thereof include rice, barley, rye, millet, sorghum, and corn.

Raph is an example of a plant of the Brassicaceae family.
anus, Brassica, Arabidopsi
plants belonging to s, Wasabia, or Capsella are included, and examples thereof include radish, rape, Arabidopsis, wasabi, and cod.

As an example of the plant of the family Rosaceae, Orunu
Examples thereof include plants belonging to s, Malus, Pynus, Fragaria, or Rosa, and include, for example, plums, peaches, apples, pears, Dutch strawberries, and roses.

As an example of a leguminous plant, Glycin
e, Vigna, Phaseolus, Pisum, V
icia, Arachis, Trifolium, Al
Phalfa or plants belonging to Medicago are mentioned, and examples thereof include soybean, adzuki bean, kidney bean, pea, broad bean, peanut, clover, horse mackerel and the like.

Examples of plants of the family Cucurbitaceae include Luffa,
Examples include plants belonging to Cucurbita or Cucumis, such as loofah, pumpkin, cucumber,
Including melon etc.

As an example of the Lamiaceae plant, Lavand
Examples thereof include plants belonging to ula, Mentha, or Perilla, and include, for example, lavender, peppermint, and perilla.

Examples of plants belonging to the family Liliaceae include All
Examples include plants belonging to ium, lilium, or Tulipa, and include, for example, leek, garlic, lily, tulip, and the like.

As an example of a plant of the family Rhinoceros, Spina
Examples of plants belonging to cia include spinach.

As an example of the plant of the family Apiaceae, Angeli
Examples thereof include plants belonging to ca, Daucus, Cryptotaenia, or Apitum, and include, for example, sardines, carrots, honeywort, and celery.

The plants used in the production method of the present invention are preferably tobacco, tomato, potato, rice, corn, radish, soybean, pea, horse mackerel, and spinach, and more preferably tobacco, tomato, potato and corn. , And soybean.

The term "enzyme capable of transferring a galactose residue to a non-reducing terminal acetylglucosamine residue" used in the present invention can transfer a galactose residue to a non-reducing terminal acetylglucosamine residue. Means enzyme. Examples of such enzymes include galactosyl transferase, lactoscinase and β-galactosidase. Such an enzyme may be derived from any species, but is preferably derived from mammals, and more preferably derived from humans.

A commercially available enzyme can be used as the “enzyme capable of transferring the galactose residue to the acetylglucosamine residue at the non-reducing end”. This is, for example, Toy
It is available from obo, Inc. (Osaka). Alternatively, it may be isolated from a source such as an animal cell, or may be obtained by expression using a known nucleotide sequence encoding the enzyme using a genetic engineering technique known to those skilled in the art.

As used herein, the term "gene" refers to a structural gene portion. The gene may be linked with regulatory sequences such as promoter, operator, and terminator, as appropriate for expression in plants.

The term “heterologous glycoprotein” used in the present invention
Refers to a glycoprotein that is not originally produced in the host used, typically a plant cell or plant. Examples of heterologous glycoproteins are enzymes, hormones, cytokines,
Antibodies, vaccines, receptors, serum proteins and the like. Examples of enzymes include horseradish peroxidase, kinase, glucocerebrosidase (gluco).
cerebrosidase), alpha-galactosidase, phytase, TPA (issue-type)
Plasminogen activator), H
MG-CoA reductase (HMG-CoA redu
ctase) and the like. Examples of hormones and cytokines include enkephalin, interferon alpha, GM-CSF, G-CSF, chorionic gonadotropin, interleukin-2, interferon-beta, interferon-gamma, erythropoietin, vascular endothelial growth factor ( Vascular en
dothelial growth factor),
Examples include human chorionic gonadotropin (HCG), luteinizing hormone (LH), thyroid stimulating hormone (TSH), prolactin, follicle stimulating hormone and the like. Examples of the antibody include IgG, scFv, secretory IgA and the like. Examples of vaccines include hepatitis B surface antigen, rotavirus antigen, Escherichia coli enterotoxin, malaria antigen, G protein of rabies virus rabies virus, HIV viral glycoprotein (eg, gp1).
20) and the like. Examples of receptors and matrix proteins are EGF receptor, fibronectin, α1-antitrypsin, coagulation factor VII
I etc. are mentioned. Examples of serum proteins include albumin, complement system proteins, plasminogen, corticosteroid binding globulin (corticoster).
liquid-binding globulin, throxine-binding globulin
ng globulin), protein C (prote
in C) and the like.

The "gene of heterologous glycoprotein" may be isolated from any cell using a nucleotide sequence known to encode the heterologous glycoprotein of interest, or may be purchased commercially. Alternatively, these may be modified and used as appropriate for expression in plants.

The gene for the heterologous glycoprotein is introduced into plant cells by a method known in the art. These genes may be introduced separately or simultaneously. Examples of methods for introducing a gene into plant cells include the Agrobacterium method, electroporation method, and gold particle method, as described above.

The gene product expressed and secreted by the plant cell into which the above-mentioned gene has been introduced can be confirmed by a method known in the art depending on the gene product expressed and secreted. Such confirmation methods include silver staining,
Western blotting, Northern hybridization, detection of enzyme activity and the like can be mentioned.

The transformed cell expressing the heterologous glycoprotein expresses and secretes the heterologous glycoprotein having a plant-type sugar chain. By culturing the transformed cells, the plant-type glycoprotein can be expressed and accumulated in the plant cells or secreted in large amounts in the culture medium.

This animal-type glycoprotein contains a core sugar chain and an outer sugar chain, and this core sugar chain comprises a plurality of manno-glycans.
It consists essentially of glucose and acetylglucosamine. The outer sugar chain of the resulting glycoprotein contains a non-reducing end sugar chain portion. The external sugar chain may have a linear structure or a branched structure. The branched sugar chain part is mono, bi,
It can be either tri- or tetra-structured. The glycoprotein produced by the transformed cell is preferably
Does not contain fucose or xylose.

The obtained transformed plant cells may be maintained in the state of cultured cells, may be differentiated into a specific tissue or organ, or may be regenerated into a complete plant body. Alternatively, it may be a part of seeds, fruits, leaves, roots, stems, flowers and the like obtained from a whole plant.

Methods of transforming plant cells are known to those skilled in the art and include: Micro injection (C
Rossway et al. , BioTechniq
ues 4: 320-334 (1986)), electroporation (Riggs et al., Pro.
c. Natl. Acad. Sci. USA 83:56
02-5606 (1986), Agrobacterium-mediated transformation (Hinchee et al., Biote.
chnology 6: 915-921 (1988);
Ishida et al. , Nature Biot
technology 14: 745-750 (June
1996) for maize transformation), direct gene transfer (Paszkowski et al., EM).
BO J. 3: 2717-2722 (1984); Ha.
yashimato et al. , Plant Ph
ysiol 93: 857-863 (1990) (rice)), Agracetus, Inc. , Madiso
n, Wis. And Dupont, Inc. , Wilm
Inton, Del. Accelerated Particle Acceleration Using a Device from Sanford (Sanford et al., US Pat. No. 4,945,050; McCave et al.,
Biotechnology 6: 923-926 (1
988)) and so on.

Weissinger et al. , A
nnual Rev. Genet. 22: 421-47
7 (1988); Sanford et al. , Pa
rticulate Science and Tec
hnology 5.27-37 91987) (onion); Svab et al. , Proc. Nat
l. Acad. Sci. USA 87: 8526-85.
30 (1990) (Tobacco chloroplast); Chri
Sto et al. , Plant Physio
l. 87: 671-674 (1988) (soybean); Mc.
Cave et al. , Bio / Technology
y 6.923-926 (1988) (soybean); Kle
in et al. , Proc. Natl. Acad.
Sci. USA, 85: 4305-4309 (198).
8) (corn); Klein et al. , Bio
/ Technology 6: 559-563 (198)
8) (corn); Klein et al. , Pl
ant Physiol. 91: 440-444 (19
88) (corn); Fromm et al. ，
Bio / Technology 8: 833-839.
(1990); and Gordon-Kammet.
al. , Plant Cell 2: 603-618
(1990) (corn); Koziel et a.
l. , Biotechnology 11: 194-2
00 (1993) (corn); Shimamoto
et al. , Nature 338: 274-27.
7 (1989) (rice); Christou et a
l. , Biotechnology 9: 957-96.
2 (1991) (rice); Datta et al. , B
iol / Technology 3: 736-740
(1990) (Rice); European Patent
Application EP 0 332 581
(Plants of the grass family such as Kamogaya); Vasil et
al. , Biotechnology 11: 1553
-1558 (1993) (Wheat); Weeks et
al. , Plant Physiol. 102: 107
7-1084 (1993) (Wheat); Wan et.
al. , Plant Physiol. 104: 37-
48 (1994) (barley); Jahne et a.
l. , Theor. Appl. Genet. 89:52
5-533 (1994) (barley); Umbeck
et al. , Bio / Technology 5: 2
63-266 (1987) (Cotton); Casas
et al. , Proc. Natl. Acad. Sc
i. USA 90: 11212-11216 (Dece
mber 1993) (Sorghum); Somers e
t al. , Bio / Technology 10: 1
589-1594 (December 1992) (Otomugi); Torbert et al. , Plan
t Cell Reports 14: 635-640.
(1995) (Otomugi); Weeks et a.
l. , Plant Physiol. 102: 1077
-1084 (1993) (Wheat); Chang et.
al. , WO94 / 13822 (Wheat) and Ne
hara et al. , The Plant Jour
See also nal 5: 285-297 (1994) (Wheat).

A particularly preferred embodiment for introducing recombinant DNA molecules into corn by particle bombardment is:
Koziel et al. , Biotechnolo
gy11: 194-200 (1993), Hill e.
t al. , Euphytica 85: 119-12.
3 (1995) and Koziel et al. , A
nnals of the New York Aca
demy of Sciences 792: 164-
171 (1996). Another preferred embodiment for maize, the protoplast transformation method, is described in EP 0 292 435. Transformation of plants can be performed using a single DNA species or multiple DNA species (ie co-transformation).

Techniques and media known in the art are used for culturing, differentiating and regenerating the transformed plant cells. Such media include, for example, Murashig.
e-Skoog (MS) medium, GaMburg B5
(B) Medium, White medium, Nitsch & Nits
ch (Nitsch) medium and the like, but not limited thereto. These media are usually used after adding an appropriate amount of a plant growth regulator (plant hormone) or the like.

In order to produce a glycoprotein having an animal-type sugar chain, as long as a transformed plant cell grows and produces a desired gene product, basically, a carbon source, a nitrogen source, and A culture medium having any composition containing micronutrients necessary for the growth of plant cells such as vitamins and salts can be used. Stabilization of the produced heterologous protein,
In addition, polyvinylpyrrolidone, proteolytic enzyme inhibitors and the like may be added to improve the efficiency of secretion of heterologous proteins.

The glycoprotein having a plant-type sugar chain produced by the transformed plant cell can be typically isolated from the culture solution of the plant cell. Isolation of glycoproteins from plant cell cultures can be performed using methods well known to those of skill in the art. For example, salting out (ammonium sulfate precipitation, sodium phosphate precipitation, etc.), solvent precipitation (protein fractionation precipitation method using acetone or ethanol), dialysis, gel filtration, ion exchange, column chromatography such as reverse phase,
Glycoproteins can be purified and isolated from the culture broth using techniques such as ultrafiltration and high performance liquid chromatography (HPLC), alone or in combination.

Alternatively, the glycoprotein of the present invention may be isolated or extracted from plant cells. Furthermore, the glycoprotein of the present invention can be edible as it is contained in a transformed cell. Since the glycoprotein of the present invention has animal-type glycosylation, it has no antigenicity and is therefore suitable for administration to animals including humans.

[0053]

EXAMPLES The present invention will be described with reference to examples. The following examples illustrate, but do not limit, the invention.

As a typical example of plant cells, tobacco culture cells Nicotiana tabacum L. cv. b
light yellow 2 (obtained as cell line name BY2 from catalog number RPC1 of Gene Cell Office Plant Cell Development Bank, RIKEN Life Science Tsukuba Research Center). The sugar chain structure of the glycoprotein produced by BY2 is shown in PCT International Publication No. WO by the present inventors.
00/34490.

The principle of the present invention is schematically shown in FIG. Cultured BY2 cells secrete hundreds of proteins extracellularly into the medium (Okushima Y et al., 1999, supra). In FIG. 3, BY2 cells are shown on the left of the figure and the structure of the glycoprotein secreted therefrom is shown on the upper right of the figure. When this glycoprotein was recovered from the medium and reacted with β1,4-galactosyltransferase, a glycoprotein having an animal type sugar chain to which the galactose residue shown in the lower right of FIG. 3 was transferred and bound was produced. can get.

1. Preparation of Glycoprotein Produced by Tobacco Cultured Cells Tobacco cultured cell BY2 strain was prepared by adding 1 L of Murashige.
-The culture was carried out for 7 days in a modified Murashige-Skoog medium prepared using a mixed salt for Skoog medium (Wako Pure Chemical Industries, Ltd.). The obtained culture solution was centrifuged at 2000 rpm for 10 minutes (room temperature) to collect the supernatant. The resulting supernatant was dialyzed against dH ₂ O (deionized water) at 4 ° C. (1 ×
10 ⁵ times dilution), and freeze-dried. The obtained freeze-dried product (500 mg) was used as a glycoprotein sample.

2. Galactose transfer to glycoprotein sugar chain in vitro 2.1. In vitro galactosyltransferase reaction for lectin blotting analysis 10 mM manganese chloride, 100 μg / ml UDP-
Galactose, 60 mU / ml human galactose transferase (manufactured by Toyobo Co .; E. coli recombinant),
And 50 mM HEPES buffer (pH 7.4) containing 1 or 10 mg / ml of the above glycoprotein sample
In the medium, the transfer galactose enzyme reaction was performed at 37 ° C. for 24 hours. The enzymatic reaction was stopped by boiling at 100 ° C. for 3 minutes, centrifugation was performed at 4 ° C. at 12,000 rpm for 5 minutes, and then the supernatant was recovered.

(Lectin blotting) 10 μl of the collected supernatant was subjected to SDS-PAGE (120 V, 2 hours) using a 12.5% polyacrylamide gel.
After the electrophoresis, the separated glycoprotein was transferred to the nitrocellulose membrane at 1 A / cm ² for 45 minutes using an SDS-PAGE running buffer containing 15% methanol. Then, the transferred nitrocellulose membrane was blocked with a 1% BSA solution at room temperature for 1 hour. After blocking
Nitro cellulosic membrane was washed with a washing buffer.
After washing with PBS solution containing 0.05% Tween-20 for 3 times for 10 minutes, horseradish peroxidase was added.
Ze labeled RCA (Ricinus communis a
Incubation was carried out at room temperature for 2 hours in a washing buffer containing gglutinin) ₁₂₀ lectin (Seikagaku Corporation). Next, the washing buffer
After washing with r for 3 times for 10 minutes, the nitrocellulose membrane was stained with POD Immunostain Kit (Wako). The RCA ₁₂₀ lectin specifically recognizes a sugar chain having β1,4-galactose linked to a terminal N-acetylglucosamine residue. The result of the lectin analysis is shown in FIG. The samples electrophoresed in each lane were obtained by enzymatic reaction in the reaction system shown in Table 1.

[0059]

[Table 1] Lane 6 is 100 μg / ml asialofetuin used as a positive control.

As shown in FIG. 4, β1,4-galactose-linked sugar chains were confirmed in the sample to which the galactosyltransferase was added (lane 1 and lane 2), and BY
It was shown that galactose residues were bound to the glycoproteins secreted by the two strains by the in vitro enzymatic reaction at β1,4 positions.

2.2. In vitro galactosyltransferase reaction for sugar chain structure analysis 10 mM manganese chloride, 100 μg / ml UDP-
Galactose, 10 mU / ml human-derived galactose transferase (manufactured by Toyobo Co .; E. coli recombinant), and 10 mg / ml glycoprotein sample,
37 mM in 50 mM HEPES buffer (pH 7.4)
The transferase reaction was carried out for 2 days. After the enzyme reaction, the enzyme reaction solution was dialyzed against DH ₂ O (deionized water) at 4 ° C. (1 ×
10 ⁵ times dilution), and freeze-dried. The obtained freeze-dried product was used as a glycoprotein sample.

The sugar chain structure is obtained by cleaving a sugar chain from a glycoprotein and subjecting the obtained sugar chain to pyridylamination (PA conversion).
This was analyzed by subjecting it to HPLC analysis, exoglycosidase digestion, and MS spectral analysis.

(Preparation of Sugar Chain from Glycoprotein) The freeze-dried glycoprotein sample was hydrolyzed at 100 ° C. for 10 hours to cut out the sugar chain. Then, excess acetone was added to the hydrazine decomposition product at 4 ° C for 100
The sugar chains were precipitated by centrifugation at 00 rpm for 20 minutes. After N-acetylating the sugar chain in the presence of a saturated aqueous sodium hydrogen carbonate solution and acetic anhydride, Dowex 50 ×
2 (Muromachi Chemical Co., Ltd.), desalted, and equilibrated with 0.1N ammonia water TSK gel Toyopearl
The sugar chain was recovered by passing through a HW-40 column (2.5 × 30 cm; manufactured by Tosoh).

(Preparation of Pyridyl Amination (PA) Sugar Chain) The recovered sugar chain was pyridylamino (PA) using 2-aminopyridine. The PA sugar chain was TSK Toyopearl HW- equilibrated with 0.1N ammonia water.
It was purified by passing through 40 columns (2.5 × 30 cM; manufactured by Tosoh).

(Purification of PA-modified sugar chain by HPLC) PA
The modified sugar chain is subjected to Reversed-Phase (RP) and Size-Fractionation (SF) HP.
It was analyzed using LC analysis. In the HPLC analysis, a Hitachi HPLC apparatus having a fluorescence detector L-7480 was used, and the fluorescence intensity was measured with excitation and fluorescence wavelengths of 310 nm and 380 nm, respectively. Cosmosil 5C18-P
column (6 x 250 mm; manufactured by Nacalai Tesque, Inc.)
In RP-HPLC analysis using P., the concentration of acetonitrile in 0.02% TFA aqueous solution was increased from 0% to 6% in 40 minutes under a flow rate of 1.2 ml / min.
The glycosylated A sugar chain was eluted. Also, Asahipak NH
The flow rate was 0.7 in the SF-HPLC analysis using 2P-50 column (4.6 × 250 mm; Showa Denko).
The PA-treated sugar chain was eluted by increasing the concentration of acetonitrile in the dH ₂ O-acetonitrile mixture solution from 26% to 50% in 25 minutes under ml / min.

(Analysis of PA-modified sugar chain by exoglycosidase digestion) β-galactosidase (Diplococ)
cus pneumoniae; manufactured by Roche) The enzymatic digestion reaction was carried out by adding 0.1 m sodium acetate buffer (pH) containing 5 mU of β-galactosidase to each PA-modified sugar chain.
5.5) in 37 ° C. for 2 days. Similarly, N-acetylglucosaminida
Ze (Diplococcus pneumoniae;
Roche) enzymatic digestion reaction, each PA sugar chain 5m
0.1 M containing U N-acetylglucosaminidase
It was carried out by incubation in a sodium acetate buffer (pH 5.5) at 37 ° C for 2 days. Also, α
The mannosidase (Almond; Sigma) enzymatic digestion reaction was carried out in a 0.1 M sodium acetate buffer (p) containing 10 mM zinc acetate and 10 μU of α-mannosidase.
H 4.0) by incubation at 37 ° C. for 2 days. Each enzyme digestion reaction is 3 at 100 ℃
It was stopped by boiling for a minute. And 12,000r
After centrifugation at pm for 5 minutes, the supernatant was subjected to HPLC. P
The structure of the A-modified sugar chain was analyzed by comparing the elution time of the sample sugar chain with the known elution time of the sugar chain.

(MS spectrum analysis) MS spectrum analysis was carried out by Voyger DE-Pro Mass spe.
ctrometry (PerSeptive Bios
systems, Framingha M MA).

(Results) FIG. 5 is a chromatogram showing the elution pattern of PA-modified sugar chains in RP-HPLC. As shown in FIG. 5, the PA sugar chain prepared was
It was separated into peak components I to IX by P-HPLC.

Next, each of these nine peak components was further separated using SF-HPLC. SF-HPL
The chromatogram of each peak component of I to IX in C is shown in FIG. The reference term (for example, PeakI) shown on the right side of each chromatogram in FIG. 6 indicates the peak component of RP-HPLC derived from the sample subjected to SF-HPLC. Next, for each peak component obtained by SF-HPLC, it was confirmed whether or not a galactose residue was bound at the β1,4 position at the non-reducing end. As a result, as described below, the eight peak components (I-4, I-
5, II-3, II-4, IV-1, V-1, V-2,
For V-3), binding of a galactose residue at the β1,4 position at the non-reducing end was confirmed.

Peak I-4 : Molecular weight of peak I-4 (m
/ Z value 1836.63) is GalGN2M3FX-.
It matched the molecular weight of PA. In addition, β1 of peak I-4,
GN2M3FX-by enzymatic digestion with 4-galactosidase
PA (the second chromatogram from the top of FIG. 7) was obtained, and M3FX-PA was further obtained by digestion with N-acetylglucosaminidase (the third chromatogram from the top of FIG. 7). In addition, in RP-HPLC, the N-acetylglucosaminidase digestion product of peak I-4 was
Elution was earlier than peak II-3. In RP-HPLC, GalGN ₁ M3FX-PA was GalGN ¹ M3.
It is known to elute earlier than FX-PA. From this, the peak I-4 is Gal ₁ GN2M3FX-.
It was estimated to be PA.

From these results, the structure of peak I-4 is β-D-GlcNAc- (1 → 2) -α-D-Man-
(1 → 6) [β-D-Gal- (1 → 4) -β-D-Glc
NAc- (1 → 2) -α-D-Man- (1 → 3)] [β-
D-Xyl- (1 → 2)] β-D-Man- (1 → 4) -β
-D-GlcNAc- (1 → 4)-[α-L-Fuc- (1
→ 3)] GlcNAc-PA (Gal ₁ GN2M3FX)
Was estimated to be.

Peak I-5 : The molecular weight of peak I-5 (m / z value 1717.97) was Gal2GN2M3.
-Consistent with the molecular weight of PA. In addition, β of peak I-5
GN2M3-by enzymatic digestion of 1,4-galactosidase
PA is obtained and further N-acetylglucosaminida
Z3-digestion gave M3-PA. From these results, the structure of peak I-5 is [β-D-Gal- (1 → 4)
-Β-D-GlcNAc- (1 → 2) -α-D-Man-
(1 → 6)] [β-D-Gal- (1 → 4) -β-D-Gl
cNAc- (1 → 2) -α-D-Man- (1 → 3)] β-
D-Man- (1 → 4) -β-D-GlcNAc- (1 →
4) -GlcNAc-PA (Gal2GN2M3-P
A) was estimated.

[0073] The molecular weight of the peak II-3 peak II-3 (m / z value 1654.85)
Was in agreement with the molecular weight of GalGNM3FX-PA. In addition, GNM3FX-PA was obtained by digesting β1,4-galactosidase of peak II-3 and further M3FX-PA was obtained by digesting N-acetylglucosaminidase. Also, as described above, RP-HPLC
In, the N-acetylglucosaminidase digestion product of peak II-3 eluted later than peak I-4.
From these results, the structure of peak II-3 is β-DG
al- (1 → 4) -β-D-GlcNAc- (1 → 2) -α
-D-Man- (1 → 6) [α-D-Man- (1 → 3)]
[Β-D-Xyl- (1 → 2)] β-D-Man- (1 →
4) -β-D-GlcNAc- (1 → 4)-[α-L-Fu
c- (1 → 3)] GlcNAc-PA (GalGN ¹ M3
FX).

[0074] The molecular weight of the peak II-4 peak II-4 (m / z value 2019.70)
Was in agreement with the molecular weight of Gal2GN2M3FX-PA. Also, β1,4-galactosidase of peak II-4
GN2M3FX-PA was obtained by enzymatic digestion (Fig. 7).
(The second chromatogram from the bottom), and M3FX-PA was obtained by digestion with N-acetylglucosaminidase (the bottom chromatogram in FIG. 7). From these results, the structure of peak II-4 is β-D-Gal- (1 → 4)
-Β-D-GlcNAc- (1 → 2) -α-D-Man-
(1 → 6) [β-D-Gal- (1 → 4) -β-D-Glc
NAc- (1 → 2) -α-D-Man- (1 → 3)] [β-
D-Xyl- (1 → 2)] β-D-Man- (1 → 4) -β
-D-GlcNAc- (1 → 4)-[α-L-Fuc- (1
→ 3)] GlcNAc-PA (Gal2GN2M3F
X) was estimated.

Peak IV-1: The molecular weight of peak IV-1 (m / z value 1486.59) was GalGNM3X.
-Consistent with the molecular weight of PA. In addition, β of peak IV-1
GNM3X-by digestion with 1,4-galactosidase enzyme
PA was obtained, and M3X-PA was obtained by digestion with N-acetylglucosaminidase. Also, RP-HP
In LC, the elution time of peak IV-1 was GalGN
₁ This was in agreement with the elution time of M3X-PA. From these results, the structure of peak IV-1 is α-D-Man- (1 →
6) [β-D-Gal- (1 → 4) -β-D-GlcNA
c- (1 → 2) -α-D-Man- (1 → 3)] [β-D-
Xyl- (1 → 2)] β-D-Man- (1 → 4) -β-D
-GlcNAc- (1 → 4) -GlcNAc-PA (Ga
lGN was assumed to be ₁ M3X-PA).

[0076] The molecular weight of the peak V-1 peak V-1 (m / z value 1487.04)
Was in agreement with the molecular weight of GalGNM3X-PA. In addition, GNM3X-PA was obtained by digesting the peak V-1 with β1,4-galactosidase enzyme, and further M3X-PA was obtained by digesting N-acetylglucosaminidase. In addition, in RP-HPLC, the elution time of peak V-1 was consistent with that of GalGN ¹ M3X-PA. From these results, the structure of peak V-1 is β-D
-Gal- (1 → 4) -β-D-GlcNAc- (1 → 2)
-Α-D-Man- (1 → 6) [α-D-Man- (1 →
3)] [β-D-Xyl- (1 → 2)] β-D-Man-
(1 → 4) -β-D-GlcNAc- (1 → 4) -GlcN
It was estimated to be Ac-PA (GalGN ¹ M3X-PA).

[0077] The molecular weight of the peak V-2 peak V-2 (m / z value 1712.21)
Was in agreement with the molecular weight of GalGN2M3X-PA. Also, GN2M3X-PA was obtained by digesting peak V-2 with β1,4-galactosidase enzyme, and further M3X-PA was obtained by digesting N-acetylglucosaminidase. Further, in RP-HPLC, the elution time of the N-acetylglucosaminidase digestion product of peak V-2 coincided with the elution time of peak IV-1 and GalGN ₁ M3X-PA. From these results, peak V-2
Has a structure of β-D-GlcNAc- (1 → 2) -α-D-
Man- (1 → 6) [β-D-Gal- (1 → 4) -β-D
-GlcNAc- (1 → 2) -α-D-Man- (1 →
3)] [β-D-Xyl- (1 → 2)] β-D-Man-
(1 → 4) -β-D-GlcNAc- (1 → 4) -GlcN
It was estimated to be Ac-PA (Gal ₁ GN2M3X-PA).

[0078] The molecular weight of the peak V-3 peak V-3 (m / z value 1851.95)
Was in agreement with the molecular weight of Gal2GN2M3X-PA.
Also, GN2M3X-PA was obtained by digesting peak V-3 with β1,4-galactosidase enzyme, and further M3X-PA was obtained by digesting N-acetylglucosaminidase. From these results, the structure of peak V-3 is β-
D-Gal- (1 → 4) -β-D-GlcNAc- (1 →
2) -α-D-Man- (1 → 6) [β-D-Gal- (1
→ 4) -β-D-GlcNAc- (1 → 2) -α-D-M
an- (1 → 3)] [β-D-Xyl- (1 → 2)] β-D
-Man- (1 → 4) -β-D-GlcNAc- (1 → 4)
-GlcNAc-PA (Gal2GN2M3X-PA)
Was estimated to be.

Summary FIG. 8 shows the sugar chain structure of the glycoprotein obtained in the above Example, which is the sugar chain structure of the glycoprotein in BY2 strain cells (Nirian Q. Palapac).
et al (1999) Biosci. Biotec
hnol. Biochem. 63 (1), 35-39)
And sugar chain structure of glycoprotein secreted extracellularly (R
yoMisaki et al (2001) Biosc
i. Biotechnol. Biochem. (Scheduled to be published in October issue). In the figure, squares represent N-acetylglucosamine, circles represent mannose, triangles represent galactose, diamonds represent sialic acid, F represents fucose, and X represents xylose. Reference terms such as PeakI-4 shown to the right of the sugar chain structure indicate peaks in the HPLC analysis from which the sugar chain structure was derived.

As shown in FIG. 8, it was shown that β1,4-bonded glass-lactose residues (indicated by triangles in the figure) are present in all the peak components.

[0081]

EFFECTS OF THE INVENTION A method for conveniently and rapidly producing a glycoprotein having a sugar chain structure that is stable in vivo, retains its original physiological activity, and does not serve as an allergen is provided. The resulting glycoprotein is useful for administration to animals because it has an animal-type sugar chain structure.

[Brief description of drawings]

FIG. 1 is a diagram showing the structures of sugar chains present in animal cells and plant cells.

FIG. 2 is a view showing a part of a sugar chain synthetic pathway in animal cells and plant cells. In the figure, squares represent N-acetylglucosamine, circles represent mannose, triangles represent galactose, diamonds represent sialic acid, F represents fucose, and X represents xylose.

FIG. 3 is a diagram schematically showing the method of the present invention.

FIG. 4 is a photograph of an electrophoresis gel showing the results of lectin analysis.

FIG. 5 is a chromatogram showing an elution pattern of sugar chains in RP-HPLC.

FIG. 6 is a chromatogram showing the elution pattern of sugar chains in SF-HPLC.

FIG. 7 is a chromatogram showing the elution pattern of sugar chains in the SF-HPLC chromatogram.

FIG. 8 is a diagram showing a sugar chain structure obtained by the method of the present invention.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Tatsuharu Seki             2-1-1-1015 Shinsenri Nishimachi, Toyonaka City, Osaka Prefecture F-term (reference) 2B030 AA02 AB03 AD08 CA06 CA17                       CA19 CB03 CD03 CD07 CD09                 4B024 AA01 BA80 CA01 DA01 GA11                       HA01                 4B064 AG01 CA11 CA21 CB30 CC24                       CD09 CE20 DA01                 4H045 AA10 BA53 CA30 EA20 FA74

Claims (8)

[Claims] 1. A method for converting a glycoprotein having a plant-type sugar chain into a glycoprotein having an animal-type sugar chain, which comprises converting a glycoprotein having a plant-type sugar chain into a non-reducing terminal acetylglucosamine residue. -A step of reacting with an enzyme capable of carrying out a transfer reaction of amino acid residues. 2. The glycoprotein having the plant type sugar chain,
The method according to claim 1, which is a glycoprotein produced by a plant cell. 3. The method of claim 2, wherein the glycoprotein produced by the plant cell is a heterologous glycoprotein. 4. The method according to claim 3, wherein the heterologous protein is obtained by a method comprising the steps of transforming a plant cell with a gene encoding the heterologous protein, and culturing the resulting transformant. Method. 5. The method according to claim 4, further comprising a step of collecting a culture solution of the transformant. 6. The glycoprotein having the animal type sugar chain,
A core sugar chain and an outer sugar chain, wherein the core sugar chain consists essentially of a plurality of mannoses and acetylglucosamine, and the outer sugar chain comprises a terminal sugar chain portion containing a non-reducing terminal galactose. The method according to Item 1. 7. The method according to claim 1, wherein the glycoprotein having an animal type sugar chain does not contain fucose or xylose. 8. A glycoprotein having an animal-type sugar chain, which is obtained by the method according to claim 1.