U.S. patent application number 12/953074 was filed with the patent office on 2011-03-24 for fucose transporter.
Invention is credited to KIYOSHI HABU, SHIGEYUKI IIJIMA, YASUO SEKIMORI, IZUMI SUGO, MASAYUKI TSUCHIYA, KENJU UENO.
Application Number | 20110070614 12/953074 |
Document ID | / |
Family ID | 34199142 |
Filed Date | 2011-03-24 |
United States Patent
Application |
20110070614 |
Kind Code |
A1 |
TSUCHIYA; MASAYUKI ; et
al. |
March 24, 2011 |
FUCOSE TRANSPORTER
Abstract
The present invention provides a gene encoding a fucose
transporter, a fucose transporter polypeptide, a method for
screening for a compound that binds to a fucose transporter or a
compound that inhibits fucose transport activity, a cell having
inhibited fucose transporter functions, and a cell wherein the
expression of the fucose transporter is inhibited. The present
invention further relates to a method for producing recombinant
protein, and specifically, to a method for producing protein by
which fucose existing within the Golgi apparatus of a host cell is
decreased, a method for inhibiting the addition of fucose to
protein by which fucose existing within the Golgi apparatus of a
host cell is decreased upon production of recombinant protein using
the host cell, a method for increasing the cytotoxic activity of an
antibody by which an antibody is produced using a cell wherein
fucose existing within the Golgi apparatus is decreased, and a cell
having a Golgi apparatus wherein fucose existing within the Golgi
apparatus is decreased.
Inventors: |
TSUCHIYA; MASAYUKI;
(SHIZUOKA, JP) ; IIJIMA; SHIGEYUKI; (SHIZUOKA,
JP) ; SUGO; IZUMI; (SHIZUOKA, JP) ; SEKIMORI;
YASUO; (SHIZUOKA, JP) ; UENO; KENJU;
(SHIZUOKA, JP) ; HABU; KIYOSHI; (IBARAKI,
JP) |
Family ID: |
34199142 |
Appl. No.: |
12/953074 |
Filed: |
November 23, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10561191 |
Dec 16, 2005 |
7863042 |
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PCT/JP2004/008956 |
Jun 18, 2004 |
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12953074 |
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Current U.S.
Class: |
435/69.6 ;
435/69.1 |
Current CPC
Class: |
G01N 2500/00 20130101;
C07K 14/47 20130101; C12P 21/005 20130101 |
Class at
Publication: |
435/69.6 ;
435/69.1 |
International
Class: |
C12P 21/00 20060101
C12P021/00 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 18, 2003 |
JP |
2003-174006 |
Jun 18, 2003 |
JP |
2003-174010 |
Jul 29, 2003 |
JP |
2003-282081 |
Jul 29, 2003 |
JP |
2003-282102 |
Claims
1. A method for producing a recombinant protein, wherein fucose
existing in the Golgi apparatus of a host cell is decreased.
2. The method for producing a recombinant protein according to
claim 1, wherein the fucose transporter functions are inhibited by
artificially suppressing the expression of the fucose transporter
in a host cell.
3. The method for producing a recombinant protein according to
claim 2, wherein the expression of the fucose transporter is
suppressed using RNAi.
4. The method for producing a recombinant protein according to
claim 1, wherein the fucose transporter functions are inhibited by
deleting a gene encoding the fucose transporter in a host cell.
5. The method for producing a recombinant protein according to
claim 1, wherein the protein is an antibody.
6. The method for producing a recombinant protein according to
claim 1, wherein a protein not having fucose added thereto is
produced.
7. The method for producing a recombinant protein according to
claim 1, wherein the host cell is a CHO cell.
Description
[0001] This application is a Divisional of copending application
Ser. No. 10/561,191 filed on Dec. 16, 2005, which is a National
Phase of PCT International Application No. PCT/JP2004/008956 filed
on Jun. 18, 2004, which claims the benefit of Application Nos.
2003-174006; 2003-174010; 2003-282081; and 2003-282102 filed in
Japan on Jun. 18, 2003; Jun. 18, 2003; Jul. 29, 2003; and Jul. 29,
2003, respectively. The entire contents of all of the above
applications are hereby incorporated by reference.
TECHNICAL FIELD
[0002] The present invention relates to a fucose transporter
polypeptide, a DNA encoding the fucose transporter polypeptide, a
cell having inhibited fucose transporter functions, and a method
for screening for a compound that binds to a fucose transporter or
a compound that inhibits fucose transport activity. The present
invention further relates to a method for producing a recombinant
protein and particularly an antibody using the cell having
inhibited fucose transporter functions.
BACKGROUND ART
[0003] Antibodies can exert anti-tumor effects via their ADCC
(antibody-dependent cell-mediated cytotoxicity) activity or CDC
(complement dependent cytotoxicity) activity. Antibodies are sugar
chain-bound glycoproteins. It is known that an antibody's cytotoxic
activity can vary depending on the types and amounts of sugar
chains that bind to the antibody. In particular, it has been
reported that the amount of fucose binding to an antibody is
strongly involved in the cytotoxic activity (Shields et al., J
Biol. Chem., 277(30), 26733-26740, 2002). Furthermore, a method for
producing a recombinant antibody not having fucose has been
reported. Such method involves preventing an enzyme that catalyzes
the binding of fucose to a sugar chain from being expressed upon
antibody production in order to obtain an antibody with enhanced
cytotoxic activity (International Patent Publication No.
WO00/61739).
SUMMARY OF THE INVENTION
[0004] An object of the present invention is to isolate a fucose
transporter gene or polypeptide intracellularly involved in fucose
transport. Furthermore, another object of the present invention is
to obtain a cell for producing a foreign protein having inhibited
fucose transporter functions. Furthermore, another object of the
present invention is to isolate a compound that can inhibit fucose
transporter functions. Furthermore, another object of the present
invention is to provide a method for easily and reliably producing
a recombinant protein wherein the binding of fucose is eliminated
or decreases. In particular, an object of the present invention is
to provide a method for producing an antibody wherein the binding
of fucose is eliminated or decreases and whose cytotoxic activity
is enhanced. Furthermore, another object of the present invention
is to provide a host cell for producing such protein.
[0005] In a mechanism by which fucose binds to an antibody within
an antibody-producing cell, it is known that GDP binds to fucose
that has been incorporated into a cell. GDP-fucose is then
incorporated into the Golgi apparatus and then the fucose of the
GDP-fucose is transferred to N-acetylglucosamine that has been
added as a sugar chain to protein within the Golgi apparatus.
Specifically, the Fc region of an antibody molecule has two sites
to which an N-glycoside-bound sugar chain binds. Fucose binds to
the N-acetylglucosamine portion of an N-glycoside-bound sugar chain
(Pate L. Smith et al., J. Cell Biol. 2002, 158, 801-815). The
present inventors have studied this mechanism and inferred that if
incorporation of GDP-fucose into the Golgi apparatus can be
inhibited, the binding of fucose to protein can be suppressed. As a
result of intensive studies concerning a substance that
incorporates GDP-fucose into the Golgi apparatus, the present
inventors isolated the substance as a fucose transporter and have
discovered that an antibody to which no fucose binds and which has
enhanced cytotoxic activity can be obtained by using a cell (having
inhibited fucose transporter functions) as a host for the
production of recombinant protein. Thus, the present inventors have
completed the present invention.
[0006] In the present invention, to satisfy conditions where the
addition of fucose to an antibody is inhibited, it is not necessary
that all the produced antibodies do not experience the addition of
fucose thereto, but the proportion of protein to which fucose has
been added should be decreased among antibody compositions.
[0007] The present invention will be described in detail as
follows.
[1] A recombinant polypeptide or a fragment thereof as shown in (a)
or (b): (a) a polypeptide comprising the amino acid sequence
represented by SEQ ID NO: 2; or (b) a polypeptide comprising an
amino acid sequence derived from the amino acid sequence
represented by SEQ ID NO: 2 by deletion, substitution, insertion,
or addition of 1 or several amino acids and being functionally
equivalent to the polypeptide (a). [2] A DNA, which encodes the
following polypeptide (a) or (b): (a) a polypeptide comprising the
amino acid sequence represented by SEQ ID NO: 2; or (b) a
polypeptide comprising an amino acid sequence derived from the
amino acid sequence represented by SEQ ID NO: 2 by deletion,
substitution, insertion, or addition of 1 or several amino acids
and being functionally equivalent to the polypeptide (a). [3] A
DNA, which comprises the following DNA (c) or (d): (c) a DNA
comprising the nucleotide sequence represented by SEQ ID NO: 1; or
(d) a DNA hybridizing under stringent conditions to a DNA
consisting of a sequence complementary to the DNA comprising the
nucleotide sequence of SEQ ID NO: 1 and encoding a polypeptide that
is functionally equivalent to a polypeptide encoded by the DNA (c).
[4] A DNA fragment, which is a fragment of the DNA according to [2]
or [3] or is a fragment of a DNA that is complementary to the DNA
according to [2] or [3] and consists of at least 15 nucleotides.
[5] A recombinant vector, which comprises the DNA according to [2]
or [3]. [6] A transformant, which comprises the recombinant vector
according to [5]. [7] A method for producing the polypeptide
according to [1], which comprises culturing the transformant
according to [6] and collecting the polypeptide from the cultured
transformant or the culture supernatant thereof. [8] An antibody,
which binds to the polypeptide according to [1]. [9] A screening
method for a compound that binds to the polypeptide according to
[1], which comprises the steps of: (a) contacting a sample to be
tested with the polypeptide; (b) detecting the binding activity
between the polypeptide and the sample to be tested; and (c)
selecting a compound having activity of binding to the polypeptide.
[10] A compound binding to the polypeptide according to [1], which
can be isolated by the method according [9]. [11] A screening
method for a compound that inhibits the GDP-fucose transport
activity of the polypeptide according [1], which comprises the
steps of: (a) contacting a sample to be tested and GDP-fucose with
the polypeptide; (b) detecting the GDP-fucose-incorporating ability
of the polypeptide; and (c) selecting a compound that inhibits the
GDP-fucose transport activity of a polypeptide. [12] A compound
that inhibits the GDP-fucose transport activity of the polypeptide
according [1], which can be isolated by the method according to
[11]. [13] A cell, which has a Golgi apparatus wherein fucose is
decreased. [14] A cell, which exhibits decreased fucose transport
ability or lacks such ability. [15] A cell, which exhibits
decreased activity of incorporating fucose into a Golgi apparatus,
or which lacks such activity. [16] The cell according to any one of
[13] to [15], which is treated with a compound that binds to a
fucose transporter or a compound that inhibits fucose transport
activity. [17] A cell, wherein the expression of a fucose
transporter is artificially suppressed. [18] The cell according to
[17], wherein the expression of a fucose transporter is suppressed
by using RNAi. [19] A cell, wherein a fucose transporter gene is
disrupted. [20] The cell according to any one of [13] to [19],
which is an animal cell. [21] The cell according to [20], wherein
the animal cell is a Chinese hamster cell. [22] The cell according
to [20], wherein the animal cell is a CHO cell. [23] The cell
according to any one of [19] to [22], wherein the gene is disrupted
by homologous recombination using a gene targeting vector. [24] A
targeting vector, which targets a gene encoding a fucose
transporter. [25] The targeting vector according to [24], wherein
the fucose transporter is a Chinese hamster fucose transporter.
[26] A method for producing a recombinant protein, wherein fucose
existing in the Golgi apparatus of a host cell is decreased. [27] A
method for producing a recombinant protein, wherein the
incorporation of fucose into the Golgi apparatus in a host cell is
inhibited. [28] A method for producing a recombinant protein,
wherein the incorporation of fucose mediated by a fucose
transporter in a host cell is inhibited. [29] A method for
producing a recombinant protein, wherein fucose transporter
functions of a host cell are inhibited. [30] The method for
producing a recombinant protein according to any one of [26] to
[29], wherein the fucose transporter functions are inhibited by
artificially suppressing the expression of the fucose transporter
in a host cell. [31] The method for producing a protein according
to [30], wherein the expression of the fucose transporter is
suppressed using RNAi. [32] The method for producing a recombinant
protein according to any one of [26] to [30], wherein the fucose
transporter functions are inhibited by deleting a gene encoding the
fucose transporter in a host cell. [33] The production method
according to any one of [26] to [32], wherein the protein is an
antibody. [34] The production method according to any one of [26]
to [33], wherein a protein not having fucose added thereto is
produced. [35] The production method according to any one of [26]
to [34], wherein the host cell is a CHO cell. [36] A method for
inhibiting the addition of fucose to a protein, wherein fucose
existing in the Golgi apparatus in a host cell is decreased when a
recombinant protein is produced using the host cell. [37] A method
for inhibiting the addition of fucose to a protein, wherein fucose
transporter functions in a host cell are inhibited when a
recombinant protein is produced using the host cell. [38] The
method for inhibiting the addition of fucose to a protein according
to [36] or [37], wherein the expression of a fucose transporter is
artificially suppressed when a recombinant protein is produced
using a host cell. [39] The method for inhibiting the addition of
fucose to a protein according to [38], wherein the expression of a
fucose transporter is suppressed using RNAi. [40] The method for
inhibiting the addition of fucose to a protein according to any one
of [36] to [38], wherein a gene encoding a fucose transporter is
deleted when a recombinant protein is produced using a host cell.
[41] A method for inhibiting the addition of fucose to a protein,
wherein the incorporation of fucose mediated by a fucose
transporter is inhibited when a recombinant protein is produced
using a host cell. [42] The method for inhibiting the addition of
fucose to a protein according to any one of [36] to [41], wherein
the protein is an antibody. [43] The method for inhibiting the
addition of fucose to a protein according to any one of [36] to
[42], wherein the host cell is a CHO cell. [44] A method for
increasing the cytotoxic activity of an antibody, wherein an
antibody is produced with a cell in which fucose existing in the
Golgi apparatus is decreased. [45] A method for increasing the
cytotoxic activity of an antibody, wherein an antibody is produced
with a host cell having inhibited fucose transporter functions.
[46] A method for increasing the cytotoxic activity of an antibody,
wherein an antibody is produced with a cell in which the expression
of a fucose transporter is artificially suppressed. [47] A method
for increasing the cytotoxic activity of an antibody, wherein an
antibody is produced with a cell that lacks a gene encoding a
fucose transporter. [48] A method for increasing the cytotoxic
activity of an antibody, wherein an antibody is produced by
inhibiting the incorporation of fucose into the Golgi apparatus.
[49] The method for increasing the cytotoxic activity of an
antibody according to any one of [44] to [48], wherein the host
cell is a CHO cell.
[0008] This specification includes part or all of the contents as
disclosed in the specification and/or drawings of Japanese Patent
Application Nos. 2003-174006, 2003-174010, 2003-282081, and
2003-282102, which are priority documents of the present
application.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 shows human (SEQ ID NO: 11) and mouse (SEQ ID NO: 12)
GDP-fucose transporter cDNA sequences and PCR primer sequences (SEQ
ID NOS: 13 and 14) for producing a probe, which have been designed
to obtain CHO-derived GDP-fucose transporter cDNA from these common
sequences.
[0010] FIG. 2 shows a CHO-cell-derived GDP-fucose transporter cDNA
sequence (SEQ ID NO: 15) obtained by cloning (lower-case letters
indicate vector-derived cloning sites). FIG. 2 also shows PCR
primer sequences (SEQ ID NO: 16-19) for producing a probe that was
used for obtaining GDP-fucose transporter genomic DNA.
[0011] FIG. 3 shows a restriction enzyme map of a GDP-fucose
transporter gene obtained by cloning. The underlined portion
indicates a sequence (lower-case letter: intron) located at the
boundary portion between an exon and an intron.
[0012] FIG. 4 shows a CHO-derived GDP-fucose transporter gene
sequence (SEQ ID NO: 1).
[0013] FIG. 5 shows the results of inhibiting a transporter gene
using siRNA.
[0014] FIG. 6 shows the structure of a targeting (KO2) vector and
an outline of PCR screening.
[0015] FIG. 7 is a photograph showing the results of PCR
screening.
[0016] FIG. 8 shows restriction enzyme maps for a wild-type
chromosome and a knockout-type chromosome.
[0017] FIG. 9 shows photographs showing the results of carrying out
the Southern blot method.
BEST MODE FOR CARRYING OUT THE INVENTION
[0018] The present invention provides a Chinese hamster (CH) fucose
transporter. Chinese hamster ovary cells (CHO cells) are currently
widely used as host cells for producing proteins such as
antibodies. Thus, the Chinese hamster fucose transporter of the
present invention is particularly useful.
[0019] The present invention also encompasses DNA encoding
polypeptides that are functionally equivalent to the CH fucose
transporter gene. Examples of such DNA include DNA encoding a
mutant, allele, variant, homolog, or the like of a CH fucose
transporter polypeptide. Here, "functionally equivalent" indicates
that a subject polypeptide has biological functions equivalent to
those of the CH fucose transporter polypeptide.
[0020] In the present invention, "biological functions equivalent
to those of the CH fucose transporter" indicates fucose transport
activity, and preferably the fucose transport activity in CHO
cells.
[0021] As a method for preparing a polypeptide that is functionally
equivalent to a polypeptide that is well known by persons skilled
in the art, a method for introducing mutation into a polypeptide is
known. For example, persons skilled in the art can prepare a
polypeptide that is functionally equivalent to the CH fucose
transporter polypeptide by appropriately introducing mutation into
amino acids of the polypeptide through the use of, for example, the
site-directed mutagenesis method (Gotoh, T. et al. (1995), Gene
152, 271-275; Zoller, M J, and Smith, M. (1983), Methods Enzymol.
100, 468-500; Kramer, W. et al. (1984), Nucleic Acids Res. 12,
9441-9456; Kramer W and Fritz H J (1987), Methods. Enzymol. 154,
350-367; Kunkel, T A (1985), Proc Natl Acad Sci U.S.A. 82, 488-492;
and Kunkel (1988), Methods Enzymol. 85, 2763-2766).
[0022] Furthermore, amino acid mutation can also take place in
nature. Thus, the present invention also includes a polypeptide
consisting of an amino acid sequence derived from the amino acid
sequence of the CH fucose transporter polypeptide by mutation of 1
or a plurality of amino acids that is functionally equivalent to
the polypeptide. The number of mutated amino acids in such a mutant
is generally 30 amino acids or less, preferably 15 amino acids or
less, further preferably 5 amino acids or less, and particularly
preferably 3 amino acids or less.
[0023] It is desirable that amino acid residues are mutated to
result in other amino acids while retaining the properties of an
amino acid side chain. Examples of such properties of an amino acid
side chain include hydrophobic amino acids (A, I, L, M, F, P, W, Y,
and V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, and
T), amino acids having aliphatic side chains (G, A, V, L, I, and
P), amino acids having hydroxyl-group-containing side chains (S, T,
and Y), amino acids having sulfur-atom-containing side chains (C
and M), amino acids having carboxylic-acid- and amide-containing
side chains (D, N, E, and Q), amino acids having base-containing
side chains (R, K, and H), and amino acids having
aromatic-containing side chains (H, F, Y, and W) (all the capital
letters in parentheses represent single-letter denotations for
amino acids).
[0024] It is already known that a polypeptide having an amino acid
sequence derived from an amino acid sequence by modification such
as by deletion of 1 or a plurality of amino acid residues, addition
of 1 or a plurality of amino acid residues, and/or substitution
with other amino acids retains its biological activity (Mark, D. F.
et al., Proc. Natl. Acad. Sci. U.S.A. (1984) 81, 5662-5666; Zoller,
M. J. & Smith, M., Nucleic Acids Research (1982) 10, 6487-6500;
Wang, A. et al., Science 224, 1431-1433; Dalbadie-McFarland, G. et
al., Proc. Natl. Acad. Sci. U.S.A. (1982) 79, 6409-6413).
[0025] Examples of a polypeptide having an amino acid sequence
derived from the amino acid sequence of the CH fucose transporter
polypeptide by addition of a plurality of amino acids include
fusion polypeptides comprising such polypeptides. Such fusion
polypeptides result from the fusion of such polypeptides with other
polypeptides. Such fusion polypeptides are also included in the
present invention. To prepare a fusion polypeptide, for example,
DNA encoding the CHO fucose transporter polypeptide is ligated to a
DNA encoding another polypeptide so that the frames match. The
resultant is then introduced into an expression vector and
expressed by a host. Examples of other polypeptides that can be
fused with the polypeptide of the present invention include, but
are not specifically limited to, known peptides such as FLAG (Hopp,
T. P. et al., BioTechnology (1988) 6, 1204-1210), 6.times.His
consisting of 6 His (histidine) residues, 10.times.His, influenza
hemagglutinin (HA), human c-myc fragments, VSV-GP fragments, p18HIV
fragments, T7-tag, HSV-tag, E-tag, SV40T antigen fragments, lck
tags, .alpha.-tubulin fragments, B-tags, and Protein C fragments.
Furthermore, examples of other polypeptides to be fused with the
polypeptide of the present invention include GST
(glutathione-S-transferase), HA (influenza hemagglutinin),
immunoglobulin constant region, .beta.-galactosidase, and MBP
(maltose-binding protein). These polypeptides and genes encoding
the polypeptides are marketed. Hence, a fusion polypeptide can be
prepared by fusing a DNA encoding such marketed polypeptide with
DNA encoding the polypeptide of the present invention and then
expressing the thus prepared fusion DNA.
[0026] Furthermore, another example of a method for preparing a DNA
encoding a polypeptide that is functionally equivalent to a
polypeptide that is well known by persons skilled in the art is a
method using hybridization techniques (Sambrook, J et al.,
Molecular Cloning 2.sup.nd ed., 9.47-9.58, Cold Spring Harbor Lab.
Press, 1989). Specifically, techniques known by persons skilled in
the art are isolation of a DNA having high homology with a DNA
sequence that encodes the CH fucose transporter polypeptide or a
portion thereof and isolation of a polypeptide functionally
equivalent to the CH fucose transporter polypeptide from the
DNA.
[0027] The present invention includes DNA hybridizing under
stringent conditions to the DNA encoding the CH fucose transporter
polypeptide and encoding a polypeptide functionally equivalent to
the CH fucose transporter polypeptide. Examples of such DNA include
homologs derived from humans or other mammals (e.g., rats, rabbits,
and cattle).
[0028] Hybridization conditions for the isolation of DNA encoding a
polypeptide that is functionally equivalent to the CH fucose
transporter polypeptide can be appropriately selected by persons
skilled in the art. Such stringent conditions for hybridization
are, for example, low stringent conditions. Such low stringent
conditions comprise, upon washing after hybridization, for example,
42.degree. C., 0.1.times.SSC, and 0.1% SDS, and preferably
50.degree. C., 0.1.times.SSC, and 0.1% SDS. More preferable
hybridization conditions are, for example, high stringent
conditions. Such high stringent conditions comprise, for example,
65.degree. C., 0.1.times.SSC, and 0.1% SDS. Under these conditions,
it can be expected that DNA having high homology as the temperature
is elevated may be efficiently obtained. However, a plurality of
factors such as temperature and salt concentration may influence
the stringency concerning hybridization. Persons skilled in the art
can realize stringency similar to the above stringency by
appropriately selecting these factors.
[0029] Moreover, DNA encoding a polypeptide functionally equivalent
to the CH fucose transporter polypeptide can also be isolated by a
gene amplification method (e.g., a polymerase chain reaction (PCR)
method) using primers that are synthesized based on the sequence
information concerning the DNA encoding the CH fucose transporter
polypeptide.
[0030] A polypeptide that is encoded by DNA isolated by these
hybridization techniques and gene amplification techniques and that
is functionally equivalent to the CH fucose transporter polypeptide
generally has high homology with the CH fucose transporter
polypeptide in terms of amino acid sequence. Examples of the
polypeptide of the present invention also include polypeptides that
are functionally equivalent to the CH fucose transporter
polypeptide and that have high homology with the amino acid
sequences of the polypeptide. Such high homology at the amino acid
level indicates generally 70% or more homology, preferably 80% or
more homology, further preferably 92% or more homology, and further
preferably 95% or more homology.
[0031] Furthermore, such high homology at the nucleotide sequence
level indicates generally 70% or more homology, preferably 80% or
more homology, further preferably 90% or more homology, and further
preferably 95% or more homology.
[0032] The homology of amino acid sequences or nucleotide sequences
can be determined by, for example, algorithm BLAST (Proc. Natl.
Acad. Sci. U.S.A. 90: 5873-5877, 1993) according to Karlin and
Altschul. Based on this algorithm, a program called BLASTN or a
program called BLASTX has been developed (Altschul et al., J. Mol.
Biol. 215: 403-410, 1990). When nucleotide sequences are analyzed
by BLASTN based on BLAST, parameters are determined to be, for
example, score=100 and word length=12. Furthermore, when amino acid
sequences are analyzed by BLASTX based on BLAST, parameters are
determined to be, for example, score=50 and word length=3. When
BLAST and Gapped BLAST programs are used, default parameters for
each program are used. Specific techniques for these analysis
methods are known.
[0033] The DNA of the present invention is used for, for example,
in vivo or in vitro production of the polypeptide of the present
invention as described later. The DNA of the present invention may
be in any form as long as it can encode the polypeptide of the
present invention. Specifically, such DNA may be cDNA synthesized
from mRNA, genomic DNA, or chemically-synthesized DNA. Furthermore,
a DNA having an arbitrary nucleotide sequence based on genetic code
degeneration is included herein, as long as it encodes the
polypeptide of the present invention.
[0034] The DNA of the present invention can be prepared by methods
known by persons skilled in the art. For example, such DNA can be
prepared by constructing a cDNA library from a cell expressing the
polypeptide of the present invention and then carrying out
hybridization using as a probe a portion of the DNA sequence of the
present invention. Such cDNA library may also be prepared by a
method described in, for example, relevant literature (Sambrook, J.
et al., Molecular Cloning, Cold Spring Harbor Laboratory Press
(1989)). Alternatively, a commercial DNA library may also be used.
Furthermore, the DNA of the present invention can also be prepared
by preparing RNA from a cell expressing the polypeptide of the
present invention, synthesizing cDNA using reverse transcriptase,
synthesizing oligo DNA based on the DNA sequence of the present
invention, carrying out PCR reaction using the resultants as
primers, and then amplifying the cDNA that encodes the polypeptide
of the present invention.
[0035] Moreover, through the determination of the nucleotide
sequence of the thus obtained cDNA, a translation region encoded by
the sequence can be determined and the amino acid sequence of the
polypeptide of the present invention can be obtained. Furthermore,
through screening of a genomic DNA library using the thus obtained
cDNA as a probe, a genomic DNA can be isolated.
[0036] Specifically, the following steps are carried out. First,
mRNA is isolated from cells, tissues, and organs expressing the
polypeptide of the present invention. To isolate mRNA, total RNA is
prepared by a known method such as guanidine ultracentrifugation
(Chirgwin, J. M. et al., Biochemistry (1979) 18, 5294-5299) or an
AGPC method (Chomczynski, P. and Sacchi, N., Anal. Biochem. (1987)
162, 156-159). mRNA is then purified from the total RNA using a
mRNA Purification Kit (Pharmacia Corporation) or the like. In
addition, mRNA can also be directly prepared using a QuickPrep mRNA
Purification Kit (Pharmacia Corporation).
[0037] cDNA is synthesized from the thus obtained mRNA using
reverse transcriptase. cDNA can also be synthesized using an AMV
Reverse Transcriptase First-strand cDNA Synthesis Kit (SEIKAGAKU
CORPORATION) or the like. Furthermore, through the use of primers
and the like described in this specification, cDNA synthesis and
cDNA amplification can be carried out according to the 5'-RACE
method (Frohman, M. A. et al., Proc. Natl. Acad. Sci. U.S.A. (1988)
85, 8998-9002; Belyaysky, A. et al., Nucleic Acids Res. (1989) 17,
2919-2932) using a 5'-Ampli FINDER RACE Kit (produced by Clontech)
and polymerase chain reaction (PCR).
[0038] A target DNA fragment is prepared from the thus obtained PCR
product and then ligated to a vector DNA. Furthermore, a
recombinant vector is constructed using the resultant, the
recombinant vector is introduced into Escherichia coli or the like,
and then colonies are selected, thereby preparing a desired
recombinant vector. The nucleotide sequence of a target DNA can be
confirmed by a known method such as a dideoxy nucleotide chain
termination method.
[0039] Furthermore, regarding the DNA of the present invention, a
nucleotide sequence having higher expression efficiency can be
designed in view of the codon usage frequency of a host to be used
for expression (Grantham, R. et al., Nucleic Acids Research (1981)
9, r43-74). Moreover, the DNA of the present invention can be
altered using a commercial kit or by a known method. Examples of
such alteration include digestion with a restriction enzyme,
insertion of a synthetic oligonucleotide or an appropriate DNA
fragment, addition of a linker, and insertion of an initiation
codon (ATG) and/or termination codon (TAA, TGA, or TAG).
[0040] The present invention provides a polypeptide encoded by the
above DNA of the present invention. The polypeptide of the present
invention may differ in amino acid sequence, molecular weight,
isoelectric point, or the sugar chain presence, absence, or form,
due to the cells or hosts that produce such polypeptide or
purification methods as described later. However, the thus obtained
polypeptide is included in the present invention, as long as it has
functions equivalent to those of the CH fucose transporter
polypeptide. For example, when the polypeptide of the present
invention is expressed in prokaryotic cells such as Escherichia
coli, a methionine residue is added to the N-terminus of the amino
acid sequence of the original polypeptide. The polypeptide of the
present invention also encompasses such polypeptide.
[0041] The polypeptide of the present invention can be prepared by
a method known by persons skilled in the art as a recombinant
polypeptide or a natural polypeptide. Such recombinant polypeptide
can be purified and prepared as follows. DNA encoding the
polypeptide of the present invention is incorporated into an
appropriate expression vector, a transformant that has been
obtained by introducing the vector into an appropriate host cell is
collected, and then an extract is obtained. Subsequently, the
resultant is subjected to chromatography such as ion exchange
chromatography, reverse phase chromatography, or gel filtration,
affinity chromatography using a column (to which an antibody
against the polypeptide of the present invention has been
immobilized), or chromatography using combination of a plurality of
such columns.
[0042] Furthermore, when the polypeptide of the present invention
is expressed as a fusion polypeptide with a
glutathione-S-transferase protein or as a recombinant polypeptide
to which a plurality of histidines have been added in host cells
(e.g., animal cells or Escherichia coli), the expressed recombinant
polypeptide can be purified using a glutathione column or a nickel
column. After purification of the fusion polypeptide, if necessary,
regions other than the target polypeptide can also be cleaved and
removed from the fusion polypeptide using thrombin or factor
Xa.
[0043] In the case of a natural polypeptide, such polypeptide can
be isolated by a method known by persons skilled in the art, such
as by causing an affinity column (to which an antibody that binds
to the polypeptide of the present invention has been bound as
described later) to act on extracts obtained from tissues or cells
(e.g., testis) expressing the polypeptide of the present invention
for purification. An antibody used herein may be a polyclonal or
monoclonal antibody.
[0044] The present invention further encompasses partial peptides
of the polypeptide of the present invention. Such partial peptides
of the present invention can be used for, for example, producing an
antibody against the polypeptide of the present invention or
screening for a compound that binds to the polypeptide of the
present invention.
[0045] When used as an immunogen, the partial peptide of the
present invention generally consists of an amino acid sequence of
at least 7 or more amino acids, preferably 8 or more amino acids,
and further preferably 9 or more amino acids. When used as a
competitive inhibitor for the polypeptide of the present invention,
such partial peptide consists of an amino acid sequence of at least
100 or more amino acids, preferably 200 or more amino acids, and
further preferably 300 or more amino acids.
[0046] Such partial peptides of the present invention can be
produced by a genetic engineering technique known as the peptide
synthesis method, or by cleaving the polypeptide of the present
invention with appropriate peptidase. Peptides may be synthesized
by, for example, either a solid-phase synthesis method or a
liquid-phase synthesis method.
[0047] The present invention also provides a vector into which the
DNA of the present invention is inserted. Such vector of the
present invention is useful for retaining the DNA of the present
invention within host cells or expressing the polypeptide of the
present invention.
[0048] For example, when Escherichia coli is used as a host, such
vector is amplified in large quantities in Escherichia coli (e.g.,
JM109, DH5.alpha., HB101, and XL1Blue) for mass preparation. Hence,
vectors used herein are not specifically limited, as long as they
have "ori" for amplification in Escherichia coli and have a gene
for the selection of transformed Escherichia coli (e.g., a drug
resistance gene that enables distinguishment by the use of a drug
such as ampicillin, tetracycline, kanamycin, or
chloramphenicol).
[0049] Examples of such vector include M13-based vectors, pUC-based
vectors, pBR322, pBluescript, and pCR-Script. Furthermore, for the
purpose of cDNA subcloning or excision of cDNA, examples of such
vector include, in addition to the above vectors, pGEM-T, pDIRECT,
and pT7.
[0050] When a vector is used for the purpose of producing the
polypeptide of the present invention, an expression vector is
particularly useful. As an expression vector, for example, when a
vector is used for expression in Escherichia coli, such vector
should have the above characteristics that enable amplification in
Escherichia coli. Furthermore, when a host is Escherichia coli such
as JM109, DH5.alpha., HB101, or XL1-Blue, such vector essentially
has a promoter that enables efficient expression in Escherichia
coli, such as a lacZ promoter (Ward et al., Nature (1989) 341,
544-546; FASEB J. (1992) 6, 2422-2427), an araB promoter (Better et
al., Science (1988) 240, 1041-1043), or a T7 promoter. Examples of
such vector include, in addition to the above vectors, pGEX-5X-1
(produced by Pharmacia Corporation), pQE used in the "QIAexpress
system" (produced by QIAGEN), pEGFP, and pET (in this case, a host
is preferably BL21 expressing T7 RNA polymerase).
[0051] Furthermore, such vector may also contain a signal sequence
for polypeptide secretion. As a signal sequence for polypeptide
secretion, when polypeptides are produced in periplasms of
Escherichia coli, a pelB signal sequence (Lei, S. P. et al., J.
Bacteriol. (1987) 169, 4379) may be used. Vectors can be introduced
into host cells using, for example, a calcium chloride method or an
electroporation method.
[0052] Microorganisms other than Escherichia coli can also be used
as hosts for producing the polypeptide of the present invention. In
this case, examples of vectors for producing the polypeptide of the
present invention include expression vectors derived from mammals
(e.g., pcDNA3 (produced by Invitrogen Corporation) and pEGF-BOS
(Nucleic Acids. Res. 1990, 18(17), p. 5322), pEF, and pCDM8),
expression vectors derived from insect cells (e.g., the "Bac-to-BAC
baculovirus expression system" (produced by GIBCO-BRL Life
Technologies Inc.) and pBacPAK8), expression vectors derived from
plants (e.g., pMH1 and pMH2), expression vectors derived from
animal viruses (e.g., pHSV, pMV, and pAdexLcw), expression vectors
derived from retroviruses (e.g., pZIPneo), expression vectors
derived from yeast (e.g., the "Pichia Expression Kit" (produced by
Invitrogen Corporation), pNV11, and SP-Q01), and expression vectors
derived from Bacillus subtilis (e.g., pPL608 and pKTH50).
[0053] For the purpose of expression in animal cells such as CHO
cells, COS cells, or NIH3T3 cells, a vector used herein essentially
has a promoter required for expression within cells, such as an
SV40 promoter (Mulligan et al., Nature (1979) 277, 108), an
MMLV-LTR promoter, an EF1.alpha. promoter (Mizushima et al.,
Nucleic Acids Res. (1990) 18, 5322), or a CMV promoter. It is
further preferable that such vector has a gene for selection of
transformed cells (e.g., a drug resistance gene that enables
distinguishment by the use of a drug (e.g., neomycin and G418)).
Examples of a vector having such properties include pMAM, pDR2,
pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
[0054] Furthermore, an example of a method for the purpose of
stable expression of a gene and amplification of the number of
copies of a gene within cells involves introducing a vector (e.g.,
pCHOI) having a complementary DHFR gene into CHO cells lacking the
nucleic acid synthesis pathway, followed by amplification using
methotrexate (MTX). Furthermore, an example of a method for the
purpose of transient expression of a gene involves transforming COS
cells having a gene that expresses SV40 T antigen on the chromosome
with a vector (e.g., pcD) having an SV40 replication origin. As a
replication initiation site, a site derived from polyoma virus,
adenovirus, bovine papilloma virus (BPV), or the like can also be
used. Furthermore, to amplify the number of copies of a gene in a
host cell system, an expression vector may contain as a selection
marker an aminoglycoside transferase (APH) gene, thymidine kinase
(TK) gene, Escherichia coli xanthine-guanine
phosphoribosyltransferase (Ecogpt) gene, dihydrofolate reductase
(dhfr) gene, or the like.
[0055] In the meantime, examples of a method for expressing in vivo
the DNA of the present invention within animals involves
incorporating the DNA of the present invention into an appropriate
vector and then introducing the vector into an animal body by, for
example, a retrovirus method, a liposome method, a cationic
liposome method, or an adenovirus method. Examples of vectors used
herein include, but are not limited to, adenovirus vectors (e.g.,
pAdexlcw) and retrovirus vectors (e.g., pZlPneo). General genetic
engineering techniques including, for example, insertion of the DNA
of the present invention into a vector can be carried out according
to a standard method (Molecular Cloning, 5.61-5.63). Administration
into living bodies may be carried out by an ex vivo method or an in
vivo method.
[0056] Furthermore, the present invention provides a host cell
having the vector of the present invention introduced therein. Such
host cell into which the vector of the present invention is
introduced is not specifically limited. For example, Escherichia
coli, various animal cells, or the like can be used. Such host cell
of the present invention can be used as, for example, a production
system for producing or expressing the polypeptide of the present
invention. As such a production system for producing a polypeptide,
in vitro and in vivo production systems can be employed. Examples
of an in vitro production system include a production system using
eukaryotic cells and a production system using prokaryotic
cells.
[0057] When eukaryotic cells are used, animal cells, plant cells,
and fungal cells, for example, can be used as hosts. As animal
cells, mammalian cells such as CHO (J. Exp. Med. (1995) 108, 945),
COS, 3T3, myeloma, BHK (baby hamster kidney), HeLa, and Vero are
known. As amphibian cells, Xenopus oocytes (Valle, et al., Nature
(1981) 291, 358-340), for example, are known. As insect cells Sf9,
Sf21, and Tn5, for example, are known. As CHO cells, in particular,
dhfr-CHO cells (Proc. Natl. Acad. Sci. U.S.A. (1980) 77, 4216-4220)
or CHO K-1 cells (Proc. Natl. Acad. Sci. U.S.A. (1968) 60, 1275),
which are deficient in a DHFR gene, can be preferably used. For the
purpose of mass-expression in animal cells, CHO cells are
particularly preferable. A vector can be introduced into a host
cell by, for example, a calcium phosphate method, a DEAF dextran
method, a method using cationic ribosome DOTAP (produced by
Boehringer Mannheim), an electroporation method, or
lipofection.
[0058] As plant cells Nicotiana tabacum-derived cells, for example,
are known to comprise a polypeptide production system. Polypeptides
can be obtained by culturing the calli of Nicotiana tabacum-derived
cells. As fungal cells, yeast such as that of the genus
Saccharomyces (e.g., Saccharomyces cerevisiae) and filamentous
bacteria such as those of the genus Aspergillus (e.g., Aspergillus
niger) are known.
[0059] When prokaryotic cells are used, a production system using
bacterial cells may be employed. Examples of bacterial cells
include Escherichia coli (E. coli) such as JM109, DH5.alpha., and
HB101. In addition, Bacillus subtilis is also known.
[0060] Polypeptides can be obtained by transforming these cells
with a target DNA and then culturing in vitro the thus transformed
cells. Culture can be carried out according to a known method. As a
culture solution for animal cells, DMEM, MEM, RPMI1640, or IMDM,
for example, can be used. At this time, a serum fluid such as fetal
calf serum (FCS) can be used together therewith. Alternatively
serum-free culture may also be carried out. pH during culture is
preferably between approximately 6 and 8. Culture is generally
carried out at approximately 30.degree. C. to 40.degree. C. for
approximately 15 to 200 hours. If necessary, exchange of media,
aeration, and agitation are carried out.
[0061] Meanwhile, examples of systems for in vivo production of
polypeptides include production systems using animals and
production systems using plants. Target DNA is introduced into
these animals or plants, polypeptides are produced in vivo within
the animals or the plants, and then the polypeptides are collected.
The term "host" in the present invention encompasses these animals
and plants.
[0062] When animals are used, there are production systems using
mammals and production systems using insects. As mammals, goats,
pigs, sheep, mice, or cattle can be used (Vicki Glaser, SPECTRUM
Biotechnology Applications, 1993). Furthermore, when mammals are
used, transgenic animals can be used.
[0063] For example, a target DNA is prepared in the form of a
fusion gene with a gene encoding a polypeptide such as goat .beta.
casein that is uniquely produced in milk. Subsequently, a DNA
fragment comprising the fusion gene is injected into a goat embryo
and then the embryo is transplanted into a female goat. A target
polypeptide can be obtained from milk that is produced by
transgenic goats born from goats that have accepted such embryos,
or from the progenies of such transgenic goats. To increase the
amount of milk containing polypeptides, which is produced by
transgenic goats, an appropriate hormone may also be used for such
transgenic goats (Ebert, K. M. et al., Bio/Technology (1994) 12,
699-702).
[0064] Furthermore, as insects, silkworm, for example, can be used.
When silkworms are used, a target polypeptide can be obtained from
the body fluid of a silkworm by infecting the silkworm with a
baculovirus wherein a DNA encoding the target polypeptide has been
inserted (Susumu, M. et al., Nature (1985) 315, 592-594).
[0065] Furthermore, when plants are used, tobacco, for example, can
be used. When tobacco is used, a DNA encoding a target polypeptide
is inserted into an expression vector for a plant, such as pMON
530, and then the vector is introduced into bacteria such as
Agrobacterium tumefaciens. Tobacco such as Nicotiana tabacum is
infected with such bacteria and then the desired polypeptide can be
obtained from the tobacco leaves (Julian K.-C. Ma et al., Eur. J.
Immunol. (1994) 24, 131-138).
[0066] The polypeptide of the present invention that is obtained by
such method can be isolated from within host cells or outside the
cells (e.g., media) and then purified as a substantially pure and
uniform polypeptide. To separate and purify polypeptides,
separation and purification methods that are generally used for
polypeptide purification may be employed and are not specifically
limited. For example, polypeptides can be separated and purified by
the use of appropriate selection and combination of a
chromatography column, a filter, ultrafiltration, salting-out,
solvent precipitation, solvent extraction, distillation,
immunoprecipitation, SDS-polyacrylamide gel electrophoresis, an
isoelectric focusing method, dialysis, recrystallization, and the
like.
[0067] Examples of chromatography include affinity chromatography,
ion exchange chromatography, hydrophobic chromatography, gel
filtration, reverse phase chromatography, and adsorption
chromatography (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual, Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press, 1996). These types of
chromatography can be carried out using liquid-phase chromatography
such as HPLC or FPLC. The present invention also encompasses
polypeptides that are purified at high levels using these
purification methods.
[0068] In addition, when a proper protein modification enzyme is
caused to act on polypeptides before or after purification,
modification can be arbitrarily carried out or peptides can be
partially removed. As protein modification enzymes, trypsin,
chymotrypsin, lysylendopeptidase, protein kinase, and glucosidase,
for example, are used.
[0069] The present invention also provides an antibody that binds
to the polypeptide of the present invention. Examples of forms of
such antibodies of the present invention include, but are not
specifically limited to, monoclonal antibodies and polyclonal
antibodies. Moreover, the origins of such antibodies are not
limited. Any antibodies may be used, such as mouse antibodies, rat
antibodies, rabbit antibodies, camel antibodies, and human
antibodies. Furthermore, chimeric antibodies or humanized
antibodies produced by genetic recombination are also included in
the antibodies of the present invention.
[0070] Polypeptides that are used as sensitizing antigens in the
present invention may be complete proteins or partial peptides of a
protein. Examples of such partial peptides of a protein include
amino group (N)-terminal fragments or carboxyl (C) terminal
fragments of a protein. "Antibody" in this specification means an
antibody that reacts with the full-length protein or a fragment
thereof.
[0071] A gene encoding the polypeptide or a fragment thereof of the
present invention is inserted into a known expression vector
system. Host cells described in this specification are transformed
with the vector and then the target polypeptide or a fragment
thereof is obtained by a known method from the inside or the
outside of the host cells. Such polypeptide or fragment may be used
as a sensitizing antigen. Moreover, a cell expressing the
polypeptide, a lysate of such cells, or a chemically synthesized
polypeptide of the present invention can also be used as a
sensitizing antigen. Preferably, a short peptide is appropriately
bound to a carrier protein such as keyhole limpet hemocyanin,
bovine serum albumin, or ovalbumin, so as to prepare an
antigen.
[0072] Mammals to be immunized with such sensitizing antigen are
not specifically limited. Preferably, mammals are selected in view
of compatibility with a parent cell to be used for cell fusion. In
general, animals of the order Rodentia, the order Lagomorpha, or
the order Primates are used.
[0073] As animals of the order Rodentia, mice, rats, and hamsters,
for example, are used. As animals of the order Lagomorpha, rabbits,
for example, are used. As animals of the order Primates, monkeys,
for example, are used. As such monkeys, monkeys of the order
Catarrhini (Monkeys of the Old World), such as crab-eating monkeys,
Rhesus monkeys, Hamadryas baboons, and chimpanzees are used.
[0074] Animals are immunized with a sensitizing antigen according
to a known method. In a general method, a sensitizing antigen is
intraperitoneally or subcutaneously injected into mammals.
Specifically, a sensitizing antigen is diluted and suspended to an
appropriate amount using PBS (Phosphate-Buffered Saline) or
physiological saline. If desired, a general adjuvant such as
Freund's complete adjuvant is mixed in an appropriate amount with
the suspension. After emulsification, the resultant is administered
to mammals. Furthermore, it is preferable to administer the
sensitizing antigen (which has been mixed in an appropriate amount
with Freund's incomplete adjuvant) several times over a period of
every 4 to 21 days. Furthermore, at the time of immunization with a
sensitizing antigen, an appropriate carrier can be used.
Immunization is carried out as described above. An increase in the
desired antibody level in serum is confirmed by a standard
method.
[0075] Here, to obtain a polyclonal antibody against the
polypeptide of the present invention, after confirmation of an
increase in the desired antibody level in serum, blood of mammals
that have been sensitized with the antigen is collected. Serum is
separated from the blood by a known method. As polyclonal
antibodies, serum containing polyclonal antibodies may also be
used. If necessary, a fraction containing polyclonal antibodies may
be further isolated from the serum and then used. For example,
immunoglobulin G or M can be prepared by obtaining a fraction for
recognition of only the polypeptide of the present invention using
an affinity column (with which the polypeptide of the present
invention has been coupled) and then purifying the fraction using a
protein A column or a protein G column.
[0076] To obtain a monoclonal antibody, after confirmation of an
increase in the desired antibody level in the serum of a mammal
that has been sensitized with the above antigen, immunocytes are
removed from the mammal and then subjected to cell fusion. At this
time, a particularly preferable example of such an immunocyte to be
used for cell fusion is a spleen cell. The other (parent) cell to
be fused with the above immunocyte is preferably a mammalian
myeloma cell and more preferably a myeloma cell that has acquired
properties for the selection of the resultant fusion cells through
the use of a drug.
[0077] Cell fusion of the above immunocyte with a myeloma cell can
be carried out according to basically a known method such as
Milstein et al.'s method (Galfre, G. and Milstein, C., Methods
Enzymol. (1981) 73, 3-46).
[0078] Hybridomas obtained by cell fusion are selected by culture
in a general culture solution for selection, such as an HAT culture
solution (culture solution containing hypoxanthine, aminopterin,
and thymidine). Culture in such an HAT culture solution is
continued for a time sufficient for cells (unfused cells) other
than the target hybridomas to die. In general, culture is carried
out for several days to several weeks. Next, a general limiting
dilution method is conducted. Then, screening for and cloning of
hybridomas that produce a target antibody are carried out.
[0079] The above hybridomas are obtained by immunizing non-human
animals with an antigen. In addition to this method, hybridomas
that produce a desired human antibody having activity of binding to
a polypeptide can also be obtained by in vitro sensitization of
human lymphocytes, such as human lymphocytes that have been
infected with the EB virus, with a polypeptide, a
polypeptide-expressing cell, or a lysate thereof, followed by
fusing of the thus sensitized lymphocytes with myeloma cells having
a human-derived ability to permanently divide, such as U266 (JP
Patent Publication (Kokai) No. 63-17688 A (1988)).
[0080] Subsequently, the thus obtained hybridomas are transplanted
into a mouse abdominal cavity and then ascite is collected from the
mouse. The thus obtained monoclonal antibody can be prepared by
purification using, for example, an ammonium sulfate precipitation,
a protein A column, a protein G column, DEAE ion exchange
chromatography, or an affinity column with which the polypeptide of
the present invention has been coupled. Such antibody of the
present invention is also used for purification or detection of the
polypeptide of the present invention, and is used as a candidate of
an agonist or an antagonist of the polypeptide of the present
invention.
[0081] For the purpose of, for example, lowering heterologous
antigenicity against humans, artificially altered gene recombinant
antibody such as a chimeric antibody or a humanized antibody can be
appropriately used. Such gene recombinant antibodies can be
produced using a known method. A chimeric antibody comprises the
variable region of the heavy and light chains of an antibody of a
non-human mammal such as a mouse and the constant region of the
heavy and light chains of a human antibody. DNA encoding the
variable region of a mouse antibody is ligated to DNA encoding the
constant region of a human antibody. The resultant is incorporated
into an expression vector, and then the vector is introduced into a
host to cause the host to produce the gene product. Thus a gene
recombinant antibody can be obtained. A humanized antibody is also
referred to as a reshaped human antibody. A humanized antibody is
obtained by transplanting the complementarity determining region
(CDR) of an antibody of a non-human mammal such as a mouse into the
complementarity determining region of a human antibody. General
gene recombination techniques therefor are also known.
Specifically, DNA sequences designed to have the CDR of a mouse
antibody ligated to the framework region (FR) of a human antibody
are synthesized by the PCR method from several oligonucleotides,
adjacent oligonucleotides of which have an overlap region at their
terminal portions. The thus obtained DNA is ligated to DNA encoding
the constant region of a human antibody, the resultant is
incorporated into an expression vector, and then the vector is
introduced into a host to cause the host to produce the gene
product, so that a gene recombinant antibody can be obtained (see
European Patent Application Publication No. EP 239400 and
International Patent Application Publication No. WO 96/02576). As
the FR of a human antibody, which is ligated via CDR, FR that
allows the formation of an antigen-binding site with a good
complementarity determining region is selected. If necessary, for
the formation of an antigen-binding site having the appropriate
complementarity determining region of a reshaped human antibody,
the amino acids of the framework region of an antibody variable
region may be substituted (Sato, K. et al., Cancer Res, 1993, 53,
851-856). Furthermore, methods for obtaining human antibodies are
also known. For example, a desired human antibody having activity
of binding to an antigen can also be obtained by sensitizing a
human lymphocyte in vitro with a desired antigen or a cell that
expresses a desired antigen and then fusing the sensitized
lymphocyte to a human myeloma cell such as U266 (see JP Patent
Publication (Kokoku) No. 1-59878 B (1989)). Moreover, a desired
human antibody can be obtained by immunizing a transgenic animal
that has all the repertories of a human antibody gene with a
desired antigen (see International Patent Application Publication
No. WO93/12227, WO92/03918, WO94/02602, WO94/25585, WO96/34096, and
WO96/33735). Furthermore, a technique is also known by which a
human antibody is obtained by panning using a human antibody
library. For example, the variable region of a human antibody is
expressed as a single chain antibody (scFv) on the surface of a
phage by a phage display method. A phage that binds to an antigen
can be selected. A DNA sequence encoding the variable region of a
human antibody that binds to the antigen can be determined by
analyzing the gene of the thus selected phage. When the DNA
sequence of scFv that binds to an antigen is revealed, an
appropriate expression vector is constructed based on the sequence,
and then a human antibody can be obtained. These methods are
already known. Concerning these methods, WO92/01047, WO92/20791,
WO93/06213, WO93/11236, WO93/19172, WO95/01438, and WO95/15388 can
be referred to.
[0082] Furthermore, the antibody of the present invention may be an
antibody fragment or a modified antibody product, as long as it
binds to the polypeptide of the present invention. Examples of such
antibody fragment include Fab, F(ab').sub.2, Fv, or single chain Fv
(scFv) wherein Fv of the H chain and Fv of the L chain are linked
using an appropriate linker (Huston, J. S. et al., Proc. Natl.
Acad. Sci. U.S.A. (1988) 85, 5879-5883), and a diabody.
Specifically, an antibody is treated with an enzyme such as papain
or pepsin to generate antibody fragments. Alternatively, a gene
encoding such antibody fragment is constructed, the gene is
introduced into an expression vector, and then the gene is
expressed in an appropriate host cell (e.g., see Co, M. S. et al.,
J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H.,
Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A.,
Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol.
(1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986)
121, 663-669; and Bird, R. E. and Walker, B. W., Trends Biotechnol.
(1991) 9, 132-137). A diabody is prepared by dimerization;
specifically, by linking two fragments (e.g., scFv), each of which
is prepared by linking two variable regions using a linker or the
like (hereinafter, referred to as a fragment composing a diabody).
Generally, a diabody contains two VLs and 2 VHs (P. Holliger et
al., Proc. Natl. Acad. Sci. U.S.A. 90, 6444-6448 (1993); EP404097;
WO93/11161; Johnson et al., Methods in Enzymology, 203, 88-98,
(1991); Holliger et al., Protein Engineering, 9, 299-305, (1996);
Perisic et al., Structure, 2, 1217-1226, (1994); John et al.,
Protein Engineering, 12(7), 597-604, (1999); Holliger et al., Proc.
Natl. Acad. Sci. U.S.A. 90, 6444-6448, (1993); and Atwell et al.,
Mol. Immunol. 33, 1301-1312, (1996)).
[0083] As a modified product of an antibody, antibodies to which
various molecules such as polyethylene glycol (PEG) have been bound
can also be used. The term "antibody" of the present invention also
encompasses such modified product of an antibody. Such modified
products of an antibody can be obtained by chemically modifying an
obtained antibody. Methods therefor have already been established
in the field.
[0084] Antibodies obtained as described above can be purified to a
uniform level. To separate and purify antibodies used in the
present invention, separation and purification methods that are
generally used for polypeptides may be used. Antibodies can be
separated and purified by appropriate selection or combination of,
for example, a column for chromatography such as affinity
chromatography, a filter, ultrafiltration, salting-out, dialysis,
SDS-polyacrylamide gel electrophoresis, and an isoelectric focusing
method (Antibodies: A Laboratory Manual, Ed Harlow and David Lane,
Cold Spring Harbor Laboratory, 1988). However, methods for
separation and purification are not limited to the above methods.
The concentrations of antibodies obtained above can be measured by
absorbance measurement, enzyme-linked immunosorbent assay (ELISA),
or the like.
[0085] Examples of a column to be used for affinity chromatography
include a protein A column and a protein G column. Examples of a
column using protein A include Hyper D, POROS, and Sepharose F. F.
(Pharmacia).
[0086] Examples of chromatography other than affinity
chromatography include ion exchange chromatography, hydrophobic
chromatography, gel filtration, reverse phase chromatography, and
adsorption chromatography (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual, Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press, 1996). These types of
chromatography can be carried out using liquid-phase chromatography
such as HPLC or FPLC.
[0087] As a method for measuring the antigen-binding activity of
the antibody of the present invention, absorbance measurement,
ELISA (enzyme-linked immunosorbent assay), EIA (enzyme-linked
immunoassay), RIA (radioimmunoassay), or a fluorescent antibody
technique can be used, for example, when ELISA is used, the
polypeptide of the present invention is added to a plate to which
the antibody of the present invention has been immobilized and then
a sample containing a target antibody, such as the culture
supernatant of antibody-producing cells or a purified antibody, is
added. A secondary antibody that recognizes an antibody labeled
with an enzyme such as alkaline phosphatase is added and then the
plate is incubated. After washing, an enzyme substrate such as
p-nitrophenyl phosphate is added and then absorbance is measured,
so that antigen-binding activity can be evaluated. As a
polypeptide, a polypeptide fragment such as a fragment comprising
the C-terminus of the polypeptide may also be used. For evaluation
of the activity of the antibody of the present invention, BIAcore
(produced by Pharmacia) can be used.
[0088] The method for detecting or measuring the polypeptide of the
present invention comprises contacting the antibody of the present
invention with a sample that presumably contains the polypeptide of
the present invention and detecting or measuring an immune complex
of the antibody and the polypeptide. Through the use of these
techniques, the method for detecting or measuring the polypeptide
of the present invention can be conducted. The method for detecting
or measuring the polypeptide of the present invention enables
specific detection or measurement of polypeptides. Hence, the
method is useful in various experiments and the like using
polypeptides.
[0089] The present invention also provides DNA encoding the CH
fucose transporter polypeptide or a polynucleotide comprising at
least 15 nucleotides complementary to a complementary strand of the
DNA.
[0090] Here "complementary strand" indicates a strand that is
complementary to the other strand when the two strands form a
double-strand nucleic acid; that is, a base pair consisting of A:T
(in the case of RNA, "U") or a base pair consisting of G:C.
Furthermore, "complementary" is not limited to a case where a
sequence is completely complementary to a sequence in terms of a
region consisting of at least 15 sequential nucleotides. A
complementary sequence that can be used herein has at least 70%,
preferably at least 80%, more preferably 90%, and further
preferably 95% or more homology in terms of nucleotide sequence. As
algorithm for the determination of homology, the algorithm
described in this specification may be used.
[0091] Antisense oligonucleotides used in the present invention may
be single-stranded, double-stranded, or greater
number-stranded.
[0092] Nucleotides may be DNA, RNA, or mixtures of DNA and RNA.
[0093] Such nucleic acid can be used for probes and primers to be
used for detecting or amplifying DNA that encodes the polypeptide
of the present invention, probes and primers to be used for
detecting the expression of such DNA, production of DNA chips, and
the like.
[0094] Moreover, nucleotides or nucleotide derivatives (e.g., an
antisense oligonucleotide, a ribozyme, or DNAs encoding them) for
controlling the expression of the polypeptide of the present
invention are included. When the expression of the polypeptide of
the present invention is inhibited, a target site therefor is not
specifically limited. A protein-coding region, a 5' untranslation
region, or the like can be targeted. For example, an antisense
oligonucleotide that inhibits the expression of the polypeptide of
the present invention can inhibit fucose transport into the Golgi
apparatus in CHO cells, so as to be able to inhibit the addition of
fucose to an antibody. Thus, such antisense oligonucleotide is
useful for production of an antibody having high cytotoxic
activity, and the like.
[0095] When used as a primer, the 3' side region is designed to be
a complementary region, and a restriction enzyme recognition
sequence, a tag, or the like can be added to the 5' side.
[0096] An example of an antisense oligonucleotide is an antisense
oligonucleotide that hybridizes to any position in the nucleotide
sequence of SEQ ID NO: 1 or a complementary sequence thereof.
Preferably, such antisense oligonucleotide corresponds to at least
15 sequential nucleotides in the nucleotide sequence of SEQ ID NO:
1. Further preferably, such antisense oligonucleotide is
characterized in that at least 15 sequential nucleotides contain a
translation initiation codon. Specific hybridization conditions
are, for example, the above conditions.
[0097] As antisense oligonucleotides, derivatives or modified
products thereof can be used. Examples of such modified products
include methylphosphonate-type or ethylphosphonate-type products
such as modified lower alkyl phosphonate, modified
phosphorothioate, and modified phosphoroamidate.
[0098] Examples of such antisense oligonucleotides include not only
antisense oligonucleotides (wherein all the nucleotides
corresponding to the nucleotides that compose a predetermined
region of DNA or mRNA form a complementary sequence), but also
include, as long as DNA or mRNA and oligonucleotides can
specifically hybridize to the nucleotide sequence represented by
SEQ ID NO: 1, oligonucleotides wherein a mismatch of 1 or a
plurality of nucleotides is present.
[0099] The antisense oligonucleotide derivative of the present
invention acts on cells that produce the polypeptide of the present
invention and binds to DNA or mRNA encoding the polypeptide,
thereby inhibiting transcription or translation and promoting mRNA
degradation. As a result of suppression of the expression of the
polypeptide of the present invention, the derivative has an effect
of suppressing the action of the polypeptide of the present
invention.
[0100] When RNA is used as an oligonucleotide, a phenomenon that is
generally referred to as RNA interference (RNAi) takes place. RNAi
is a phenomenon whereby when double-strand RNA (dsRNA) is
introduced into a cell, intracellular mRNA corresponding to the RNA
sequence is specifically degraded so that the gene is not expressed
as protein. In the case of RNAi, double-strand RNA is generally
used, but RNAi is not limited thereto. For example, double strands
that are formed in self-complementary single-strand RNAs can also
be used. Regarding regions where double strands are formed, double
strands may be formed in all the regions or single strands or the
like may be formed in partial regions (e.g., both ends or one end).
The length of oligo RNA to be used for RNAi is not limited. The
length of the oligo RNA of the present invention is, for example, 5
to 1000 nucleotides (in the case of double strands, 5 to 1000 bp),
preferably 10 to 100 nucleotides (in the case of double strands, 10
to 100 bp), further preferably 15 to 25 nucleotides (in the case of
double strands, 15 to 25 bp), and particularly preferably 19 to 23
nucleotides (in the case of double strands, 19 to 23 bp).
[0101] Furthermore, the present invention provides a method for
screening for a compound that binds to the polypeptide of the
present invention. Such method comprises contacting the polypeptide
of the present invention with a sample to be tested that presumably
contains a compound binding to the polypeptide, detecting binding
activity between the polypeptide and the sample to be tested, and
then selecting the compound that has activity of binding to the
polypeptide of the present invention.
[0102] Furthermore, the present invention provides a method for
screening for a substance that inhibits the fucose transport
activity, and particularly the GDP-fucose transport activity, of
the polypeptide of the present invention. Such method comprises
contacting the polypeptide of the present invention with a sample
to be tested, detecting the fucose transport activity of the
polypeptide of the present invention, and then selecting a compound
that inhibits the fucose transport activity of the polypeptide of
the present invention.
[0103] The polypeptide of the present invention that is used for
screening may be a recombinant polypeptide, a polypeptide derived
from nature, or a partial peptide. Furthermore, the polypeptide of
the present invention to be used for screening may be in a form
whereby it is expressed on the cell surface or the form of a
membrane fraction. Examples of a sample to be tested are not
specifically limited and include cell extracts, cell culture
supernatants, products of fermentation microorganisms, extracts of
marine organisms, plant extracts, purified or crudely purified
polypeptides, non-peptide compounds, synthetic low molecular weight
compounds, and natural compounds. The polypeptide of the present
invention (that is contacted with such sample to be tested) can be
contacted in the form of, for example, a purified polypeptide, a
soluble polypeptide, a polypeptide bound to a carrier, a
polypeptide that is fused with another polypeptide, a polypeptide
expressed on the cell membrane, or a membrane fraction, with a
sample to be tested.
[0104] As a method for screening for a polypeptide that binds to
the polypeptide of the present invention, many methods known by
persons skilled in the art can be used. Such screening can be
carried out by an immunoprecipitation method, for example.
Specifically, such screening can be carried out as follows. A gene
encoding the polypeptide of the present invention is inserted into
a vector for expressing a foreign gene, such as pSV2neo, pcDNA I,
or pCD8, so that the gene is expressed in animal cells or the like.
As promoters to be used for expression, any promoters that can be
generally used can be used. Examples of such promoter include an
SV40 early promoter (Rigby In Williamson (ed.), Genetic
Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)), an
EF-1.alpha. promoter (Kim et al., Gene 91, pp. 217-223 (1990)), a
CAG promoter (Niwa et al. Gene 108, pp. 193-200 (1991)), an RSV LTR
promoter (Cullen Methods in Enzymology 152, pp. 684-704 (1987)), an
SR.alpha. promoter (Takebe et al. Mol. Cell. Biol. 8, p. 466
(1988)), a CMV immediate early promoter (Seed and Aruffo Proc.
Natl. Acad. Sci. U.S.A. 84, pp. 3365-3369 (1987)), an SV40 late
promoter (Gheysen and Fiers, J. Mol. Appl. Genet. 1, pp. 385-394
(1982)), an Adenovirus late promoter (Kaufman et al. Mol. Cell.
Biol. 9, p. 946 (1989)), and an HSV TK promoter.
[0105] Examples of a method for expressing a foreign gene by
introducing the gene into animal cells include an electroporation
method (Chu, G. et al., Nucl. Acid Res. 15, 1311-1326 (1987)), a
calcium phosphate method (Chen, C. and Okayama, H. Mol. Cell. Biol.
7, 2745-2752 (1987)), a DEAE dextran method (Lopata, M. A. et al.,
Nucl. Acids Res. 12, 5707-5717 (1984); Sussman, D. J. and Milman,
G., Mol. Cell. Biol. 4, 1642-1643 (1985)), and a lipofectin method
(Derijard, B., Cell 7, 1025-1037 (1994); Lamb, B. T. et al., Nature
Genetics 5, 22-30 (1993); Rabindran, S. K. et al., Science 259,
230-234 (1993)). Any of these methods may be employed.
[0106] The polypeptide of the present invention can be expressed as
a fusion polypeptide by introducing a recognition site (epitope) of
a monoclonal antibody, the specificity of which has been clarified,
into the N- or C-terminus of the polypeptide of the present
invention. As an epitope-antibody system to be used herein, a
commercial system can be used (Experimental Medicine 13, 85-90
(1995)). A vector that enables expression of a fusion polypeptide
with .beta.-galactosidase, a maltose-binding protein,
glutathione-S-transferase, a green fluorescent protein (GFP), or
the like via a multi-cloning site is marketed.
[0107] To keep the properties of the polypeptide of the present
invention unchanged as far as possible when it is prepared in the
form of a fusion polypeptide, a method has been reported wherein
only a small epitope portion consisting of several to more than a
dozen amino acids is introduced, so as to prepare a fusion
polypeptide. For example, epitopes such as polyhistidine (His-tag),
influenza hemagglutinin HA, human c-myc, FLAG, a vesicular
stomatitis virus glycoprotein (VSV-GP), a T7 gene 10 protein
(T7-tag), a human herpes simplex virus glycoprotein (HSV-tag), and
an E-tag (an epitope on a monoclonal phage) and monoclonal
antibodies that recognize such epitopes can be used as
epitope-antibody systems for screening for polypeptides that bind
to the polypeptide of the present invention (Experimental Medicine
13, 85-90 (1995)).
[0108] In immunoprecipitation, such antibody is added to a cell
lysis solution prepared using an appropriate surfactant, so as to
form an immune complex. The immune complex comprises the
polypeptide of the present invention, a polypeptide capable of
binding thereto, and an antibody. In addition to the use of an
antibody against the above epitope, immunoprecipitation can also be
carried out using an antibody against the polypeptide of the
present invention. Such antibody against the polypeptide of the
present invention can be prepared by, for example, introducing a
gene encoding the polypeptide of the present invention into an
appropriate Escherichia coli expression vector for expression
within Escherichia coli, purifying the thus expressed polypeptide,
and then immunizing rabbits, mice, rats, goats, chickens, or the
like with the polypeptide. Moreover, such antibodies can also be
prepared by immunizing a partial peptide of the synthesized
polypeptide of the present invention with the above animals.
[0109] Immune complexes can be precipitated using Protein A
Sepharose or Protein G Sepharose, for example, if antibodies are
mouse IgG antibodies. Furthermore, when the polypeptide of the
present invention is prepared as, for example, a fusion polypeptide
with an epitope such as GST, an immune complex can also be formed
using a substance such as glutathione Sepharose 4B that
specifically binds to such epitope in a manner similar to that in a
case where an antibody of the polypeptide of the present invention
is used.
[0110] General methods for immunoprecipitation can be carried out
by or according to, for example, a method described in literature
(Harlow, E. and Lane, D.: Antibodies, pp. 511-552, Cold Spring
Harbor Laboratory Publications, New York (1988)).
[0111] SDS-PAGE is generally employed for the analysis of
immunoprecipitated polypeptides. Through the use of gel with an
appropriate concentration, a bound polypeptide can be analyzed
based on the molecular weight of the polypeptide. At this time, it
is generally difficult to detect such polypeptide that has bound to
the polypeptide of the present invention by a general staining
method for polypeptides, such as Coomassie staining or silver
staining. Detection sensitivity can be improved by culturing cells
in a culture solution containing .sup.35S-methionine or
.sup.35S-cysteine, which is a radioactive isotope, so as to label
polypeptides within the cells and then detecting them. If the
molecular amount of a polypeptide is revealed, such target
polypeptide can be directly purified from SDS-polyacrylamide gel
and then the sequence thereof can also be determined.
[0112] Furthermore, as a method for isolating a polypeptide that
binds to the polypeptide of the present invention, a West western
blotting method (Skolnik, E. Y. et al., Cell (1991) 65, 83-90), for
example, can be employed. Specifically, a cDNA library is
constructed from cells, tissues, or organs (e.g., testis) that are
predicted to express a polypeptide that binds to the polypeptide of
the present invention using a phage vector (e.g., .lamda.gt11 and
ZAP). The resultant is then expressed on LB-agarose and then the
expressed polypeptide is immobilized on a filter. The purified and
labeled polypeptide of the present invention is caused to react
with the above filter. Subsequently, plaques expressing
polypeptides binding to the polypeptide of the present invention
are detected based on the labels. Examples of a method for labeling
the polypeptide of the present invention include a method using
binding between biotin and avidin, a method using an antibody that
specifically binds to the polypeptide of the present invention or a
polypeptide (e.g., GST) fusing with the polypeptide of the present
invention, a method using a radioisotope, and a method using
fluorescence.
[0113] Another embodiment of the screening method of the present
invention is a method that is conducted using a 2-hybrid system
using cells (Fields, S., and Sternglanz, R., Trends. Genet. (1994)
10, 286-292; Dalton S, and Treisman R (1992), Characterization of
SAP-1, a protein recruited by serum response factor to the c-fos
serum response element., Cell 68, 597-612; "MATCHMAKER Two-Hybrid
System," "Mammalian MATCHMAKER Two-Hybrid Assay Kit," "MATCHMAKER
One-Hybrid System" (these systems and kits are all produced by
Clontech), and "HybriZAP Two-Hybrid Vector System" (produced by
Stratagene Corp.)).
[0114] In the 2-hybrid system, the polypeptide of the present
invention or a partial peptide thereof is fused with an SRF
DNA-binding region or a GAL4 DNA-binding region and then the
product is expressed in yeast cells. A cDNA library that is
expressed while being fused with a VP16 or GAL4 transcription
activation region is constructed from cells that are predicted to
express a polypeptide binding to the polypeptide of the present
invention. The cDNA library is then introduced into the above yeast
cells. A library-derived cDNA is isolated from a detected positive
clone. (A positive clone can be confirmed when a polypeptide that
binds to the polypeptide of the present invention is expressed
within a yeast cell, following which a reporter gene is activated
because of the binding of the two.) By introducing the isolated
cDNA into Escherichia coli for expression, the polypeptide encoded
by the cDNA can be obtained. Accordingly, a polypeptide that binds
to the polypeptide of the present invention or the gene thereof can
be prepared.
[0115] Examples of a reporter gene to be used in the 2-hybrid
system include, but are not limited to, in addition to an HIS3
gene, an Ade2 gene, a LacZ gene, a CAT gene, a luciferase gene, and
a PAI-1 (Plasminogen activator inhibitor type 1) gene. Screening by
the 2-hybrid method can also be carried out using mammalian cells
in addition to yeast.
[0116] A compound that binds to the polypeptide of the present
invention can also be screened for using affinity chromatography.
For example, the polypeptide of the present invention is
immobilized to a carrier of an affinity column and then a sample to
be tested, which is predicted to express a polypeptide that binds
to the polypeptide of the present invention, is applied. Examples
of a sample to be tested in this case include a cell extract and a
cell lysate. After application of a sample to be tested, the column
is washed, and then a polypeptide that has bound to the polypeptide
of the present invention can be prepared.
[0117] The amino acid sequence of the thus obtained polypeptide is
analyzed and then an oligo DNA is synthesized based on the
sequence. A DNA encoding the polypeptide can be obtained by
screening a cDNA library using the DNA as a probe.
[0118] Furthermore, an example of a method for isolating not only a
polypeptide but also a compound (including agonists and
antagonists) that binds to the polypeptide of the present
invention, which is known by persons skilled in the art, is a
method that involves causing a synthetic compound, a natural
product bank, or a random phage peptide display library to act on
the immobilized polypeptide of the present invention and then
screening for a molecule that binds to the polypeptide of the
present invention, or a screening method using high throughput
based on combinatorial chemistry technology (Wrighton N C; Farrell
F X; Chang R; Kashyap A K; Barbone F P; Mulcahy L S; Johnson D L;
Barrett R W; Jolliffe L K; Dower W J., Small peptides as potent
mimetics of the protein hormone erythropoietin, Science (UNITED
STATES) Jul. 26 1996, 273, pp. 458-64; Verdine G L., The
combinatorial chemistry of nature, Nature (ENGLAND) Nov. 7 1996,
384, pp. 11-13; Hogan J C Jr., Directed combinatorial chemistry,
Nature (ENGLAND) Nov. 7 1996, 384, pp. 17-9).
[0119] In the present invention, a biosensor using the surface
plasmon resonance phenomenon can also be used as a means for
detecting or measuring bound compounds. With such a biosensor,
interaction between the polypeptide of the present invention and a
compound to be tested can be observed in real time as surface
plasmon resonance signals using a fine amount of polypeptides
without labeling them (e.g., produced by BIAcore or Pharmacia).
Hence, by the use of a biosensor produced by BIAcore or the like,
binding between the polypeptide of the present invention and a
compound to be tested can be evaluated.
[0120] A method for screening for a substance that inhibits the
fucose transport activity of the polypeptide of the present
invention can be carried out by a method known by persons skilled
in the art. For example, the polypeptide of the present invention
is expressed on a membrane (e.g., cell membrane, Golgi apparatus
membrane, or viral membrane). Fucose labeled with a fluorescent
substance or the like is contacted with a substance to be tested
and then the amount of incorporated fucose is measured. Thus, a
substance that inhibits the fucose transport activity of the
polypeptide of the present invention can be screened for.
[0121] A compound that can be isolated by the screening according
to the present invention is a candidate for regulating the activity
of the polypeptide of the present invention and may be applied for
the production of an antibody with high cytotoxic activity.
[0122] "Fucose transporter" in the present invention means a
polypeptide having fucose transport activity. For example, when a
fucose transporter is expressed on the cell membrane, it generally
incorporates fucose into the cells. When a fucose transporter is
expressed on the Golgi membrane, it generally incorporates fucose
into the Golgi apparatus. In the present invention, a preferable
fucose transporter is a Chinese hamster fucose transporter, and a
more preferable example is a fucose transporter having the amino
acid sequence represented by SEQ ID NO: 2. SEQ ID NO: 1 shows the
nucleotide sequence of the Chinese hamster fucose transporter
gene.
[0123] A method for decreasing fucose existing in the Golgi
apparatus is not specifically limited. That is, such fucose can be
decreased by any method. An example of such method is a method by
which the amount of fucose to be incorporated into the Golgi
apparatus is decreased.
[0124] The Golgi apparatus incorporates fucose into itself mainly
via fucose transporters existing on the Golgi membrane. Through
inhibition of the fucose transporter functions, incorporation of
fucose into the Golgi apparatus can be inhibited and the amount of
fucose to be incorporated into the Golgi apparatus can be
decreased.
[0125] To inhibit fucose transporter functions of cells means to
cause a decrease or disappearance of the fucose transport activity
of a fucose transporter.
[0126] The fucose transporter functions of cells may be inhibited
by any method, that is, by a method known by persons skilled in the
art. Specific examples of such method include a method by which the
number of fucose transporters is decreased by inhibiting fucose
transporter expression, or the like, and a method by which the
fucose transport ability of a fucose transporter is lowered by the
use of, for example, an antagonist for the fucose transporter.
[0127] A method for inhibiting fucose transporter expression is not
specifically limited, as long as the number of fucose transporters
having normal transport ability decreases. Fucose transporter
expression can be inhibited by, for example, removal of a fucose
transporter gene contained in a genome, inhibition of the process
for transcription to mRNA, mRNA degradation, or inhibition of the
process for translation into protein. Specific examples of a method
for inhibiting the expression of a fucose transporter gene include
a method that involves deleting (knockout) a gene that encodes a
fucose transporter using a targeting vector or the like that
targets the fucose transporter, a method that uses an antisense DNA
for a gene encoding a fucose transporter, or a method that uses RNA
interference (RNAi). Cells having inhibited fucose transporters may
be cells having fucose transporter functions that have been
inhibited by any method. For use in production of pharmaceutical
products and the like, cells for which no Cre-loxp is used are
preferable as cells having highly stable chromosomes (Schumidt E.
E. et al., PNAS 97, 13702-13707 (February 2001)).
[0128] Protein produced by the production method of the present
invention may be any protein. In general, such protein is a
glycoprotein and preferably an antibody.
[0129] Types of antibody that are produced by the method of the
present invention are not specifically limited. For example, mouse
antibodies, rat antibodies, rabbit antibodies, sheep antibodies,
camel antibodies, human antibodies, and artificially altered (for
the purpose of for example, lowering heterologous antigenicity
against humans) gene recombinant antibody such as a chimeric
antibody or a humanized antibody can be appropriately used. Such
gene recombinant antibodies can be produced using a known method. A
chimeric antibody comprises the variable region of the heavy and
light chains of an antibody of a non-human mammal such as a mouse
and the constant region of the heavy and light chains of a human
antibody. DNA encoding the variable region of a mouse antibody is
ligated to DNA encoding the constant region of a human antibody.
The resultant is incorporated into an expression vector, and then
the vector is introduced into a host to cause the host to produce
the gene product. Thus a gene recombinant antibody can be obtained.
A humanized antibody is also referred to as a reshaped human
antibody. A humanized antibody is obtained by transplanting the
complementarity determining region (CDR) of an antibody of a
non-human mammal such as a mouse into the complementarity
determining region of a human antibody. General gene recombination
techniques therefor are also known. Specifically, DNA sequences
designed to have the CDR of a mouse antibody ligated to the
framework region (FR) of a human antibody are synthesized by the
PCR method from several oligonucleotides, adjacent oligonucleotides
of which have an overlap region at their terminal portions. The
thus obtained DNA is ligated to DNA encoding the constant region of
a human antibody, the resultant is incorporated into an expression
vector, and then the vector is introduced into a host to cause the
host to produce the gene product, so that a gene recombinant
antibody can be obtained (see European Patent Application
Publication No. EP 239400 and International Patent Application
Publication No. WO 96/02576). As the FR of a human antibody, which
is ligated via CDR, FR that allows the formation of an
antigen-binding site with a good complementarity determining region
is selected. If necessary, for the formation of an antigen-binding
site having the appropriate complementarity determining region of a
reshaped human antibody, the amino acids of the framework region of
an antibody variable region may be substituted (Sato, K. et al.,
Cancer Res, 1993, 53, 851-856). Furthermore, methods for obtaining
human antibodies are also known. For example, a desired human
antibody having activity of binding to an antigen can also be
obtained by sensitizing a human lymphocyte in vitro with a desired
antigen or a cell that expresses a desired antigen and then fusing
the sensitized lymphocyte to a human myeloma cell such as U266 (see
JP Patent Publication (Kokoku) No. 1-59878 B (1989)). Moreover, a
desired human antibody can be obtained by immunizing a transgenic
animal that has all the repertories of a human antibody gene with a
desired antigen (see International Patent Application Publication
No. WO93/12227, WO92/03918, WO94/02602, WO94/2558, WO96/34096, and
WO96/33735). Furthermore, a technique is also known by which a
human antibody is obtained by panning using a human antibody
library. For example, the variable region of a human antibody is
expressed as a single chain antibody (scFv) on the surface of a
phage by a phage display method. A phage that binds to an antigen
can be selected. A DNA sequence encoding the variable region of a
human antibody that binds to the antigen can be determined by
analyzing the gene of the thus selected phage. When the DNA
sequence of scFv that binds to an antigen is revealed, an
appropriate expression vector is constructed based on the sequence,
and then a human antibody can be obtained. These methods are
already known. Concerning these methods, WO92/01047, WO92/20791,
WO93/06213, WO93/11236, WO93/19172, WO95/01438, and WO95/15388 can
be referred to.
[0130] Furthermore, the antibody of the present invention may be a
lower molecular weight antibody such as an antibody fragment or a
modified product of the antibody, as long as such antibody can bind
to an antigen. Examples of such antibody fragment include Fab,
F(ab').sub.2, Fv, or single chain Fv (scFv) wherein Fv of the H
chain and Fv of the L chain are linked using an appropriate linker
(Huston, J. S. et al., Proc. Natl. Acad. Sci. U.S.A. (1988) 85,
5879-5883), and a diabody. To obtain such antibody fragment, a gene
encoding such antibody fragment is constructed, the gene is
introduced into an expression vector, and then the gene is
expressed in an appropriate host cell (e.g., see Co, M. S. et al.,
J. Immunol. (1994) 152, 2968-2976; Better, M. and Horwitz, A. H.,
Methods Enzymol. (1989) 178, 476-496; Pluckthun, A. and Skerra, A.,
Methods Enzymol. (1989) 178, 497-515; Lamoyi, E., Methods Enzymol.
(1986) 121, 652-663; Rousseaux, J. et al., Methods Enzymol. (1986)
121, 663-669; and Bird, R. E. and Walker, B. W., Trends Biotechnol.
(1991) 9, 132-137). A diabody is prepared by dimerization;
specifically, by linking two fragments (e.g., scFv), each of which
is prepared by linking two variable regions using a linker or the
like (hereinafter, referred to as a fragment composing a diabody).
Generally, a diabody contains two VLs and 2 VHs (P. Holliger et
al., Proc. Natl. Acad. Sci. U.S.A. 90, 6444-6448 (1993); EP404097;
WO93/11161; Johnson et al., Methods in Enzymology, 203, 88-98,
(1991); Holliger et al., Protein Engineering, 9, 299-305, (1996);
Perisic et al., Structure, 2, 1217-1226, (1994); John et al.,
Protein Engineering, 12(7), 597-604, (1999); Holliger et al., Proc.
Natl. Acad. Sci. U.S.A. 90, 6444-6448, (1993); and Atwell et al.,
Mol. Immunol. 33, 1301-1312, (1996)).
[0131] As a modified product of an antibody, antibodies to which
various molecules such as polyethylene glycol (PEG) have been bound
can also be used. Furthermore, a radioactive isotope, a chemical
therapeutic agent, a cytotoxic substance such as toxin derived from
bacteria, or the like can be bound to an antibody. In particular, a
radiolabeled antibody is useful. Such modified product of an
antibody can be obtained by chemically modifying an obtained
antibody. In addition, methods for modifying antibodies have
already been established in the field.
[0132] A recombinant polypeptide can be produced by a method known
by persons skilled in the art. In general, such recombinant
polypeptide can be purified and prepared as follows. DNA encoding a
polypeptide is incorporated into an appropriate expression vector,
a transformant that has been obtained by introducing the vector
into an appropriate host cell is collected, and then an extract is
obtained. Subsequently, the resultant is subjected to
chromatography such as ion exchange chromatography, reverse phase
chromatography, or gel filtration, affinity chromatography using a
column (to which an antibody against the polypeptide of the present
invention has been immobilized), or chromatography using
combination of a plurality of such columns.
[0133] Furthermore, when protein is expressed as a fusion
polypeptide with a glutathione-S-transferase protein or as a
recombinant polypeptide to which a plurality of histidines have
been added in host cells (e.g., animal cells or Escherichia coli),
the expressed recombinant polypeptide can be purified using a
glutathione column or a nickel column. After purification of the
fusion polypeptide, if necessary, regions other than the target
polypeptide can also be cleaved and removed from the fusion
polypeptide using thrombin or factor Xa.
[0134] Protein to be produced by the production method of the
present invention is preferably an antibody with cytotoxic activity
that is affected by fucose binding thereto.
[0135] A method for producing an antibody using genetic
recombination techniques, which is well known by persons skilled in
the art, involves incorporating an antibody gene into an
appropriate vector, introducing the vector into a host, and thus
causing the production of the antibody using genetic recombination
techniques (e.g., see Carl, A. K. Borrebaeck, James, W. Larrick,
THERAPEUTIC MONOCLONAL ANTIBODIES, Published in the United Kingdom
by MACMILLAN PUBLISHERS LTD, 1990).
[0136] Furthermore, the present invention encompasses a host cell
that can produce a foreign protein, wherein no fucose is added to
the foreign protein.
[0137] Such cells are characterized in that fucose existing in the
cells, such as in the Golgi apparatus, is decreased. A method for
decreasing fucose existing in the Golgi apparatus is not
specifically limited. That is, such fucose can be decreased by any
method. An example of such method is a method by which the amount
of fucose to be incorporated into the Golgi apparatus is decreased.
The Golgi apparatus incorporates fucose into itself mainly via
fucose transporters existing on the Golgi membrane. Through
inhibition of the fucose transporter functions, incorporation of
fucose into the Golgi apparatus can be inhibited and the amount of
fucose to be incorporated into the Golgi apparatus can be
decreased.
[0138] To inhibit fucose transporter functions of cells means to
cause a decrease or disappearance of the fucose transport activity
of a fucose transporter.
[0139] The fucose transporter functions of cells may be inhibited
by any method, that is, by a method known by persons skilled in the
art. Specific examples of such method include a method by which the
number of fucose transporters is decreased by inhibiting fucose
transporter expression, or the like, and a method by which the
fucose transport ability of a fucose transporter is lowered by the
use of, for example, an antagonist for the fucose transporter.
[0140] A method for inhibiting fucose transporter expression is not
specifically limited, as long as the number of fucose transporters
having normal transport ability decreases. Fucose transporter
expression can be inhibited by, for example, removal of a fucose
transporter gene contained in a genome, inhibition of the process
for transcription to mRNA, mRNA degradation, or inhibition of the
process for translation into protein. Specific examples of a method
for inhibiting the expression of a fucose transporter gene include
a method that involves deleting (knockout) a gene that encodes a
fucose transporter using a targeting vector or the like that
targets the fucose transporter, a method that uses an antisense DNA
for a gene encoding a fucose transporter, or a method that uses RNA
interference (RNAi). These methods will be described later.
[0141] Protein having no fucose binding thereto can be obtained by
expressing a foreign protein using such cells having inhibited
fucose transporter functions as host cells. Here, a foreign protein
means a protein not derived from the cell itself. Host cells are
not specifically limited. For example, cells wherein sugar is added
to a recombinant protein when the protein is expressed can be used.
More specifically, various animal cells or the like can be used.
Preferably CHO cells can be used. In the present invention, in
particular, CHO cells wherein a fucose transporter gene has been
knocked out can be appropriately used. As animal cells, mammalian
cells such as CHO (J. Exp. Med. (1995) 108, 945), COS, 3T3,
myeloma, BHK (baby hamster kidney), HeLa, and Vero are known. As
amphibian cells, Xenopus oocytes (Valle, et al., Nature (1981) 291,
358-340), for example, are known. As insect cells Sf9, Sf21, and
Tn5, for example, are known. Examples of CHO cells include dhfr-CHO
cells (Proc. Natl. Acad. Sci. U.S.A. (1980) 77, 4216-4220) and CHO
K-1 cells (Proc. Natl. Acad. Sci. U.S.A. (1968) 60, 1275), which
are deficient in a DHFR gene. For the purpose of mass-expression in
animal cells, CHO cells are particularly preferable.
[0142] Protein having no fucose binding thereto can be obtained by
incorporating a gene encoding a foreign protein such as an antibody
to be produced into an expression vector and then incorporating the
expression vector into host cells capable of producing such foreign
protein; that is, cells having inhibited fucose transporter
functions. Examples of vectors include expression vectors derived
from mammals (e.g., pcDNA3 (produced by Invitrogen Corporation) and
pEGF-BOS (Nucleic Acids. Res. 1990, 18(17), p. 5322), pEF, and
pCDM8), expression vectors derived from insect cells (e.g., the
"Bac-to-BAC baculovirus expression system" (produced by GIBCO-BRL
Life Technologies Inc.) and pBacPAK8), expression vectors derived
from plants (e.g., pMH1 and pMH2), expression vectors derived from
animal viruses (e.g., pHSV, pMV, and pAdexLcw), expression vectors
derived from retroviruses (e.g., pZlPneo), expression vectors
derived from yeast (e.g., the "Pichia Expression Kit" (produced by
Invitrogen Corporation), pNV11, and SP-Q01), and expression vectors
derived from Bacillus subtilis (e.g., pPL608 and pKTH50). When CHO
cells are used as host cells, it is preferable to use a vector
derived from a mammal.
[0143] For the purpose of expression in animal cells such as CHO
cells, COS cells, or NIH3T3 cells, generally a vector used herein
has a promoter required for expression within cells, such as an
SV40 promoter (Mulligan et al., Nature (1979) 277, 108), an
MMLV-LTR promoter, an EF1.alpha. promoter (Mizushima et al.,
Nucleic Acids Res. (1990) 18, 5322), or a CMV promoter. It is
further preferable that such vector has a gene for selection of
transformed cells (e.g., a drug resistance gene that enables
distinguishment by the use of a drug (e.g., neomycin and G418)).
Examples of a vector having such properties include pMAM, pDR2,
pBK-RSV, pBK-CMV, pOPRSV, and pOP13.
[0144] Furthermore, an example of a method for the purpose of
stable expression of a gene and amplification of the number of
copies of a gene within cells involves introducing a vector (e.g.,
pCHOI) having a complementary DHFR gene into CHO cells that are
deficient in the nucleic acid synthesis pathway, followed by
amplification using methotrexate (MTX). Furthermore, an example of
a method for the purpose of transient expression of a gene involves
transforming COS cells having a gene that expresses SV40 T antigen
on the chromosome with a vector (e.g., pcD) having an SV40
replication origin. As a replication initiation site, a site
derived from polyoma virus, adenovirus, bovine papilloma virus
(BPV), or the like can also be used. Furthermore, to amplify the
number of copies of a gene in a host cell system, an expression
vector may contain as a selection marker an aminoglycoside
transferase (APH) gene, thymidine kinase (TK) gene, Escherichia
coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene,
dihydrofolate reductase (dhfr) gene, or the like.
[0145] A vector can be introduced into a host cell by, for example,
a calcium phosphate method, a DEAE dextran method, a method using
cationic ribosome DOTAP (produced by Boehringer Mannheim), an
electroporation method, or lipofection.
[0146] Cell culture can be carried out according to a known method.
As a culture solution for animal cells, DMEM, MEM, RPMI1640, or
IMDM, for example, can be used. At this time, a serum fluid such as
fetal calf serum (FCS) can be used together therewith.
Alternatively serum-free culture may also be carried out. pH during
culture is preferably between approximately 6 and 8. Culture is
generally carried out at approximately 30.degree. C. to 40.degree.
C. for approximately 15 to 200 hours. If necessary, exchange of
media, aeration, and agitation are carried out.
[0147] Protein produced by the host cells of the present invention
may be any protein. In general, such protein is a glycoprotein and
preferably an antibody.
[0148] Types of antibody that are produced by the method of the
present invention are not specifically limited. For example, mouse
antibodies, rat antibodies, rabbit antibodies, sheep antibodies,
camel antibodies, human antibodies, and artificially altered (for
the purpose of, for example, lowering heterologous antigenicity
against humans) gene recombinant antibody such as a chimeric
antibody or a humanized antibody can be appropriately used. Such
gene recombinant antibodies can be produced using a known method.
Information about antibodies has already been described above.
[0149] An example of a cell wherein fucose transporter expression
is inhibited is a cell wherein a gene encoding a fucose transporter
is disrupted. "Disruption of a gene" means the suppression of the
expression of the gene by partial deletion, substitution,
insertion, addition, or the like conducted for the nucleotide
sequence of the gene. "Disruption of a gene" of the present
invention includes not only a case where gene expression is
completely suppressed, but also a case wherein gene expression is
partially suppressed. "Deletion (knockout) of a gene" and
"inactivation of a gene" are also used so as to have a meaning
equivalent to that of "disruption of a gene." Furthermore, cells
having a gene disrupted by homologous recombination using gene
targeting are referred to as gene-knock out cells. A cell wherein a
gene encoding a fucose transporter is disrupted is an example of a
cell wherein fucose transporter expression is artificially
suppressed. A cell wherein a gene encoding a fucose transporter is
disrupted is a cell wherein the amount of fucose existing in the
Golgi apparatus is significantly decreased compared with that in a
cell wherein a fucose transporter gene is not disrupted, a cell
wherein intracellular fucose transport ability is lowered or
deleted, or a cell wherein intracellular activity to incorporate
fucose into the Golgi apparatus is lowered or eliminated. The
amount of fucose in the Golgi apparatus can be measured by
isolating the Golgi apparatus from a cell, extracting sugar, and
then carrying out an antigen antibody reaction, a binding reaction
between sugar and lectin, liquid chromatography, electrophoresis,
or the like. Moreover, intracellular fucose transport ability and
intracellular activity to incorporate fucose into the Golgi
apparatus can be measured by, for example, using fucose labeled
with a fluorescent substance, a radioisotope, or the like.
[0150] A gene can be disrupted by, for example, a homologous
recombination method.
[0151] Such homologous recombination method means a method by which
only the target gene is arbitrarily altered by homologous gene
recombination between a gene on a chromosome and a foreign DNA.
Another DNA sequence is inserted into an exon of a gene for the
purpose of dividing a sequence encoding a protein. To facilitate
identification of a cell having a gene targeting vector, a
selection marker such as a neomycin resistance gene derived from a
bacterium is generally used as a sequence to divide the gene. A
targeting vector is designed and produced based on the sequence
information of the fucose transporter gene described in this
specification and the fucose transporter gene to be disrupted is
then subjected to homologous recombination using the targeting
vector. For example, a substitution vector can contain a homologous
region that has been ligated to the 5' and the 3' side of mutation
to be introduced, a positive selection marker, a restriction enzyme
site for linearizing the vector outside the homologous region, a
negative selection marker arranged outside the homologous region, a
restriction enzyme cleavage site for detecting mutation, and the
like. Targeting vectors can be produced according to methods
described in, for example, edited by Kenichi Yamamura et al.,
Transgenic Animal, KYORITSU SHUPPAN CO., LTD., Mar. 1, 1997;
Shinichi Aizawa, Gene Targeting, Production of Mutant Mice using ES
cells, Bio Manual Series 8, YODOSHA CO., LTD., 1995; Hogan et al.,
Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press
(1944); Joyner, A. L., Gene Targeting, A Practical Approach Series,
IRL Press (1993); and edited by Masami Matsumura et al.,
Experimental Medicine, Separate Volume, New Genetic Engineering
Handbook (3rd revised version), YODOSHA CO., LTD., 1999. Both
insertion and substitution targeting vectors may be used.
Furthermore, recombination can also be caused by targeting using a
Cre-lox system. Targeting using the Cre-lox system can be carried
out according to a method described in, for example, JP Patent
Publication (Kohyo) NO. 11-503015 A (1999). As a method for
selecting homologous recombinants that have experienced homologous
recombination, a known selection method such as positive selection,
promoter selection, negative selection, or polyA selection may be
used. For identification of a homologous recombinant, both the PCR
method and the Southern blotting method can be used.
[0152] In addition, examples of a method for producing a cell
wherein fucose transporter expression is inhibited include an
antisense method, a ribozyme method, a method using a retrovirus, a
method using a transposon, an RNAi method, an RDO method, a TFO
method, and a method for obtaining established cells from mammals
wherein a fucose transporter gene has been knocked out.
[0153] The antisense method is a method for inhibiting fucose
transporter translation in cells using an antisense oligonucleotide
of the fucose transporter gene of the present invention. An example
of an antisense oligonucleotide is an antisense oligonucleotide
that hybridizes to any position in the nucleotide sequence of SEQ
ID NO: 1 or a complementary sequence thereof. For example, such
antisense oligonucleotide generally corresponds to at least 15
sequential nucleotides in the nucleotide sequence of SEQ ID NO: 1.
Further preferably, such antisense oligonucleotide is characterized
in that at least 15 sequential nucleotides contain a translation
initiation codon. Examples of such antisense oligonucleotides
include not only antisense oligonucleotides (wherein all the
nucleotides corresponding to the nucleotides that compose a
predetermined region of DNA or mRNA form a complementary sequence),
but also include, as long as DNA or mRNA and oligonucleotides can
specifically hybridize to the nucleotide sequence represented by
SEQ ID NO: 1, oligonucleotides wherein a mismatch of 1 or a
plurality of nucleotides is present. Specific conditions for
hybridization are, for example, low stringent conditions. Such low
stringent conditions comprise, upon washing after hybridization,
for example, 42.degree. C., 0.1.times.SSC, and 0.1% SDS, and
preferably 50.degree. C., 0.1.times.SSC, and 0.1% SDS. More
preferable hybridization conditions are, for example, high
stringent conditions. Such high stringent conditions comprise, for
example, 65.degree. C., 5.times.SSC, and 0.1% SDS. However, a
plurality of factors such as temperature and salt concentration may
influence the stringency concerning hybridization. Persons skilled
in the art can realize stringency similar to the above stringency
by appropriately selecting these factors.
[0154] As antisense oligonucleotides, derivatives or modified
products thereof can be used. Examples of such modified products
include methylphosphonate-type or ethylphosphonate-type products
such as modified lower alkyl phosphonate, modified
phosphorothioate, and modified phosphoroamidate.
[0155] Such antisense oligonucleotide acts on cells that produce
the polypeptide of the present invention and binds to DNA or mRNA
encoding the polypeptide, thereby inhibiting transcription or
translation and promoting mRNA degradation. As a result of
suppression of the expression of the polypeptide of the present
invention, the antisense oligonucleotide has an effect of
suppressing the action of the polypeptide of the present
invention.
[0156] An antisense oligonucleotide is inserted downstream of a
promoter of an appropriate expression vector, and then host cells
can also be transformed with the expression vector.
[0157] A ribozyme method involves cleaving mRNA of a fucose
transporter gene in cells using RNA having activity to cleave
nucleic acids, so as to avoid the expression of the gene. A
ribozyme comprises a recognition site complementary to a substrate
RNA, a loop-shaped enzyme active site, and a stem II region
accompanying the enzyme active site. Such recognition site may be
designed so as to become complementary to a part of the fucose
transporter gene of the present invention. In a manner similar to
that in the above antisense method, a ribozyme is inserted
downstream of a promoter of an appropriate expression vector that
enables expression of the ribozyme. Host cells are then transformed
with the expression vector. The ribozyme method can be carried out
according to the descriptions in Cell Technology, 12, 239 (1993);
BIO/TECHNOLOGY, 17, 1097 (1999); Hum. Mol. Genet., 5, 1083 (1995);
Cell Technology, 13, 255 (1994); and Proc. Natl. Acad. Sci, U.S.A.
96, 1886 (1999). Screening for cells that have become unable to
produce any fucose transporter because of the antisense method or
the ribozyme method may be carried out using fucose transporter
activity as an index. Alternatively, such screening can also be
carried out by Western blotting or Northern blotting using fucose
transporter gene transcription or expression as an index.
[0158] Furthermore, a fucose transporter gene can be disrupted
using a retrovirus. A retrovirus is introduced into host cells by
infecting the host cells with the retrovirus. Then, cells having
disrupted fucose transporter genes are screened for. Thus, cells
not having fucose transporter activity can be obtained. Cells may
be screened for using fucose transporter activity as an index.
Alternatively, cells may also be screened for by Western blotting
or Northern blotting using fucose transporter gene transcription or
expression as an index.
[0159] Furthermore, a fucose transporter gene is disrupted in a
similar manner using a transposon. Then, cells having disrupted
fucose transporter genes are obtained by screening. The thus
obtained cells may also be used for antibody production. A
transposon system may be constructed according to a method
described in NatureGent, 25, 35, (2000) or the like.
[0160] Furthermore, cells wherein the expression of the fucose
transporter of the present invention is inhibited can also be
obtained using RNA interference (RNAi). RNAi is a phenomenon
whereby when double-strand RNA (dsRNA) is introduced into a cell,
intracellular mRNA corresponding to the RNA sequence is
specifically degraded so that the gene is not expressed as protein.
In the case of RNAi, double-strand RNA is generally used, but RNAi
is not limited thereto. For example, double strands that are formed
in self-complementary single-strand RNAs can also be used.
Regarding regions where double strands are formed, double strands
may be formed in all the regions or single strands or the like may
be formed in partial regions (e.g., both ends or one end). The
length of oligo RNA to be used for RNAi is not limited. The length
of the oligo RNA of the present invention is, for example, 5 to
1000 nucleotides (in the case of double strands, 5 to 1000 bp),
preferably 10 to 100 nucleotides (in the case of double strands, 10
to 100 bp), further preferably 15 to 25 nucleotides (in the case of
double strands, 15 to 25 bp), and particularly preferably 19 to 23
nucleotides (in the case of double strands, 19 to 23 bp).
[0161] As described above, the RNAi method makes use of a
phenomenon whereby a double-strand RNA (dsRNA) being homologous to
a gene and consisting of sense RNA and antisense RNA disrupts a
homologous portion of a gene transcription product (mRNA). A
double-strand RNA corresponding to the full-length sequence of a
fucose transporter gene to be used herein may be used or a short
(e.g., 21 to 23 b) dsRNA (small interfering RNA; siRNA)
corresponding to a partial sequence may also be used. A
double-strand RNA may be directly incorporated into a cell.
Alternatively, a vector producing a double-strand RNA is
constructed, the vector is introduced into a host cell, and then
the double-strand RNA may be produced within the cell. For example,
the whole or a portion of the DNA encoding the fucose transporter
of the present invention is incorporated into a vector such that it
becomes an inverted repeat sequence, and then the vector may be
introduced into a host cell. The RNAi method can be conducted
according to the descriptions in Nature, 391, 806, (1998); Proc.
Natl. Acsd. Sci. U.S.A. 95, 15502 (1998); Nature, 395, 854, (1998);
Proc. Natl. Acsd. Sci. U.S.A. 96, 5049, (1999); Cell, 95, 1017,
(1998); Proc. Natl. Acsd. Sci. U.S.A. 96, 1451, (1999); Proc. Natl.
Acsd. Sci. U.S.A. 95, 13959, (1998); Nature Cell Biol., 2, 70,
(2000); and the like. Screening for cells that have become unable
to produce any fucose transporter as a result of the use of the
RNAi method may be carried out using fucose transporter activity as
an index. Alternatively, such screening can also be carried out by
Western blotting or Northern blotting using fucose transporter gene
transcription or expression as an index.
[0162] The fucose transporter of the present invention can also be
disrupted by an RDO method or a TFO method. RDO (chimeric RNA-DNA
oligonucleotide) is a double strand formed by binding a DNA strand
to an RNA strand, and is characterized by having a GC clamp and a T
loop. Through the use of RDO corresponding to a fucose transporter
gene, mutation can be introduced into the fucose transporter gene
and the gene can be disrupted. RDO can be constructed according to
the descriptions in Science, 273, 1386, (1996); Nature Medicine, 4,
285, (1998); Hepatology, 25, 1462, (1997); Gene Therapy, 5, 1960,
(1999); J. Mol. Med., 75, 829, (1997); Proc. Natl. Acsd. Sci.
U.S.A. 96, 8774, (1999); Proc. Natl. Acsd. Sci. U.S.A. 96, 8768,
(1999); Nuc. Acids. Res., 27, 1323, (1999); Invest, Dematol., 111,
1172, (1998); Nature Biotech., 16, 1343, (1998), Nature Biotech.,
18, 43, (2000); Nature Biotech., 18, 555, (2000); J. Mol. Med., 80,
620, (2002); and the like. A triplex-forming oligonucleotide (TFO)
is a short single-strand DNA segment that can bind to a specific
site of double-strand genomic DNA and can induce mutation at its
binding site. TFO can be constructed according to the descriptions
in J. Mol. Med., 80, 620, (2002) and the like.
[0163] Furthermore, a cell wherein the fucose transporter gene of
the present invention is disrupted can also be obtained by randomly
introducing mutation into a cell. Examples of a method for randomly
introducing mutation into a cell include a method that involves
randomly introducing a gene disruption vector containing a marker
into the genome of a cell and then screening for a cell having a
disrupted fucose transporter gene, and a method that involves
randomly introducing mutation using an chemical mutagen such as ENU
(N-ethyl-N-nitrosourea) and then screening for such cell having a
disrupted fucose transporter gene. Screening for cells that have
become unable to produce any fucose transporter may be carried out
using fucose transporter activity as an index. Alternatively, such
screening can also be carried out by Western blotting or Northern
blotting using fucose transporter gene transcription or expression
as an index.
[0164] Furthermore, the cell of the present invention having a
disrupted fucose transporter gene can also be obtained from an
animal having a knocked-out fucose transporter gene. Such animal
having a knocked-out fucose transporter gene can be produced by
disrupting a fucose transporter of an ES cell by the above method
and then producing from the ES cell according to, for example, a
method disclosed in WO02/33054 Publication. Examples of animals
that are used in this case include, but are not limited to, goats,
pigs, sheep, cattle, mice, hamsters, and rats. Established cells
having no fucose transporter genes can be obtained by producing
such established cells from animals having a knocked-out fucose
transporter gene.
[0165] Cells wherein fucose transport ability is lowered or
disappears can be obtained by various methods. For example, such
cells can be obtained by inhibiting fucose transporter functions
using a compound (specifically, an antagonist for the fucose
transporter) that binds to the fucose transporter and then inhibits
fucose transport from the cytoplasm into the Golgi apparatus. The
present invention also encompasses a method for inhibiting cellular
fucose transporter functions using such compound and cells wherein
fucose transporter functions are inhibited by such compound. Cells
wherein fucose transporter functions are inhibited are cells
wherein the amount of fucose existing in the Golgi apparatus is
significantly decreased compared with cells wherein fucose
transporter functions are not inhibited. Furthermore, cells wherein
fucose transporter functions are inhibited are cells wherein fucose
transport ability is lowered or eliminated in the Golgi membrane.
Such cells also mean cells wherein intracellular activity to
incorporate fucose into the Golgi apparatus is lowered or
eliminated. Examples of such compound that inhibits fucose
transporter functions include a compound that is isolated by the
above screening method and an antibody that binds to fucose
transporter activity. Such compound may be added to a medium for
host cells that are caused to produce a recombinant protein.
Moreover, when such compound is protein, DNA encoding the protein
is introduced into an appropriate expression vector, host cells are
transformed with the expression vector, and then the protein can be
expressed and produced in host cells.
[0166] When foreign recombinant protein is produced in host cells
having disrupted fucose transporter genes or the fucose transport
activity of the fucose transporter of the present invention is
inhibited, intracellular fucose is not transported into the Golgi
apparatus. Thus, fucose is not added to protein. In the case of
such recombinant protein produced in host cells having disrupted
fucose transporter genes, the amount of bound fucose is
significantly lower or preferably unable to be detected, compared
with the case of recombinant protein produced in host cells having
an undisrupted fucose transporter gene. When a foreign protein is
an antibody, a product can be obtained wherein no fucose is binding
to an N-glycoside-bound sugar chain binding to 2
sugar-chain-binding sites existing in 1 molecule of an antibody;
that is, existing in the Fc region composed of 2 H chains. Such
antibody having no fucose binding thereto has enhanced cytotoxic
activity. Incorporation of an antibody gene in a cell can be
carried out by a general genetic engineering technique. In
addition, when an antibody for which the addition of fucose thereto
is inhibited is produced using the cell of the present invention,
it is not necessary that all the produced antibodies experience the
addition of fucose thereto. The proportion of protein to which
fucose has been added in antibody compositions should be
reduced.
[0167] Furthermore, the present invention also encompasses animals
(excluding humans) wherein fucose transporter gene expression is
inhibited. A recombinant polypeptide can be produced in vivo using
such animals. An example of such animal wherein fucose transporter
gene expression is inhibited is the above fucose
transporter-knockout animal. Production of knockout animals wherein
a specific gene is knocked-out as described above is already a
well-known technique. Persons skilled in the art can appropriately
produce such fucose transporter gene-knockout animals. Moreover,
animals wherein fucose transporter gene expression is inhibited can
be produced by, for example, introducing a gene expressing an
antisense oligonucleotide for a fucose transporter.
[0168] DNA encoding target protein is introduced into these
animals, polypeptides are produced in vivo within the animals, and
then the polypeptides are collected. The term "host" in the present
invention encompasses these animals and the like. When animals are
used, there are production systems using mammals and production
systems using insects. As mammals, goats, pigs, sheep, mice, or
cattle can be used (Vicki Glaser, SPECTRUM Biotechnology
Applications, 1993).
[0169] For example, a target DNA is prepared in the form of a
fusion gene with a gene encoding a polypeptide such as goat 13
casein that is uniquely produced in milk. Subsequently, a DNA
fragment comprising the fusion gene is injected into a goat embryo
and then the embryo is transplanted into a female goat. A target
polypeptide can be obtained from milk that is produced by
transgenic goats born from goats that have accepted such embryos or
from the progenies of such transgenic goats. To increase the amount
of milk containing polypeptides, which is produced by transgenic
goats, an appropriate hormone may also be used for such transgenic
goats (Ebert, K. M. et al., Bio/Technology (1994) 12, 699-702).
[0170] The thus obtained polypeptide can be isolated from within
host cells or outside the cells (e.g., media) and then purified as
a substantially pure and uniform polypeptide. To separate and
purify polypeptides, separation and purification methods that are
generally used for polypeptide purification may be employed and are
not specifically limited. For example, polypeptides can be
separated and purified by the use of appropriate selection and
combination of a chromatography column, a filter, ultrafiltration,
salting-out, solvent precipitation, solvent extraction,
distillation, immunoprecipitation, SDS-polyacrylamide gel
electrophoresis, an isoelectric focusing method, dialysis,
recrystallization, and the like.
[0171] Examples of chromatography include affinity chromatography,
ion exchange chromatography, hydrophobic chromatography, gel
filtration, reverse phase chromatography, and adsorption
chromatography (Strategies for Protein Purification and
Characterization: A Laboratory Course Manual, Ed Daniel R. Marshak
et al., Cold Spring Harbor Laboratory Press, 1996). These types of
chromatography can be carried out using liquid-phase chromatography
such as HPLC or FPLC.
[0172] In addition, when a proper protein modification enzyme is
caused to act on polypeptides before or after purification,
modification can be arbitrarily carried out or peptides can be
partially removed. As protein modification enzymes, trypsin,
chymotrypsin, lysylendopeptidase, protein kinase, and glucosidase,
for example, are used.
[0173] A known sequence can also be used for a gene encoding the H
chain or the L chain of an antibody that is produced by the
production method of the present invention. Furthermore, such gene
can also be obtained by a method known by persons skilled in the
art. For example, a gene encoding an antibody can be cloned and
obtained from a hybridoma producing a monoclonal antibody or can
also be obtained from an antibody library. Hybridomas can be
produced basically using a known technique as described below.
Specifically, a hybridoma can be produced by using as a sensitizing
antigen a desired antigen or a cell that expresses a desired
antigen, carrying out immunization according to a general
immunization method using such antigen, fusing the thus obtained
immunocyte with a known parent cell by a general cell fusion
method, and then screening for a monoclonal antibody-producing cell
(hybridoma) by a general screening method. The cDNA of an antibody
variable region (V region) is synthesized from the mRNA of the thus
obtained hybridoma using reverse transcriptase. The cDNA is ligated
to DNA encoding a desired antibody constant region (C region), so
that a gene encoding the H chain or the L chain can be obtained. A
sensitizing antigen upon immunization is not specifically limited.
For example, the full-length protein of a target receptor, a
partial peptide (e.g., extracellular region), and the like can be
used. Antigens can be prepared by a method known by persons skilled
in the art. For example, antigens can be prepared according to a
method using baculovirus (e.g., WO98/46777). Hybridomas can be
produced according to, for example, Milstein et al's method
(Kohler, G., and Milstein, C., Methods Enzymol. 1981, 73, 3-46) or
the like. When the immunogenicity of an antigen is low, such
antigen is bound to a macromolecule having immunogenicity, such as
albumin, and then immunization is carried out.
[0174] Regarding an antibody library, many antibody libraries are
already known. In addition, a production method for an antibody
library is known. Hence, persons skilled in the art can
appropriately obtain an antibody library.
[0175] Antibodies that are expressed and produced as described
above can be purified by a known method that is used for general
protein purification. Antibodies can be separated and purified by
appropriate selection or combination of, for example, an affinity
column (e.g., protein A column), a chromatography column, a filter,
ultrafiltration, salting-out, and dialysis (Antibodies: A
Laboratory Manual, Ed Harlow and David Lane, Cold Spring Harbor
Laboratory, 1988).
[0176] A known means can be used for measuring the antigen-binding
activity (Antibodies A Laboratory Manual, Ed Harlow, David Lane,
Cold Spring Harbor Laboratory, 1988). For example, ELISA
(enzyme-linked immunosorbent assay), EIA (enzyme-linked
immunoassay), RIA (radioimmunoassay), or a fluorescent immunoassay
can be used.
[0177] The sugar chain structure of protein produced using the cell
of the present invention can be analyzed by a method described in a
2-dimensional sugar chain mapping method (Anal. Biochem, 171, 73
(1988); Biochemical Experimental Methods 23-Methods for Studying
Glycoprotein Sugar Chains, edited by Reiko Takahashi, Center for
Academic Publications Japan (1989)). Moreover, sugar chains can
also be analyzed by mass spectrometry such as MALDI-TOF-MS.
[0178] A compound that binds to a fucose transporter and then
inhibits fucose transport from the cytoplasm into the Golgi
apparatus can be screened for by the following method.
Specifically, a fucose transporter is contacted with a sample to be
tested that presumably contains a compound binding to the fucose
transporter, and then the binding activity between the polypeptide
and the sample to be tested is detected, so that a compound having
activity of binding to the fucose transporter can be selected.
[0179] Furthermore, a fucose transporter is contacted with a sample
to be tested and then the fucose transport activity of the fucose
transporter is detected, so that a compound that inhibits the
fucose transport activity of the fucose transporter can be
selected.
[0180] The polypeptide of the present invention that is used for
screening may be a recombinant polypeptide, a polypeptide derived
from nature, or a partial peptide. Furthermore, the polypeptide of
the present invention to be used for screening may be in a form
whereby it is expressed on the cell surface or the form of a
membrane fraction. Examples of a sample to be tested are not
specifically limited and include cell extracts, cell culture
supernatants, products of fermentation microorganisms, extracts of
marine organisms, plant extracts, purified or crudely purified
polypeptides, non-peptide compounds, synthetic low molecular weight
compounds, and natural compounds. The polypeptide of the present
invention (that is contacted with such sample to be tested) can be
contacted in the form of, for example, a purified polypeptide, a
soluble polypeptide, a polypeptide bound to a carrier, a
polypeptide that is fused with another polypeptide, a polypeptide
expressed on the cell membrane, or a membrane fraction, with a
sample to be tested.
[0181] As a method for screening for a polypeptide that binds to
the polypeptide of the present invention, many methods known by
persons skilled in the art can be used. Such screening can be
carried out by an immunoprecipitation method, for example.
Specifically, such screening can be carried out as follows. A gene
encoding the polypeptide of the present invention is inserted into
a vector for expressing a foreign gene, such as pSV2neo, pcDNA I,
or pCD8, so that the gene is expressed in animal cells or the like.
As promoters to be used for expression, any promoters that can be
generally used can be used. Examples of such promoter include an
SV40 early promoter (Rigby In Williamson (ed.), Genetic
Engineering, Vol. 3. Academic Press, London, p. 83-141 (1982)), an
EF-1.alpha. promoter (Kim et al., Gene 91, pp. 217-223 (1990)), a
CAG promoter (Niwa et al. Gene 108, pp. 193-200 (1991)), an RSV LTR
promoter (Cullen Methods in Enzymology 152, pp. 684-704 (1987)), an
SR.alpha. promoter (Takebe et al. Mol. Cell. Biol. 8, p. 466
(1988)), a CMV immediate early promoter (Seed and Aruffo Proc.
Natl. Acad. Sci. U.S.A. 84, pp. 3365-3369 (1987)), an SV40 late
promoter (Gheysen and Fiers, J. Mol. Appl. Genet. 1, pp. 385-394
(1982)), an Adenovirus late promoter (Kaufman et al. Mol. Cell.
Biol. 9, p. 946 (1989)), and an HSV TK promoter.
[0182] Examples of a method for expressing a foreign gene by
introducing the gene into animal cells include an electroporation
method (Chu, G. et al., Nucl. Acid Res. 15, 1311-1326 (1987)), a
calcium phosphate method (Chen, C. and Okayama, H., Mol. Cell.
Biol. 7, 2745-2752 (1987)), a DEAE dextran method (Lopata, M. A. et
al. Nucl. Acids Res. 12, 5707-5717 (1984); Sussman, D. J. and
Milman, G., Mol. Cell. Biol. 4, 1642-1643 (1985)), and a lipofectin
method (Derijard, B., Cell 7, 1025-1037 (1994); Lamb, B. T. et al.,
Nature Genetics 5, 22-30 (1993); Rabindran, S. K. et al., Science
259, 230-234 (1993)). Any of these methods may be employed.
[0183] The polypeptide of the present invention can be expressed as
a fusion polypeptide by introducing a recognition site (epitope) of
a monoclonal antibody, the specificity of which has been clarified,
into the N- or C-terminus of the polypeptide of the present
invention. As an epitope-antibody system to be used herein, a
commercial system can be used (Experimental Medicine 13, 85-90
(1995)). A vector that enables expression of a fusion polypeptide
with .beta.-galactosidase, a maltose-binding protein,
glutathione-S-transferase, a green fluorescent protein (GFP), or
the like via a multi-cloning site is marketed.
[0184] To keep the properties of the polypeptide of the present
invention unchanged as far as possible when it is prepared in the
form of a fusion polypeptide, a method has been reported wherein
only a small epitope portion consisting of several to more than a
dozen amino acids is introduced, so as to prepare a fusion
polypeptide. For example, epitopes such as polyhistidine (His-tag),
influenza hemagglutinin HA, human c-myc, FLAG, a vesicular
stomatitis virus glycoprotein (VSV-GP), a T7 gene 10 protein
(T7-tag), a human herpes simplex virus glycoprotein (HSV-tag), and
an E-tag (an epitope on a monoclonal phage) and monoclonal
antibodies that recognize such epitopes can be used as
epitope-antibody systems for screening for polypeptides that bind
to the polypeptide of the present invention (Experimental Medicine
13, 85-90 (1995)).
[0185] In immunoprecipitation, such antibody is added to a cell
lysis solution prepared using an appropriate surfactant, so as to
form an immune complex. The immune complex comprises the
polypeptide of the present invention, a polypeptide capable of
binding thereto, and an antibody. In addition to the use of an
antibody against the above epitope, immunoprecipitation can also be
carried out using an antibody against the polypeptide of the
present invention. Such antibody against the polypeptide of the
present invention can be prepared by, for example, introducing a
gene encoding the polypeptide of the present invention into an
appropriate Escherichia coli expression vector for expression
within Escherichia coli, purifying the thus expressed polypeptide,
and then immunizing rabbits, mice, rats, goats, chickens, or the
like with the polypeptide. Moreover, such antibodies can also be
prepared by immunizing a partial peptide of the synthesized
polypeptide of the present invention with the above animals.
[0186] Immune complexes can be precipitated using Protein A
Sepharose or Protein G Sepharose, for example, if antibodies are
mouse IgG antibodies. Furthermore, when the polypeptide of the
present invention is prepared as, for example, a fusion polypeptide
with an epitope such as GST, an immune complex can also be formed
using a substance such as glutathione Sepharose 4B that
specifically binds to such epitope in a manner similar to that in a
case where an antibody of the polypeptide of the present invention
is used.
[0187] General methods for immunoprecipitation can be carried out
by or according to, for example, a method described in literature
(Harlow, E. and Lane, D.: Antibodies, pp. 511-552, Cold Spring
Harbor Laboratory Publications, New York (1988)).
[0188] SDS-PAGE is generally employed for the analysis of
immunoprecipitated polypeptides. Through the use of gel with an
appropriate concentration, a bound polypeptide can be analyzed
based on the molecular weight of the polypeptide. At this time, it
is generally difficult to detect such polypeptide that has bound to
the polypeptide of the present invention by a general staining
method for polypeptides, such as Coomassie staining or silver
staining. Detection sensitivity can be improved by culturing cells
in a culture solution containing .sup.35S-methionine or
.sup.35S-cysteine, which is a radioactive isotope, so as to label
polypeptides within the cells and then detecting them. If the
molecular amount of a polypeptide is revealed, such target
polypeptide can be directly purified from SDS-polyacrylamide gel
and then the sequence thereof can also be determined.
[0189] Furthermore, as a method for isolating a polypeptide that
binds to the polypeptide of the present invention, an West western
blotting method (Skolnik, E. Y. et al., Cell (1991) 65, 83-90), for
example, can be employed. Specifically, a cDNA library is
constructed from cells, tissues, or organs (e.g., testis) that are
predicted to express a polypeptide that binds to the polypeptide of
the present invention using a phage vector (e.g., .lamda.gt11 and
ZAP). The resultant is then expressed on LB-agarose and then the
expressed polypeptide is immobilized on a filter. The purified and
labeled polypeptide of the present invention is caused to react
with the above filter. Subsequently, plaques expressing
polypeptides binding to the polypeptide of the present invention
are detected based on the labels. Examples of a method for labeling
the polypeptide of the present invention include a method using
binding between biotin and avidin, a method using an antibody that
specifically binds to the polypeptide of the present invention or a
polypeptide (e.g., GST) fusing with the polypeptide of the present
invention, a method using a radioisotope, and a method using
fluorescence.
[0190] Another embodiment of the screening method of the present
invention is a method that is conducted using a 2-hybrid system
using cells (Fields, S., and Sternglanz, R., Trends. Genet. (1994)
10, 286-292; Dalton S, and Treisman R (1992), Characterization of
SAP-1, a protein recruited by serum response factor to the c-fos
serum response element., Cell 68, 597-612; "MATCHMAKER Two-Hybrid
System," "Mammalian MATCHMAKER Two-Hybrid Assay Kit," "MATCHMAKER
One-Hybrid System" (these systems and kits are all produced by
Clontech), and "HybriZAP Two-Hybrid Vector System" (produced by
Stratagene Corp.)).
[0191] In the 2-hybrid system, the polypeptide of the present
invention or a partial peptide thereof is fused with an SRF
DNA-binding region or a GAL4 DNA-binding region and then the
product is expressed in yeast cells. A cDNA library that is
expressed while being fused with a VP16 or GAL4 transcription
activation region is constructed from cells that are predicted to
express a polypeptide binding to the polypeptide of the present
invention. The cDNA library is then introduced into the above yeast
cells. A library-derived cDNA is isolated from a detected positive
clone. (A positive clone can be confirmed when a polypeptide that
binds to the polypeptide of the present invention is expressed
within a yeast cell, following which a reporter gene is activated
because of the binding of the two.) By introducing the isolated
cDNA into Escherichia coli for expression, the polypeptide encoded
by the cDNA can be obtained. Accordingly, a polypeptide that binds
to the polypeptide of the present invention or the gene thereof can
be prepared.
[0192] Examples of a reporter gene to be used in the 2-hybrid
system include, but are not limited to, in addition to an HISS
gene, an Ade2 gene, a LacZ gene, a CAT gene, a luciferase gene, and
a PAI-1 (Plasminogen activator inhibitor type 1) gene. Screening by
the 2-hybrid method can also be carried out using mammalian cells
in addition to yeast.
[0193] A compound that binds to the polypeptide of the present
invention can also be screened for using affinity chromatography.
For example, the polypeptide of the present invention is
immobilized to a carrier of an affinity column and then a sample to
be tested, which is predicted to express a polypeptide that binds
to the polypeptide of the present invention, is applied. Examples
of a sample to be tested in this case include a cell extract and a
cell lysate. After application of a sample to be tested, the column
is washed, and then a polypeptide that has bound to the polypeptide
of the present invention can be prepared.
[0194] The amino acid sequence of the thus obtained polypeptide is
analyzed and then an oligo DNA is synthesized based on the
sequence. A DNA encoding the polypeptide can be obtained by
screening a cDNA library using the DNA as a probe.
[0195] Furthermore, an example of a method for isolating not only a
polypeptide but also a compound (including agonists and
antagonists) that binds to the polypeptide of the present
invention, which is known by persons skilled in the art, is a
method that involves causing a synthetic compound, a natural
product bank, or a random phage peptide display library to act on
the immobilized polypeptide of the present invention and then
screening for a molecule that binds to the polypeptide of the
present invention, or a screening method using high throughput
based on combinatorial chemistry technology (Wrighton N C; Farrell
F X; Chang R; Kashyap A K; Barbone F P; Mulcahy L S; Johnson D L;
Barrett R W; Jolliffe L K; Dower W J., Small peptides as potent
mimetics of the protein hormone erythropoietin, Science (UNITED
STATES) Jul. 26 1996, 273 pp. 458-64; Verdine G L., The
combinatorial chemistry of nature, Nature (ENGLAND) Nov. 7 1996,
384, pp. 11-13; Hogan J C Jr., Directed combinatorial chemistry,
Nature (ENGLAND) Nov. 7 1996, 384, pp. 17-9).
[0196] In the present invention, a biosensor using the surface
plasmon resonance phenomenon can also be used as a means for
detecting or measuring bound compounds. With such a biosensor,
interaction between the polypeptide of the present invention and a
compound to be tested can be observed in real time as surface
plasmon resonance signals using a fine amount of polypeptides
without labeling them (e.g., produced by BIAcore or Pharmacia).
Hence, by the use of a biosensor produced by BIAcore or the like,
binding between the polypeptide of the present invention and a
compound to be tested can be evaluated.
[0197] A method for screening for a substance that inhibits the
fucose transport activity of the polypeptide of the present
invention can be carried out by a method known by persons skilled
in the art. For example, the polypeptide of the present invention
is expressed on a membrane (e.g., cell membrane, Golgi apparatus
membrane, or viral membrane). Fucose labeled with a fluorescent
substance or the like is contacted with a substance to be tested
and then the amount of incorporated fucose is measured. Thus, a
substance that inhibits the fucose transport activity of the
polypeptide of the present invention can be screened for.
[0198] A compound that can be isolated by the screening according
to the present invention is a candidate for regulating the activity
of the polypeptide of the present invention and may be applied for
the production of an antibody with high cytotoxic activity.
Cytotoxic Activity of Antibody
[0199] Antibodies produced by the method of the present invention
have enhanced cytotoxic activity.
[0200] Examples of cytotoxic activity in the present invention
include antibody-dependent cell-mediated cytotoxicity (ADCC)
activity and complement-dependent cytotoxicity (CDC) activity. In
the present invention, CDC activity means cytotoxic activity of a
complement system. ADCC activity means activity to damage a target
cell. Specifically, when a specific antibody attaches to a cell
surface antigen of a target cell, an Fc.gamma. receptor-retaining
cell (e.g., an immunocyte) binds to the Fc portion of the antibody
via an Fey receptor, damaging the target cell.
[0201] Whether or not an antibody has ADCC activity or has CDC
activity can be measured by a known method (e.g., Current protocols
in Immunology, Chapter 7, Immunologic studies in humans, Editor
John E. Coligan et al., John Wiley & Sons, Inc., (1993)).
[0202] Specifically, effector cells, a complement solution, and
target cells are prepared.
(1) Preparation of Effector Cells
[0203] A spleen is extracted from a CBA/N mouse or the like, and
then spleen cells are separated in an RPMI1640 medium (produced by
GIBCO). After washing with the same medium containing 10% fetal
bovine serum (FBS, produced by HyClone), the cells are prepared to
a concentration of 5.times.10.sup.6/ml, thereby preparing effector
cells.
(2) Preparation of a Complement Solution
[0204] Baby Rabbit Complement (produced by CEDARLANE LABORATORIES
LIMITED) is diluted 10-fold in a 10% FBS-containing medium
(produced by GIBCO), thereby preparing a complement solution.
(3) Preparation of Target Cells
[0205] Pancreatic cancer cell lines (e.g., AsPC-1 and Capan-2) are
radiolabeled by culturing the cell lines with 0.2 mCi
.sup.51Cr-sodium chromate (produced by Amersham Pharmacia Biotech)
in a 10% FBS-containing DMEM medium at 37.degree. C. for 1 hour.
After radiolabeling, the cells are washed three times in a 10%
FBS-containing RPMI1640 medium and then prepared to a cell
concentration of 2.times.10.sup.5/ml, thereby preparing target
cells.
[0206] Subsequently, ADCC activity or CDC activity are measured. To
measure ADCC activity, target cells and antibodies are added in
amounts of 50 .mu.l each to a 96-well U-bottomed plate (produced by
Beckton Dickinson) and are then allowed to react on ice for 15
minutes. Next, 100 .mu.l of effector cells is added, followed by 4
hours of culture in a carbon dioxide gas incubator. The final
antibody concentration is 0 or 10 .mu.g/ml. After culture, 100
.mu.l of the supernatant is collected, and then radioactivity is
measured using a gamma counter (COBRAIIAUTO-GMMA, MODEL D5005,
produced by Packard Instrument Company). Cytotoxic activity (%) can
be calculated by (A-C)/(B-C).times.100. "A" denotes radioactivity
(cpm) in each sample, "B" denotes radioactivity (cpm) in a sample
supplemented with 1% NP-40 (produced by NACALAI TESQUE, INC.), and
"C" denotes radioactivity (cpm) in a sample containing only target
cells.
[0207] Furthermore, to measure CDC activity, target cells and
anti-PepT antibodies are added in amounts of 50 .mu.l each to a
96-well flat-bottomed plate (produced by Becton Dickinson) and are
then allowed to react on ice for 15 minutes. Subsequently, 100
.mu.l of a complement solution is added, followed by 4 hours of
culture in a carbon dioxide gas incubator. The final antibody
concentration is 0 or 3 .mu.g/ml. After culture, 100 .mu.l of the
supernatant is collected, and then radioactivity is measured using
a gamma counter. Cytotoxic activity can be calculated in a manner
similar to that used for measurement of ADCC activity.
EXAMPLES
[0208] The present invention will be specifically described in the
following examples. However, the examples are not intended to limit
the technical scope of the present invention.
Example 1
Obtainment of GDP-Fucose Transporter Gene Fragment from Chinese
Hamster Ovary (CHO) Cell Line
[0209] The cDNA sequence of human GDP-fucose transporter (accession
#AF326199) and the same of mouse GDP-fucose transporter (accession
#AK050311) were obtained from the NCBI database. After the
sequences were analyzed using GENETYX-SV/RC, primers were designed
using portions of sequences (FIG. 1) where high homology was noted
between the human and mouse sequences. RT-PCR was then carried out
using polyA+ RNA extracted from a CHO cell line (DXB11) using an
RNA extraction kit (TAKARA: catrimox 14 RNA isolation kit) as a
template and an RT-PCR kit (TOYOBO: RT-PCRHigh). The thus obtained
fragment was subcloned into pBluescriptSK+. After the sequence
thereof was confirmed, it was excised with an appropriate
restriction enzyme and then used as a probe for library
screening.
Example 2
Cloning of the Full-Length cDNA of GDP-Fucose Transporter
[0210] The GDP-fucose transporter fragment obtained in Example 1
was labeled with .alpha.32P dCTP using a Random Prime labeling
system (Amersham). The CHO-cell-derived cDNA library used herein
was specifically derived from the CHO-K1 cell line (LambdaZAP-CMV
XR Library: Stratagene Corp.). Screening was carried out basically
according to the manuals. Specifically, in primary screening,
Escherichia coli (XL-1-Blue MRF') was infected with 10.sup.6 phages
and then inoculated together with soft agar on 10 plates. The thus
obtained plaques were transferred to nylon membranes (Hybond NX:
Amersham). The membranes were subjected to alkaline and
neutralization treatment according to a standard method. After
UVcrosslinking, hybridization was carried out using the above
probe. Secondary screening and tertiary screening were carried out
for the obtained positive clones. Finally, 9 purified positive
clones were obtained. Escherichia coli (XLOLR) was infected with
each clone and a helper phage (ExAsist interference-resistant
helper phage: Stratagene Corp.). The resultants were collected as
plasmids (pCMV-Script EX) and then the sequences were examined
(FIG. 2). FIG. 1 shows comparison between the mouse transporter
gene sequence and the human transporter gene sequence. When
compared with the CHO-cell-derived sequence, homology with the
human sequence was found to be 85.3% and the homology with the
mouse sequence was found to be 91.5%.
Example 3
Cloning of Genomic DNA of GDP-Fucose Transporter
[0211] To prepare a gene fragment on the 5' side and the same on
the 3' side as probes, primers were designed using the GDP-fucose
transporter fragment obtained in Example 1 as a template. The 5'
side fragment and the 3' side fragment were obtained by PCR.
Regarding primers, a combination (FIG. 3) of the primer for RT-PCR
used in Example 1 and a primer (with which sequences corresponding
to exon 1 and exon 2 as predicted from a mouse genome can be
obtained) was designed. Labeling of the 5' and 3' probes and
screening were carried out by the method as shown in Example 2
using a CHO-cell-derived genomic DNA library (CHO-K1 cell
line-derived Lambda FIX II Library: Stratagene Corp.). Finally, 11
positive clones were obtained. Of these clones, 7 clones were used
for infecting Escherichia coli (XL-1 Blue MRA). Phages were
collected from 100 mL of a liquid culture and then phage DNA was
collected using a QIAGEN Lambda kit (QIAGEN). The thus collected
phage DNA was digested with an appropriate restriction enzyme,
followed by selection and mapping of independent clones by Southern
blot hybridization.
Example 4
Determination of Genomic DNA Sequence of GDP-Fucose Transporter of
CHO-K1 Cell
[0212] The DNA comprising the CHO genomic gene obtained in Example
3 was digested with various restriction enzymes and then subjected
to 0.8% agarose gel electrophoresis. Subsequently, according to a
standard method, a restriction enzyme map (FIG. 3) was produced by
Southern blotting using the 5' side and the 3' side fragments of
the GDP-fucose transporter cDNA obtained in Example 2 as probes.
Bands respectively hybridizing to these fragments were excised and
then collected using a QIAquick Gel Extraction Kit (QIAGEN)
according to the attached manuals. The collected DNA fragments were
ligated to pBluescriptSK+ using a Rapid DNA ligation kit (Roche)
and then the resultant was introduced into Escherichia coli
DH5.alpha. strain (TOYOBO CO., LTD.). Plasmids were collected from
the thus obtained recombinant Escherichia coli and then analyzed
using an ABI3100 Genetic Analyzer (FIG. 4, SEQ ID NO: 1).
Example 5
Suppression of GDP-Fucose Transporter Expression in CHO Cell Using
RNAi 5.times.10.sup.5 DG44 cells were suspended in an IMDM medium
(Invitrogen Corp.) containing 5 mL of 10% FCS (MOREGATE BIOTECH),
200 won Geneticin (Invitrogen Corp.), and 200 nmol/L MTX. The
resultant was then inoculated in a Falcon 25 cm.sup.2 culture
bottle. 24 hours later, synthetic siRNA (sense strand UAA CCU CUG
CCU CAA GUA CdTdT (SEQ ID NO: 3) and antisense strand GUA CUU GAG
GCA GAG GUU AdTdT (SEQ ID NO: 4)) (B-Bridge International Inc.) for
a GDP-fucose transporter were transfected (2 nM to 500 nM) using
lipofectamine 2000 (Invitrogen Corp.). At 48 hours after
transfection, the cells were collected. RNA was extracted from the
cells using an SV total RNA isolation system (Promega Corp.).
RT-PCR reaction was carried out using a TaqMan PCR Corereagent kit
and TaqMan Reverse transcription reagents (Applied Biosystems).
GDP-fucose transporter expression was quantified using PRISM7700
(Applied Biosystems). As a result, GDP-fucose transporter gene
expression was suppressed at the mRNA level.
[0213] FIG. 5 shows the results. A graph in FIG. 5 shows relative
values when GDP-fucose transporter gene expression at 48 hours
after transfection of PBS was determined to be 100.
Example 6
Disruption of Fucose Transporter Gene in CHO Cell
Construction of Targeting Vector
[0214] A mouse pgk-1 gene promoter was excised with EcoR I-Pst I
from a pKJ2 vector (Popo H, Biochemical Genetics vol. 28, pp.
299-308, 1990) and then cloned into the EcoR I-Pst I site of
pBluescript (Stratagene Corp.), thereby producing pBSK-pgk-1. A
hygromycin resistance gene (Hyg.sup.r) was subjected to PCR using
pcDNA3.1/Hygro (Invitrogen Corp.) and Hyg5-AV and Hyg3-BH primers.
Thus, an Eco T22I site and a Kozak sequence were added to the 5'
side of Hyg.sup.r and a BamH I site was added to the 3' side
comprising a region extending to an SV40 polyA addition signal,
thereby extracting Hyg.sup.r.
TABLE-US-00001 Forward primer Hyg5-AY (SEQ ID NO: 5) 5'-ATG CAT GCC
ACC ATG AAA AAG CCT GAA CTC ACC-3' Reverse primer Hyg3-BH (SEQ ID
NO: 6) 5'-GGA TCC CAG GCT TTA CAC TTT ATG CTT C-3'
[0215] The Hyg.sup.r (Eco T221-BamH I) fragment was inserted into
the Pst I-BamH I site of pBSK-PGK, thereby producing
pBSK-pgk-1-Hyg.sup.r. A targeting vector (hereinafter referred to
as the KO2 vector) for a fucose transporter was constructed (FIG.
6) by respectively inserting the 5' side (ranging from No. 2780 Sma
I to No. 4232 BamH I in SEQ ID NO: 1), 3' side (ranging from No.
4284 to No. 10934 Sac I), and pgk-1-Hyg.sup.r fragments of the
fucose transporter into a pMC1DT-A vector (Yagi T, Proc. Natl.
Acad. Sci. U.S.A. vol. 87, pp. 9918-9922, 1990). The KO2 vector was
cleaved with Not I and then introduced into cells. By the use of
the KO2 vector, the fucose transporter will lack 46 base pairs of
exon 1 comprising the initiation codon and lose the relevant
functions.
Introduction into CHO Cells
[0216] HT Supplement (100.times.) (Invitrogen Corp. cat. 11067-030)
and penicillin streptomycin (Invitrogen Corp.: cat. 15140-122) were
each added to CHO-S-SFMII HT-(Invitrogen Corp.: cat 12052-098) in a
volume one-hundredth of the volume of CHO-S-SFMII HT-. The solution
was used as media for culture (hereinafter referred to as SFMII
(+)). The DXB11 cell line of CHO cells was subcultured and cells
after gene transfer were also cultured in SFMII (+).
8.times.10.sup.6 CHO cells were suspended in 0.8 mL of Dulbecco's
phosphate buffer (hereinafter abbreviated as "PBS"; Invitrogen
Corp.: cat 14190-144). 30 .mu.g of the targeting vector (KO2
vector) was added to the cell suspension. The cell suspension was
transferred to a Gene Pulser Cuvette (4 mm) (Bio-Rad Laboratories
Inc.: cat. 1652088). After the suspension was allowed to stand on
ice for 10 minutes, the vector was introduced into the cells by an
electroporation method using GENE-PULSER II (Bio-Rad Laboratories
Inc.: code No. 340BR) under the condition of 1.5 kV and 25 .mu.FD.
After introduction of the vector, the cells were suspended in 200
ml of SFMII(+) medium. The cells were then inoculated at 100
.mu.l/well to twenty 96-well flat-bottomed plates (IWAKI & CO.,
LTD.: cat. 1860-096). The cells in the plates were cultured in a
CO.sub.2 incubator for 24 hours at 37.degree. C., followed by
selection using hygromycin B (Invitrogen Corp.: cat. 10687-010).
Hygromycin B was dissolved in SFMII(+) to a concentration of 300
.mu.g/ml and then added at 100 .mu.l/well.
[0217] Homologous recombinants were screened for by the PCR method.
CHO cells used in screening were cultured in a 96-well
flat-bottomed plate. After removal of culture supernatants, a
buffer for cell lysis was added at 50 .mu.l/well. After incubation
at 55.degree. C. for 2 hours, proteinase K was inactivated by
subsequent heating at 95.degree. C. for 15 minutes, so as to
prepare a template for PCR. The buffer for cell lysis per well was
composed of 5 .mu.l of 10.times.LA buffer II (attached to TaKaRa LA
Taq), 2.5 .mu.l of 10% NP-40 (Roche: cat. 1 332 473), 4 .mu.l of
proteinase K (20 mg/ml and TaKaRa: cat. 9033), and 38.5 .mu.l of
distilled water (NACALAI TESQUE, INC.: cat. 36421-35). A PCR
reaction mixture was determined to contain 1 .mu.l of the above PCR
sample, 5 .mu.l of 10.times.LA buffer, 5 .mu.l of MgCl.sub.2 (25
mM), 5 .mu.l of dNTP (2.5 mM), 2 ml of each primer (10 .mu.M each),
0.5 .mu.l of LA Taq (5 IU/.mu.l and cat. RR002B), and 29.5 .mu.l of
distilled water (total 50 .mu.l). Moreover, PCR conditions consist
of pre-heating at 95.degree. C. for 1 minute, 40 amplification
cycles (each cycle consisting of 95.degree. C. for 30 seconds and
70.degree. C. for 3 minutes), and additional heating at 70.degree.
C. for 7 minutes.
[0218] Primers are as shown below. In CHO cell samples wherein
homologous recombination has taken place, an approximately 2.0-kb
band was amplified. Regarding the primers, TP-F4 was located in the
5' side fucose transporter genomic region outside the KO2 vector
and THygro-R1 was located within a hygromycin resistance gene in
the KO2 vector.
TABLE-US-00002 Forward primer TP-F4 (SEQ ID NO: 7) 5'-GGA ATG CAG
CTT CCT CAA GGG ACT CGC-3' Reverse primer THygro-F1 (SEQ ID NO: 8)
5'-GCA CTC GTC CGA GGG CAA AGG AAT AGC-3'
[0219] The number of the thus analyzed CHO cells was 537. Of these
cells, 17 were considered to be homologous recombinants (homologous
recombination efficiency was approximately 3.2% at this time).
[0220] FIG. 7 shows data resulting when the PCR products in the
screening were subjected to 1% agarose gel electrophoresis. 2.0-kb
bands appeared in samples indicated with ".largecircle.." These
clones were considered to be homologous recombinants. Next, 2.0-kb
bands were excised from the gel and then purified using a Mag
Extractor (TOYOBO CO., LTD.: cat. NPK-601). 200 ng of the purified
PCR product was subjected to direct sequencing using TP-F4 and
THygro-F1. Thus, the PCR product was confirmed to have a nucleotide
sequence resulting from homologous recombination, resulting in the
conclusion that the product was a homologous recombinant.
[0221] Such confirmation was also carried out by the Southern blot
method. The cells cultured in a 24-well plate were collected,
genomic DNA was prepared according to a standard method, and then
Southern blot was carried out. A 387-bp probe was prepared from the
region ranging from No. 2, 113 to No. 2, 500 of SEQ ID NO: 1 by the
PCR method using the following 2 types of primer. The thus prepared
probe was used for confirmation by the Southern blot method. The
genomic DNA was cleaved with Bgl II or EcoR I.
TABLE-US-00003 Forward primer (SEQ ID NO: 9) Bgl-F: 5'-TGT GCT GGG
AAT TGA ACC CAG GAC-3' Reverse primer (SEQ ID NO: 10) Bgl-R: 5'-CTA
CTT GTC TGT GCT TTC TTC C-3'
[0222] As a result of cleavage with Bgl II, an approximately 3.0-kb
band appeared from the chromosome of the original fucose
transporter and an approximately 5.0-kb band appeared from the
knocked-out chromosome. Furthermore, as a result of cleavage with
EcoR I, an approximately 8.7-kb band appeared from the chromosome
of the original fucose transporter and an approximately 4.5-kb band
appeared from the knocked-out chromosome (FIG. 8). FIG. 9 shows the
data when Southern blot was actually carried out.
Sequence Listing Free Text
[0223] SEQ ID NOS: 3 and 4: synthetic RNA
INDUSTRIAL APPLICABILITY
[0224] According to the present invention, a fucose transporter
polypeptide and a fucose transporter gene can be obtained.
Furthermore, a recombinant protein-producing cell having a
disrupted fucose transporter gene is obtained. When recombinant
protein is produced in the cell, a recombinant protein wherein the
addition of fucose is lowered or is eliminated can be produced.
Particularly when protein is an antibody, cytotoxic activity is
enhanced due to lowered fucose addition or elimination of fucose
addition. Hence, such protein is useful as an antibody
pharmaceutical having an anti-tumor effect.
[0225] All publications cited herein are incorporated herein in
their entirety. A person skilled in the art would easily understand
that various modifications and changes of the present invention are
feasible within the technical idea and the scope of the invention
as disclosed in the attached claims. The present invention is
intended to include such modifications and changes.
Sequence CWU 1
1
10110939DNACricetulus griseus 1gagctcaatt aaccctcact aaagggagtc
gactcgatcc tttacagaaa acttgcaaac 60cctcttggag tagaaaagta gtagtatctg
acacaagtat cagcaaaatg caaacttctc 120cccatcccca gaaaaccatt
ataaaaaccc ccatatctta tgcccaactg tagtgatata 180ttatttatga
tttattaaaa cttgcttaag gattcagaaa gcaaagtcag ccttaagcta
240tagagaccag gcagtcagtg gtggtacaca cctttaatcc caggactcag
gattaagaag 300tagacggacc tctgttagtt caagtctacc attacctaca
caagagtgaa gagtaaccga 360tctcatgcct ttgatcccag cagctgggat
catgtgcatt caatcccagc attcgggagt 420tatataagac aggagcaagg
tctcagagct ggcattcatt ctccagccac attgaggata 480ggaaaacatt
gaagtgtcag gatgctgagg agaggcagca gtttgaggtt tggtagaacc
540aggatcacct tttggtctga ggtagagtaa gaactgtggc tggctgcttt
gcttttctga 600tcttcagctt gaagcttgaa ctccaatatt tgtctctggg
tctattatta tcatgttaca 660cctaacttta aagctgattt acgcaagaca
gttgtaggtg gacctttctt tcctgcccac 720cagttcccaa ataactgaca
cggagactca atattaatta taaatgattg gttaatagct 780cagtcttgtt
actggctaac tcttacattt taaattaact catttccatc cctttacttg
840ctgccatgtg gttcatggct tgttcaagtc ctgcttcttc tgtctctggc
tggtgatgcc 900tctggttctg ccctttatcc cagaattctc ctagtctggc
tctcctgccc agctataggc 960cagtcagctg tttattaacc aatgagaata
atacatattt atagtgtaca aagattgctc 1020ctcaacaccc aattttttat
gtgcaacctg agaatctgga ctcattgccc tcatgcttgc 1080agaggcggca
cccttaccca ctaagccacc tttctagccc tgttgctttt gttttttgag
1140acaggttcca ctatgtagcc caggctggcc tcaaactgac cattctcctg
cctaaacctc 1200ccgaacactg gaattatagt caaggcctac ctgccctggc
attttcacac ttttatttcc 1260tggctgagtc cattgacttt acactcatca
aggttgaacc agttggagtt taattacagt 1320gccaatcgca ctgaatccca
cataatcaaa caacttcaag gaagcaaaaa accagttttt 1380cctgaagatc
aatgtcagct tgcctgattc agaatagacc cccgaaaaaa ggcaaatgct
1440tgataaccaa tttcttctta ttgttcaatc ccctgctgct gtgtgtaagc
tcctgagaaa 1500ggacagtaag gggacattca tgatcagaga aagagcccca
actccccccc cagccccacc 1560cccaccctgt ccacagtctg ttggtttggt
ttccccctgg ctgacaccca gaaatcacaa 1620cataatcacc taggtcactg
taacaagttc ctttctggaa aatgctacaa atgatattgg 1680taacatgagt
aatgaataat gcctggagtc caactccctt gtgacccagc aatgttttcc
1740gtgggtgctc ccttccccag ctgcaggcct gacatgtacc ttaaaaagcc
tcccctggag 1800gacagaattt tgtgggtact atagtgttct cacaaatact
tcccctaata cccttactta 1860gttaccataa ataacatgca gcccctggtg
aggcacacag ggctccaatg tacagcttct 1920cagacactgc aggaaccttc
ctctcctaat gcagcactgg tctcttcagg ctggacagca 1980ggaacccata
ccactccaat cctagtgtgg agtagagctg tctacgaaaa ccagcagatc
2040tatagctaaa tgtgtttcaa ttttatgctt tgacaaattg tactgacccc
acccccaccc 2100cttccccctt gctgtgctgg gaattgaacc caggaccttg
tgcatgccag gcaagtactc 2160taacactgag ctatagcccc aatctttcat
ccaagtctct atgtgtgccc acactcgctt 2220tttattttga gacaaaaggt
tcttattttg agataaggtc tcactatgtt gccttgactt 2280tttttttttt
ttttttttga acttttgacc ttcctacctc agctgagact acaagtcttt
2340taccatcagg cccggctgat ggtaaaataa cagtatttga aatagtttaa
acacatcatc 2400ttaatggtca accacacaat ttccgaaatg ttgctggctc
agtctggggc aaacctgtcc 2460gccccaacat tggtgctagg aagaaagcac
agacaagtag ccctcccagc tcaggagtaa 2520aagacctgga gggggtggcc
cacttcggtc aagttcacgg gatggggagg ggtaccctcc 2580tccagtagtg
gtggtatttg gcagttcctc caccgacgcc ctctggaagc acctgcttgg
2640acccgcaaag ccaggaatgc agcttcctca agggactcgc cagcgagggt
aacaggacag 2700aggcgtccca agagggctgg ggcggaaggg ggaagacagg
gtcggcctta gatagggcaa 2760agggccttct ggctgtgttc ccggggtaac
cgccccacca cgcctggagc ccgacgtggc 2820gagcgatggg gacagcgagc
aggaagtcgt actggggagg gccgcgtagc agatgcagcc 2880gagggcggcg
ctgccaggta cacccgaggg caccgcgggg gtgagcgcca ggtccctgaa
2940ccagccaggc ctccagagcc gagtccggcg gaccgacggt acgttctgga
atgggaaggg 3000atccgggaca ccgaattgct gcattgaggg gctcagaggt
tctgatgtgg gagtccagaa 3060agggttttat ctaccggagg tgatgtgact
tccggcctct ggaagtgctg ttggagtctc 3120tgggaccttg ggtcctctcg
actaggtttg gaaggggtga aataggggta gggagaaagg 3180agaggactgc
agcaatgtct tcccgaacga cctgggttcg ggaggggtcg aaggacaagg
3240ggctgttgtg gggggtcttc agacgcggag gggtggtatt ctattttctg
ggaagatggt 3300gtcgatgcac ttgaccaagt ctagtcgatc tgaagaggct
aggggaacag acagtgagag 3360aggatggtgg agggagtggc agaacccttc
cagaaactgg gagaggctct agcacctgca 3420accccttccc tggcctccgg
ggagtcccag aagagggcag gaccatggac acaggtgcat 3480tcgtgccggc
gcgctccggc ctggcgaagg tgcgcgctct tggaggccgc gggagggcca
3540gacgcgcgcc cggagagctg gccctttaag gctacccgga ggcgtgtcag
gaaatgcgcc 3600ctgagcccgc ccctcccgga acgcggcccg agacctggca
agctgagacg gaactcggaa 3660ctagcactcg gctcgcggcc tcggtgaggc
cttgcgcccg ccatgcctct gtcattgccc 3720ctcgggccgc ctccctgaac
ctccgtgacc gccctgcagt cctccctccc ccccttcgac 3780tcggcgggcg
cttccgggcg ctcccgcagc ccgccctcca cgtagcccac acctccctct
3840cggcgctccg cttcccacgc ggtccccgac ctgttctttc ctcctccacc
ctgcccttct 3900gtccctctcc cttcctttct cccctcgact cgtccctatt
aggcaacagc ccctgtggtc 3960cagccggcca tggctgtcaa ggctcacacc
cttagctagg ccccttctcc cttccctggg 4020tcttgtctca tgaccccctg
ccccgcccgg gagcgagcgc gatgtggagc agtgcctctg 4080gcaagcagaa
cttcacccaa gccatgtgac aattgaaggc tgtaccccca gaccctaaca
4140tcttggagcc ctgtagacca gggagtgctt ctggccgtgg ggtgacctag
ctcttctacc 4200accatgaaca gggcccctct gaagcggtcc aggatcctgc
gcatggcgct gactggaggc 4260tccactgcct ctgaggaggc agatgaagac
agcaggaaca agccgtttct gctgcgggcg 4320ctgcagatcg cgctggtcgt
ctctctctac tgggtcacct ccatctccat ggtattcctc 4380aacaagtacc
tgctggacag cccctccctg cagctggata cccctatctt cgtcactttc
4440taccaatgcc tggtgacctc tctgctgtgc aagggcctca gcactctggc
cacctgctgc 4500cctggcaccg ttgacttccc caccctgaac ctggacctta
aggtggcccg cagcgtgctg 4560ccactgtcgg tagtcttcat tggcatgata
agtttcaata acctctgcct caagtacgta 4620ggggtggcct tctacaacgt
ggggcgctcg ctcaccaccg tgttcaatgt gcttctgtcc 4680tacctgctgc
tcaaacagac cacttccttc tatgccctgc tcacatgtgg catcatcatt
4740ggtgagtggg gcccgggggc tgtgggagca ggatgggcat cgaactgaag
ccctaaaggt 4800caacactgta ggtaccttta cttactgtcc caggtccctt
gcatcagcag ttacaggaag 4860agccctgtag aaaacaaata acttccttat
ggtcattcaa caagttaggg acccagccag 4920ggtgaaaata atgttagcag
caactacagc aaagatggct ctcgccactt gcatgattaa 4980aatgtgccag
gtactcagat ctaagcattg gatccacatt aactcaacta atccctatta
5040caaggtaaaa tatatccgaa ttttacagag ggaaaaccaa ggcacagaga
ggctaagtag 5100cttgaccagg atcacacagc taataatcac tgacatagct
gggatttaaa cataagcagt 5160tacctccata gatcacacta tgaccaccat
gccactgttc cttctcaaga gttccaggat 5220cctgtctgtc cagttctctt
taaagaggac aacacatctg acattgctac cttgaggtaa 5280catttgaaat
agtgggtaga catatgtttt aagttttatt cttacttttt atgtgtgtgt
5340gtttgggggg ccaccacagt gtatgggtgg agataagggg acaacttaag
aattggtcct 5400ttctcccacc acatgggtgc tgaggtctga actcaggtca
tcaggattgg cacaaatccc 5460tttacccact gagccatttc actggtccaa
tatatgtgtg cttttaagag gctttaacta 5520ttttcccaga tgtgaatgtc
ctgctgatca ttatcccctt ttacccggaa gccctctggg 5580aggtgccatc
cctgtggtcg tctgcataca aatggggaaa ctgcaactca gagaaacaag
5640gctacttgcc agggccccac aagtaagata ggctgggatg ccatcccaga
ctggccacac 5700tccctggcct gtgcttcaag ccagtttact ttgttcctgc
ccattggaag ttagcatgtt 5760gcagtcaaac acaataacta caggccaaaa
gtgcttttaa attaaagtca gatgaacttt 5820taaacatcca gagctcctca
actgcaggag ttacaacctg attctgcaac catctttgca 5880gtgcccggta
gtcatatgta gctagaggct cttggctagg acagcatgtg ttaggaaaca
5940tctggccctg agatcattga attgagtgac tgctgggtga caaagaccaa
ggcatccgtt 6000ccctgagagt cctgggcaag cagcaatgtg accttcattt
gtacctactc aggttcttta 6060tctgtcctgt ttgacctact tagtctcctc
tggtgtctca gaggcccagg ctgggtactc 6120tggatgtcag gatcaggcca
atgcgcacat ctgccctaga aatgtccccc tggttgagca 6180gctcctgaat
ccatcggtaa agggtctgga ccagggagga gtcagataaa aagctgacag
6240cactggggga ctccatgggg aactcccacc tgcccccaca catccatcct
aagagaactg 6300gtattccttg tttcctcttt gtcctacaag gcaccctggg
atcccacttc agtctcccag 6360ccttgccagg gttagagggc atgagcctcc
ttgtggggaa tttagatgca agaaggtaca 6420gtcactagag aacctgagct
cagatcccca aagtaaccag tacctgatag tgaggcagct 6480gagaaccgca
gcagcctgcc tgagtggctg aactctgcgg cctccggaac tggccccaac
6540tgttgggtct cctcttcctt cctcctgtga gggagggccc atctctgata
agtgctgtgg 6600ggactctaga gtagggagga ggaggagcaa tctaagcagg
ccttactgag aagtccttgc 6660tggcatgtgg ctgcctgagg agtacagact
gggaacaccc atttgaatga gtaaggtttt 6720tcctgaaggc catggggagc
cacggaggaa aatcatttta gttacaagac aaagagtaga 6780ttggttaaca
tgggagcaag gacatggccc caattttcat agatgaagga aattggaact
6840cagagaggtt aagtaacttc tcccaaatag ctcagcttca aaatcacaga
acagtcagag 6900tctagatctc tctgatgcct gtgatggtcc tgccattcca
tgttgctgat ccctgtggca 6960tcagtaagcc tctaccttgt gggaatgcag
gatctaaatg aagagaggaa gtgctggccc 7020catgctgtgg tctggaaagc
tatgcaggct ctttgagcag agagtgaccc acaagtgaat 7080agagtcctat
gagactcaaa gcaacatcca cccttaagca gctctaacca aatgctcaca
7140ctgagggagc caaagccaag ttagagtcct gtgcttgccc aaggtcactt
tgcctggccc 7200tcctcctata gcacccgtgt tatcttatag ccctcattac
agtgattaca attataatta 7260gagaggtaac agggccacac tgtccttaca
cattcccctg ctagattgta gctgggagag 7320ggggagatgt aggtggctgg
gggagtggga gggaagatgc agattttcat tctgggctct 7380actccctcag
ccattttttg gtgtgggagt tagactttgg atatgttgat gatgaggtaa
7440gggccacaga acagtctgaa ctgtggtatc agaatcctgt ccctctccct
ctctcctcat 7500ccctcttcac cttgtcactc ctctgtctgc tacaggtggt
ttctggctgg gtatagacca 7560agagggagct gagggcaccc tgtccctcat
aggcaccatc ttcggggtgc tggccagcct 7620ctgcgtctcc ctcaatgcca
tctataccaa gaaggtgctc ccagcagtgg acaacagcat 7680ctggcgccta
accttctata acaatgtcaa tgcctgtgtg ctcttcttgc ccctgatggt
7740tctgctgggt gagctccgtg ccctccttga ctttgctcat ctgtacagtg
cccacttctg 7800gctcatgatg acgctgggtg gcctcttcgg ctttgccatt
ggctatgtga caggactgca 7860gatcaaattc accagtcccc tgacccacaa
tgtatcaggc acagccaagg cctgtgcgca 7920gacagtgctg gccgtgctct
actatgaaga gactaagagc ttcctgtggt ggacaagcaa 7980cctgatggtg
ctgggtggct cctcagccta tacctgggtc aggggctggg agatgcagaa
8040gacccaagag gaccccagct ccaaagaggg tgagaagagt gctattgggg
tgtgagcttc 8100ttcagggacc tgggactgaa cccaagtggg gcctacacag
cactgaaggc ttcccatgga 8160gctagccagt gtggccctga gcaatactgt
ttacatcctc cttggaatat gatctaagag 8220gagccagggt ctttcctggt
aatgtcagaa agctgccaaa tctcctgtct gccccatctt 8280gttttgggaa
aaccctacca ggaatggcac ccctacctgc ctcctcctag agcctgtcta
8340cctccatatc atctctgggg ttgggaccag ctgcagcctt aaggggctgg
attgatgaag 8400tgatgtcttc tacacaaggg agatgggttg tgatcccact
aattgaaggg atttgggtga 8460ccccacacct ctgggatcca gggcaggtag
agtagtagct taggtgctat taacatcagg 8520aacacctcag cctgcctttg
aagggaagtg ggagcttggc caagggagga aatggccatt 8580ctgccctctt
cagtgtggat gagtatggca gacctgttca tggcagctgc accctggggt
8640ggctgataag aaaacattca cctctgcatt tcatatttgc agctctagaa
cgggggagag 8700ccacacatct tttacgggtt aagtagggtg atgagctcct
ccgcagtccc taaccccagc 8760tttacctgcc tggcttccct tggcccagct
acctagctgt actccctttc tgtactcttc 8820tcttctccgt catggcctcc
cccaacacct ccatctgcag gcaggaagtg gagtccactt 8880gtaacctctg
ttcccatgac agagcccttt gaatacctga acccctcatg acagtaagag
8940acatttatgt tctctggggc tggggctgaa ggagcccact ggttctcact
tagcctatct 9000ggctcctgtc acaaaaaaaa aaaaagaaaa aaaaaaagca
taaaccaagt tactaagaac 9060agaagttggt ttataacgtt ctggggcagc
aaagcccaga tgaagggacc catcgaccct 9120ctctgtccat atcctcatgc
tgcagaagta caggcaagct cctttaagcc tcatatagga 9180acactagcct
cactcatgag ggttttactc catgacctgt caacctcaaa gccttcaaca
9240tgaggactcc aacgtaaatt tggggacaga agcactcaga ccatacccca
gcaccacacc 9300ctcctaacct cagggtagct gtcattctcc tagtctcctc
tcttgggcct ttagaacccc 9360catttccttg gggtaatgtc tgatgttttt
gtccctgtca taaaaagatg gagagactgt 9420gtccagcctt tgattcctac
ttcctacaat cccaggttct aatgaagttt gtggggcctg 9480atgccctgag
ttgtatgtga tttaataata aaaaagcaag atacagcatg tgtgtggact
9540gagtgagggc cacagggatc taaaagccaa gtgtgagggg acccagctac
agcaggcagc 9600atcctgagcc tggaatctct tcaggacaag aattctccat
atacctacct actctgggga 9660gtaggtggcc agagttcaag cttcccttag
taccaactac cactggctgt gctcttactg 9720aaggcagaca tggcactgag
tgctgtccat ctgtcactca tctccacagc cattcctaat 9780gtgtggggtg
ggagccatca ccaaacccca ttttcagata aggacacagg ctcagagagg
9840cttgtgtgga gaaaagtagc agcagaattc agagagctgg gtctcctgca
gcaccttgga 9900ctgccagcag ccacagtgct tgtcacacag cacatactca
aaagaatgcc agccccctca 9960gcctagagtg cctggccttt ctttcagatg
aggaagaggg tcaaagctgt tagcttgccc 10020accatatgac cacatacatg
accaacagct tgagggaggg aggattactg tggctcccag 10080cctgagaggt
gggacaccca aatgtattag gtccttgaat cagggctgac cttgtgattc
10140agtcactcct accagaatgc tggggaatgg ggatgccaaa ggcaaaggag
gctttctaag 10200gtgtggtgta agataggcat ttctgcttcc atgtacacct
gtgagcagag taggaaggcc 10260ctgtggagaa tatatcccac aaaccagtag
cccttcctgg cagtgggtga atactgccac 10320cctatacccc tatgcaaggc
cagtagaacc acccaaccca caacatctag agaaattaca 10380ggtcatctta
agcctctaaa ttgtggagaa actcgacatg cgcacgattc ctaacctgct
10440agcctagggt gcggggtgga taatttaagg aaactggggt ttcttataga
atcggaggct 10500ccatgaagtc accctgacaa gaggtcagca atagccagca
gcagtggcta ctcctaagcc 10560tccagacaga gcaccctgtg aatgtacctt
attctcacat ctgggtgtct ataggtgtga 10620ctgggtcaga tgtcacccag
gccattgcaa tgggccctta gccccatggg gtgttgggat 10680agcagccaag
cagctcccat gctgagatac tgcctgcagt agactgatgg ataagaaaac
10740aaggcccaaa atgttttctt tccagacttg atctttcttt gttcaaaaat
gctgttttcc 10800cttaaacttg cccaaaccca ttgttttgca gttgaggaaa
ataaggcata gaaagattaa 10860aggaagtttc tgaggttaca gagcaaagta
ctggcttcac ctgaaataga caggtgtgcc 10920ctgatcctga tttgagctc
109392352PRTCricetulus griseus 2Met Ala Leu Thr Gly Gly Ser Thr Ala
Ser Glu Glu Ala Asp Glu Asp1 5 10 15Ser Arg Asn Lys Pro Phe Leu Leu
Arg Ala Leu Gln Ile Ala Leu Val 20 25 30Val Ser Leu Tyr Trp Val Thr
Ser Ile Ser Met Val Phe Leu Asn Lys 35 40 45Tyr Leu Leu Asp Ser Pro
Ser Leu Gln Leu Asp Thr Pro Ile Phe Val 50 55 60Thr Phe Tyr Gln Cys
Leu Val Thr Ser Leu Leu Cys Lys Gly Leu Ser65 70 75 80Thr Leu Ala
Thr Cys Cys Pro Gly Thr Val Asp Phe Pro Thr Leu Asn 85 90 95Leu Asp
Leu Lys Val Ala Arg Ser Val Leu Pro Leu Ser Val Val Phe 100 105
110Ile Gly Met Ile Ser Phe Asn Asn Leu Cys Leu Lys Tyr Val Gly Val
115 120 125Ala Phe Tyr Asn Val Gly Arg Ser Leu Thr Thr Val Phe Asn
Val Leu 130 135 140Leu Ser Tyr Leu Leu Leu Lys Gln Thr Thr Ser Phe
Tyr Ala Leu Leu145 150 155 160Thr Cys Gly Ile Ile Ile Gly Gly Phe
Trp Leu Gly Ile Asp Gln Glu 165 170 175Gly Ala Glu Gly Thr Leu Ser
Leu Ile Gly Thr Ile Phe Gly Val Leu 180 185 190Ala Ser Leu Cys Val
Ser Leu Asn Ala Ile Tyr Thr Lys Lys Val Leu 195 200 205Pro Ala Val
Asp Asn Ser Ile Trp Arg Leu Thr Phe Tyr Asn Asn Val 210 215 220Asn
Ala Cys Val Leu Phe Leu Pro Leu Met Val Leu Leu Gly Glu Leu225 230
235 240Arg Ala Leu Leu Asp Phe Ala His Leu Tyr Ser Ala His Phe Trp
Leu 245 250 255Met Met Thr Leu Gly Gly Leu Phe Gly Phe Ala Ile Gly
Tyr Val Thr 260 265 270Gly Leu Gln Ile Lys Phe Thr Ser Pro Leu Thr
His Asn Val Ser Gly 275 280 285Thr Ala Lys Ala Cys Ala Gln Thr Val
Leu Ala Val Leu Tyr Tyr Glu 290 295 300Glu Thr Lys Ser Phe Leu Trp
Trp Thr Ser Asn Leu Met Val Leu Gly305 310 315 320Gly Ser Ser Ala
Tyr Thr Trp Val Arg Gly Trp Glu Met Gln Lys Thr 325 330 335Gln Glu
Asp Pro Ser Ser Lys Glu Gly Glu Lys Ser Ala Ile Gly Val 340 345
350322RNAArtificial SequenceDescription of Artificial
SequenceSynthetic RNA 3uaaccucugc cucaaguaca gc 22419RNAArtificial
SequenceDescription of Artificial SequenceSynthetic RNA 4guacuugagg
cagagguua 19533DNAArtificial SequenceDescription of Artificial
SequencePrimer 5atgcatgcca ccatgaaaaa gcctgaactc acc
33628DNAArtificial SequenceDescription of Artificial SequencePrimer
6ggatcccagg ctttacactt tatgcttc 28727DNAArtificial
SequenceDescription of Artificial SequencePrimer 7ggaatgcagc
ttcctcaagg gactcgc 27827DNAArtificial SequenceDescription of
Artificial SequencePrimer 8gcactcgtcc gagggcaaag gaatagc
27924DNAArtificial SequenceDescription of Artificial SequencePrimer
9tgtgctggga attgaaccca ggac 241022DNAArtificial SequenceDescription
of Artificial SequencePrimer 10ctacttgtct gtgctttctt cc 22
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