U.S. patent application number 10/806422 was filed with the patent office on 2004-09-02 for il-6 receptor il-6 direct fusion protein.
This patent application is currently assigned to TOSOH CORPORATION. Invention is credited to Ekida, Teiji, Ide, Teruhiko, Iida, Hiroshi, Tsuchiya, Shigeo, Yagame, Harutaka, Yasukawa, Kiyoshi.
Application Number | 20040170604 10/806422 |
Document ID | / |
Family ID | 32913108 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040170604 |
Kind Code |
A1 |
Ekida, Teiji ; et
al. |
September 2, 2004 |
IL-6 receptor IL-6 direct fusion protein
Abstract
The present invention intends to provide an IL-6R.cndot.IL-6
fusion protein and the like in which IL-6R and IL-6 are directly
linked without a linker. The IL-6 receptor.cndot.IL-6 fusion
protein of the present invention has a structure in which one amino
acid residue constituting IL-6 receptor and one amino acid residue
constituting IL-6 are directly bonded.
Inventors: |
Ekida, Teiji; (Kanagawa,
JP) ; Yagame, Harutaka; (Iwate, JP) ; Iida,
Hiroshi; (Kanagawa, JP) ; Yasukawa, Kiyoshi;
(Kanagawa, JP) ; Tsuchiya, Shigeo; (Kanagawa,
JP) ; Ide, Teruhiko; (Tokyo, JP) |
Correspondence
Address: |
SUGHRUE MION, PLLC
2100 PENNSYLVANIA AVENUE, N.W.
SUITE 800
WASHINGTON
DC
20037
US
|
Assignee: |
TOSOH CORPORATION
|
Family ID: |
32913108 |
Appl. No.: |
10/806422 |
Filed: |
March 23, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10806422 |
Mar 23, 2004 |
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09743239 |
Jan 5, 2001 |
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09743239 |
Jan 5, 2001 |
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PCT/JP99/03554 |
Jul 1, 1999 |
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Current U.S.
Class: |
424/85.2 ;
435/254.23; 435/483; 435/69.52; 530/351; 536/23.5 |
Current CPC
Class: |
C07K 2319/00 20130101;
C07K 14/5412 20130101; C07K 14/7155 20130101; A61K 38/00 20130101;
C12N 5/0647 20130101; C12N 2501/125 20130101 |
Class at
Publication: |
424/085.2 ;
435/254.23; 530/351; 435/069.52; 435/483; 536/023.5 |
International
Class: |
A61K 038/20; C07H
021/04; C12P 021/04; C12N 001/18; C12N 015/74 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 6, 1998 |
JP |
HEI 10-190597 |
Jan 29, 1999 |
JP |
HEI 11-21788 |
Apr 30, 1999 |
JP |
HEI 11-123411 |
Claims
1. An IL-6 receptor.cndot.IL-6 fusion protein, in which one amino
acid residue constituting IL-6 receptor and one amino acid residue
constituting IL-6 are directly linked.
2. An IL-6 receptor.cndot.IL-6 fusion protein, in which C-terminal
of any one of 39 amino acid residues of from N-terminal 323th
alanine residue to N-terminal 361th serine residue of IL-6 receptor
is linked to an N-terminal amino acid residue of IL-6.
3. A gene for coding for IL-6 receptor.cndot.IL-6 fusion protein in
which one amino acid residue of IL-6R and one amino acid residue of
IL-6 are linked directly.
4. The gene according to claim 3, wherein the gene codes for IL-6
receptor.cndot.IL-6 fusion protein in which C-terminal of any one
of 39 amino acid residues of from N-terminal 323th alanine residue
to N-terminal 361th serine residue of IL-6 receptor is linked to an
N-terminal amino acid residue of IL-6.
5. A yeast of Pichia pastoris species which is transformed by an
expression vector containing a gene for coding for IL-6
receptor.cndot.IL-6 fusion protein in which one amino acid residue
of IL-6 receptor and one amino acid residue of IL-6 are linked
directly.
6. The yeast of Pichia pastoris species according to claim 5, which
is transformed by an expression vector containing a gene for coding
for IL-6R.cndot.IL-6 fusion protein in which C-terminal of any one
of 39 amino acid residues of from N-terminal 323th alanine residue
to N-terminal 361th serine residue of IL-6 receptor is linked to an
N-terminal amino acid residue of IL-6.
7. A process for producing an IL-6 receptor.cndot.IL-6 fusion
protein, comprising a step of cultivating in a culture medium a
yeast of a Pichia pastoris species having been transformed by an
expression vector containing a gene for coding for an IL-6
receptor.cndot.IL-6 fusion protein in which one amino acid residue
of IL-6 receptor and one amino acid residue of IL-6 are linked
directly; and a step of collecting the IL-6 receptor.cndot.IL-6
fusion protein as a secretory protein from the culture medium.
8. A process for producing an IL-6 receptor.cndot.IL-6 fusion
protein, comprising a step of cultivating in a culture medium a
yeast of a Pichia pastoris species set forth in claim 5 having been
transformed by an expression vector containing a gene for coding
for an IL-6 receptor.cndot.IL-6 fusion protein in which C-terminal
of any one of 39 amino acid residues of from N-terminal 323th
alanine residue to N-terminal 361th serine residue of IL-6 receptor
is linked to an N-terminal amino acid residue of IL-6; and a step
of collecting the IL-6 receptor.cndot.IL-6 fusion protein as a
secretory protein from the culture medium.
9. A process for producing an IL-6 receptor.cndot.IL-6 fusion
protein, comprising a step of cultivating a yeast of a Pichia
pastoris species having been transformed by an expression vector
containing a gene for coding for an IL-6 receptor.cndot.IL-6 fusion
protein in which one amino acid residue of IL-6 receptor and one
amino acid residue of IL-6 are linked directly, in a culture medium
of natural origin containing a carbon source and no methanol, and
adding methanol during progress of the cultivation.
10. A process for producing an IL-6 receptor.cndot.IL-6 fusion
protein, comprising a step of cultivating a yeast of a Pichia
pastoris species having been transformed by an expression vector
containing a gene for coding for an IL-6 receptor.cndot.IL-6 fusion
protein in which C-terminal of any one of 39 amino acid residues of
from N-terminal 323th alanine residue to N-terminal 361th serine
residue of IL-6 receptor is linked to an N-terminal amino acid
residue of IL-6.
11. A process for producing an IL-6 receptor.cndot.IL-6 fusion
protein, comprising collecting the IL-6 receptor.cndot.IL-6 fusion
protein by treating a solution containing IL-6 receptor.cndot.IL-6
fusion protein, in which one amino acid residue of IL-6 receptor
and one amino acid residue of IL-6 are linked directly, by three
kinds of chromatography including ion-exchange chromatography,
hydrophobic chromatography, and gel-filtration chromatography to
collect the IL-6 receptor.cndot.IL-6 fusion protein.
12. A process for producing an IL-6 receptor.cndot.IL-6 fusion
protein, comprising collecting the IL-6 receptor.cndot.IL-6 fusion
protein by treating a solution containing IL-6 receptor.cndot.IL-6
fusion protein, in which C-terminal of any one of 39 amino acid
residues of from N-terminal 323th alanine residue to N-terminal
361th serine residue of IL-6 receptor is linked to an N-terminal
amino acid residue of IL-6, by three kinds of chromatography
including ion-exchange chromatography, hydrophobic chromatography,
and gel-filtration chromatography to collect the IL-6
receptor.cndot.IL-6 fusion protein.
13. An ex vivo amplifier for a hematopoietic stem cells, comprising
an IL-6 receptor.cndot.IL-6 fusion protein in which one amino acid
residue of IL-6 receptor and one amino acid residue of IL-6 are
directly linked.
14. An ex vivo amplifier for a hematopoietic stem cells, comprising
an IL-6 receptor.cndot.IL-6 fusion protein in which C-terminal of
any one of 39 amino acid residues of from N-terminal 323th alanine
residue to N-terminal 361th serine residue of IL-6 receptor is
linked to an N-terminal amino acid residue of IL-6.
15. A blood platelet proliferating agent containing as a main
component an IL-6 receptor.cndot.IL-6 fusion protein in which one
amino acid residue of IL-6 receptor and one amino acid residue of
IL-6 are directly linked.
16. A blood platelet proliferating agent containing as a main
component an IL-6 receptor.cndot.IL-6 fusion protein in which
C-terminal of any one of 39 amino acid residues of from N-terminal
323th alanine residue to N-terminal 361th serine residue of IL-6
receptor is linked to an N-terminal amino acid residue of IL-6.
Description
TECHNICAL FIELD
[0001] The present invention relates to a fusion protein composed
of an interleukin-6 receptor (hereinafter referred to as IL-6R) and
an interleukin-6 (hereinafter referred to as IL-6) directly linked
without a linker sequence; a gene for coding for the fusion
protein; a host transformed by an expression vector containing the
gene; a method of cultivating the host; a process for purifying the
fusion protein derived from the culture of the host; a novel ex
vivo amplifier containing the fusion protein for hematopoietic stem
cells; and a novel blood platelet-proliferating agent.
BACKGROUND TECHNIQUE
[0002] IL-6, IL-11, ciliary neurotropic factors, leukemia
inhibitory factors, oncostatin-M, and cardiotropin-1 belonging to
IL-6 type cytokines are known to transmit signals through a
receptor complex containing at least one signal-transmitting
protein gp130. For example, IL-6 links to IL-6R, and the resulting
IL-6.cndot.IL-6R complex contains gp130.
[0003] The hematopoiesis system is one of the living body
protection systems in which IL-6 plays an important role. The
process for expression of IL-6R in hematopoietic cells is different
between human cells and mouse cells. Undifferentiated human
hematopoietic precursor cells which can form a granulocyte
macrophage colony, an erythroblast colony, a megakaryocyte colony,
and a mixed colony thereof on a methylcellulose culture do not
express a sufficient amount of IL-6R (Tajima et al.: J.Exp.Med.
184, 1996). Therefore, the undifferentiated human hematopoietic
precursor cell is almost unreactive to IL-6, whereas it is strongly
reactive to an IL-6.cndot.IL-6R complex. In contrast, the
undifferentiated mouse hematopoietic precursor cell expresses a
sufficient amount of IL-6R. Nakahata, and the inventors of the
present invention found that the human megakaryocte precursor cell
does not express a sufficient amount of IL-6 (Japanese Patent
Application No. 9-325847). These findings are consistent with the
fact that IL-6 administered to a mouse increases significantly the
number of blood platelets and the IL-6 administered to a human body
is limited in its effect.
[0004] The linking constant between IL-6 and soluble IL-6R is
reported to be 5.times.10.sup.-9 M (Yasukawa et al.: J.Biochem.
vol.108, p.673, 1990). This means, that in a mixture of 200 ng/mL
(1.times.10.sup.-8 M) of IL-6 (mw: 20000) with 500 ng/mL
(1.times.10.sup.-8 M) of soluble IL-6R (mw: 50000), half of the
molecules will exist in an unlinked state. Actually, soluble IL-6R
is required in an amount of 1000 ng/mL or more to react it with
cells not expressing IL-6R.
[0005] By genetic engineering, two kinds of proteins existing
independently in nature can be fused into a fusion protein of one
polypeptide chain. When two kinds of proteins linkable with each
other are expressed as a fusion protein, presumably the bonding is
strong and dissociation is less liable to occur as long as the
respective proteins in the fused state can keep the inherent
structures (biologically active structure)
[0006] For two kinds of proteins in a fused protein to take the
inherent structures, steric hindrance should not be caused between
the two proteins. Further, the two proteins in the fused protein
having the inherent structures should have freedom degrees for
contacting each other. Therefore, a linker is employed
conventionally for fusion of the proteins: the linker being a
sequence of 5-20 amino acid residues such as a glycine residue and
a serine residue having a high freedom degree for linking of two
proteins. By such indirect fusion of proteins through a linker not
related to the fused proteins, the steric hindrance between the two
proteins is avoided and the freedom degree for the linking is
achieved.
[0007] For example, for expression of a fusion protein by bonding a
V domain of an H chain with a V domain of an L chain of antibodies
affinitive for bonding, a linker sequence is disclosed which has a
residue sequence of GGGGSGGGGSGGGGS (G: glycine residue, S: serine
residue) (SEQ ID NO:61) (Houston et al.: Proc.Natl.Acad.Sci. USA,
85, p.5879, 1988).
[0008] Fusion proteins were prepared recently from IL-6 and IL-6R
linkable to each other with a linker (illustrated in FIG. 1(a)).
One is a fusion protein prepared in a manner such that an
irrelevant linker RGGGGSGGGGSVE (SEQ ID NO:60) not contained in
IL-6 and IL-6R is bonded to the C-terminal 323th alanine residue in
IL-6R, and IL-6 is bonded to its C terminal side (Fisher et al.:
Nature Biotech, 15, p.142, 1997). A second one is a fusion protein
prepared in a manner such that a linker EFM (E: glutamic acid
residue, F: phenylalanine residue, and M: methionine residue) is
bonded to the C-terminal 356th valine residue of IL-6R, and further
IL-6 is bonded to its C terminal side (Chebath et al.: Eur.
Cytokine Netw, 8, p.359, 1997). The inventors of the present
invention discovered a fusion protein prepared by bonding a linker
SSELV (L: leucine residue, V: valine residue) (SEQ ID NO:62) to the
C-terminal 334th leucine residue of IL-6R, and further bonding IL-6
to the C-terminal thereof (Japanese patent Application No.
10-2921).
[0009] Generally, it is known that a foreign protein expressed by
genetic recombination can be cleaved by a naturally expressed
protease in the host. Therefore, the fusion protein
IL-6R.cndot.IL-6 having expressed may also be cleaved by protease
in the host. In particular, the yeast Pichia pastoris is known to
express various proteases. However, the aforementioned reports do
not describe this matter. If the fusion protein IL-6R.cndot.IL-6 is
cleaved by a protease expressed in the host, preparation of a
fusion protein IL-6R.cndot.IL-6 resistant to the cleavage enables
production of a larger amount of the fusion protein
IL-6R.cndot.IL-6 from the culture of the yeast Pichia pastoris or
other host.
[0010] A fusion protein like IL-6R.cndot.IL-6, which can maintains
firmly the fused state of two proteins, is expected to be
particularly effective in a fused state as a medicine in signal
transmission system of IL-6 in a human body or a like animal.
[0011] In development of a fusion protein as a periodically dosed
medicine, high possibility of the aforementioned immune reaction is
a problem, since the linker is not contained in the proteins and
irrelevant to them, and has an independent steric structure.
Therefore, the linker has preferably a sequence as short as
possible, or nonuse of the linker is desirable.
[0012] As described above, for constituting the fusion protein, the
two constituting proteins should not cause steric hindrance between
them, and should have freedom for linking together. For example,
for direct fusion of IL-6R and IL-6 without using a linker without
causing steric hindrance with a freedom degree for linking
together, many factors should be decided such as the order of the
proteins in fusion, the amino acid residue in the N-terminal side
protein and the that in the C-terminal side protein.
[0013] As the fusion protein of IL-6R with IL-6, only three
examples mentioned above have been reported which employ a linker,
and the fusion necessarily requires a linker. FIG. 1(a) illustrates
a fusion protein in which two proteins are linked together through
a linker. The direct linking without the linker has not been
reported yet.
[0014] The present invention intends to provide a IL-6R.cndot.IL-6
fusion protein in which IL-6R and IL-6 are linked directly without
a linker as illustrated in FIG. 1(b). The present invention intends
also to provide an IL-6R.cndot.IL-6 fusion protein which is
resistant to cleaving action of the protease expressed by a host,
especially a Pichia pastoris type yeast, and a gene for coding for
the fusion protein.
DISCLOSURE OF THE INVENTION
[0015] To achieve the above objects, after comprehensive
investigation on IL-6R.cndot.IL-6 fusion protein, the inventors of
the present invention completed a fusion protein having IL-6R at
the N-terminal side and IL-6 at the C-terminal side linked without
employing a linker therebetween. The present invention relates to
an IL-6R.cndot.IL-6 fusion protein in which one amino acid residue
constituting IL-6R and one amino acid residue constituting IL-6 are
directly bonded. Further, the inventors of the present invention
found that lysine of the 37th residue from the N-terminal of IL-6
can be cleaved by protease at its C-terminal side, and has
completed a protease-resistant IL-6R.cndot.IL-6 fusion protein by
modification at the protease-cleavable site, especially an
IL-6R.cndot.IL-6 fusion protein in which IL-6 with deletion of 10
amino acid residues from 28th alanine residue to 37th lysine
residue of the N-terminal side of the IL-6R is linked to C-terminal
of the IL-6R, and also completed a gene for coding for it.
[0016] The present invention provides also a gene for coding for
IL-6R.cndot.IL-6 fusion protein in which one amino acid residue of
IL-6R and one amino acid residue of IL-6 are linked directly.
[0017] The present invention further provides a yeast of Pichia
pastoris species which is transformed by an expression vector
containing a gene for coding for IL-6R.cndot.IL-6 fusion protein in
which one amino acid residue of IL-6R and one amino acid residue of
IL-6 are linked directly.
[0018] The present invention still further provides a process for
producing an IL-6R.cndot.IL-6 fusion protein, comprising a step of
cultivating in a culture medium a yeast of a Pichia pastoris
species having been transformed by an expression vector containing
a gene for coding for an IL-6R.cndot.IL-6 fusion protein in which
one amino acid residue of IL-6R and one amino acid residue of IL-6
are linked directly; and a step of collecting the IL-6R.cndot.IL-6
fusion protein as a secretory protein from the culture medium.
[0019] The present invention still further provides a process for
producing an IL-6R.cndot.IL-6 fusion protein, comprising
cultivating a yeast of a Pichia pastoris species having been
transformed by an expression vector containing a gene for coding
for an IL-6R.cndot.IL-6 fusion protein in which one amino acid
residue of IL-6R and one amino acid residue of IL-6 are linked
directly, in a culture medium of natural origin containing a carbon
source and no methanol, and adding methanol during progress of the
cultivation.
[0020] The present invention still further provides a process for
producing an IL-6R.cndot.IL-6 fusion protein, comprising subjecting
a solution containing an IL-6R.cndot.IL-6 fusion protein, in which
one amino acid residue of IL-6R and one amino acid residue of IL-6
are directly linked, to three steps of chromatography including
ion-exchange chromatography, hydrophobic chromatography, and
gel-filtration chromatography to collect the IL-6R.cndot.IL-6
fusion protein.
[0021] The present invention still further provides an ex vivo
amplifier for a hematopoietic stem cells, comprising an
IL-6R.cndot.IL-6 fusion protein in which one amino acid residue of
IL-6R and one amino acid residue of IL-6 are directly linked.
[0022] The present invention still further provides a blood
platelet growing agent containing as a main component an
IL-6R.cndot.IL-6 fusion protein in which one amino acid residue of
IL-6R and one amino acid residue of IL-6 are directly linked. The
present invention is described below in detail.
[0023] The fusion protein of the present invention is characterized
in that IL-6R is placed at the N-terminal side of the fusion
protein, and IL-6 is placed at the C-terminal side thereof, and the
two proteins are linked directly without a linker. For improving
the resistance to the protease as mentioned above, the two protein
may be linked by a polypeptide linker. However, in view of lower
antigenicity in medicine dose, preferred is the direct linking
without polypeptide linker. When the useful linker includes known
linker sequences (Fisher et al.: Nature Biotech, 15, p.142, 1997;
and Chebath et al.: Eur. Cytokine Netw, 8, p.142, 1997), or in
another example thereof, to the C-terminal 344th leucine residue
position of IL-6R, a linker composed of serine residue/serine
residue/glutamic acid residue/leucine residue/valine residue is
linked, and further to the C-terminal side thereof, IL-6 is linked.
The IL-6R constituting the IL-6R.cndot.IL-6 of the present
invention is a membrane protein having 468 amino acid residues in
full length (SEQ ID NO:63), and comprises a signal region, an
extracellular region, a transmembrane region, and an intracellular
region (Yamasaki et al.: Science, 241, p.825, 1988). In human
IL-6R, it is presumed that the signal region ranges from the
methionine residue at the first position of the N-terminal to about
the alanine residue at 19th position; the extracellular region
ranges from about the leucine residue at the 20th position to about
the aspartic acid residue at the 358th position; the transmembrane
region ranges from about the serine residue at 359th position to
about the leucine residue at the 386th position; and the
intracellular region ranges from about arginine residue at the
387th position to about the arginine residue at 468th position. The
extracellular region is classified into an immunoglobulin-like
domain and a cytokine receptor domain, presumably the
immunoglobulinlike domain ranging from about the leucine residue at
the 20th position to the aspartic acid residue at the 111th
position, and the cytokine receptor domain ranging from about the
valine residue at about the 112th position to about the alanine
residue at the 323th position.
[0024] In the IL-6R, it is known that the domain essential to the
linking to IL-6 is the cytokine receptor domain and the
immunoglobulin-like domain is not necessary. The cytokine receptor
domain is a structure constituted of two short barrel-shaped
structure, each composed of seven .beta.-sheets (Yawata et al.:
EMBO J., 12, p.1705, 1993).
[0025] In the present invention, not only the IL-6R in full length
is useful, but also the entire of the extracellular region, or the
cytokine receptor region, namely a part of the IL-6R is useful.
This is because the cytokine region can constitute the signal
transmission system by combining with the IL-6, and the
extracellular region includes that domain.
[0026] According to the information acquired by the inventors of
the present invention, specific examples of the N-terminal of IL-6R
are the leucine residue at the 20th position, the valine residue at
the 112th position, and the glutamic acid residue at the 116th
position from the N-terminal.
[0027] Further, according to the information acquired by the
inventors of the present invention, specific examples of the
C-terminal of IL-6R are any one of 39 amino acid residues in the
range from the N-terminal 323th alanine residue to the 361th serine
residue; preferably any one of the six amino acid of the 323th
alanine residue, the 333th alanine residue, the 334th leucine
residue, the 335th threonine residue, the lysine 338th residue, and
the 343th isoleucine residue. To the C-terminal side of the
fraction of the IL-6R, the N-terminal of IL-6 is linked. The
N-terminal of the IL-6R may be deleted appropriately in
consideration of the effects in the signal transmission of the
fusion protein.
[0028] The IL-6 constituting the IL-6R.cndot.IL-6 fusion protein is
a secretory protein composed of 212 amino acid residues in full
length (SEQ ID NO:64) having four .alpha.-helixes (Hirano et al.:
Nature, 324, vol. 731, 1986). The four .alpha.-helixes are known to
be all necessary for the activity of the IL-6. Therefore, the IL-6
used in the present invention is not specially limited, provided
that it has all of the four helixes. In other words, not only the
full length of the IL-6 is useful, but also a partial IL-6 in which
a part of amino acids at the N-terminal or the C-terminal are
deleted can be useful. An example of the partial IL-6 is the one
having a sequence from the N-terminal 28th alanine residue or the
29th proline residue to the N-terminal 212th methionine residue,
which is known as the structure of the secretory IL-6. Otherwise, a
sequence of the partial IL-6 may be decided by reference to
examples of expression of IL-6 (e.g., Yasukawa et al.: Biotech.
Lett., 12, p.419, 1990), or examples of expression of
IL-6R.cndot.IL-6 fusion protein (Fischer et al.: Nature Biotech.,
15, p.142, 1997).
[0029] With the fusion protein made protease-resistant by
introducing modification at the protease-cleaving site in the
primary structure, the modification and the site thereof is decided
suitably to meet the kind of the protease having different cleaving
sites: the site of deletion of the amino acid, the site of addition
of foreign amino acid residue, or the site of substitution of the
amino acid to be resistant to the cleavage by the protease. In the
modification by substitution, the inherent steric configuration is
changed by substitution not to be cleaved or to be less liable to
be cleaved by a protease. In the substitution, the intended amino
acid is substituted preferably by a smaller-sized amino acid such
as glycine and serine. In the modification by the deletion, the
protein molecule is modified by deletion of the intended amino acid
to prevent the recognition of the cleavage site by the protease, or
to change the inherent steric configuration not to be cleaved or to
be less liable to be cleaved.
[0030] The modification can be introduced by genetic engineering to
a gene for coding for the IL-6R.cndot.IL-6 fusion protein. The
modification may be any of deletion of an amino acid, insertion of
an amino acid, and substitution of an amino acid. Of the
modifications, deletion of an amino acid or amino acid sequence at
or around the protease cleavage site is particularly preferred,
since the operation therefor is relatively easy and the influence
of substitution of the non-inherent amino acid on the antigenicity
or other properties is considered to be less.
[0031] In genetic production of the IL-6R.cndot.IL-6 fusion protein
of the present invention, the specific standard of the selection is
impartation of resistance to protease secreted by the host cell
employed. More specifically, as shown later in Examples, the
IL-6R.cndot.IL-6 fusion protein to be produced genetically is
expressed in a selected host cells, and the expressed product is
subjected to purification by a conventional liquid chromatography
or other purification process. In the purification process, the
substance of the peak which seems to be the decomposition product
by the protease is collected, or in an SDS-PAGE method, the
substance of the band which seems to be the decomposition product
by the protease is collected. The N-terminal amino acid sequence is
analyzed to find the cleaved site of the IL-6R.cndot.IL-6 fusion
protein cleaved by the protease secreted by the selected host
cells.
[0032] After detection of the site cleaved by the protease of the
IL-6R.cndot.IL-6 fusion protein in the primary structure, the
IL-6R.cndot.IL-6 fusion protein which is resistant to the protease
can be obtained by deletion of the amino acid residues around the
cleaved site or other modification method so as not to be cleaved
or to be less liable to be cleaved by the protease.
[0033] Further, the protease-resistant IL-6R.cndot.IL-6 fusion
protein of the present invention is useful for retaining the
biological activity of IL-6R.cndot.IL-6 fusion protein in a
medicine for a longer time in the presence of a protease. For
producing the IL-6R.cndot.IL-6 fusion protein of this purpose, the
protease which can maintain the activity in the environment for
exhibiting a biological activity of the IL-6R.cndot.IL-6 fusion
protein is obtained, the site of cleavage of the unmodified
IL-6R.cndot.IL-6 fusion protein by the protease is detected, and
the site is modified as described above.
[0034] The IL-6R.cndot.IL-6 fusion protein modified as above is
preferably tested for confirmation of the retention of its
biological activity after the modification. For the confirmation,
the modified IL-6R.cndot.IL-6 fusion protein is prepared, and is
tested, for instance, as shown later in Example by use of a BAF130
cell strain. If the modified IL-6R.cndot.IL-6 fusion protein is
found to be not biologically active in this confirmation, another
modification is introduced, and the confirmation is conducted again
in the same manner as above.
[0035] Further, the modified IL-6R.cndot.IL-6 fusion protein is
preferably tested for confirmation of its resistance to the
protease. For this confirmation, the modified IL-6R.cndot.IL-6
fusion protein is allowed to coexist with the protease for a
certain time, and the protein is analyzed, for instance, by
combination of the SDS-PAGE and the western blotting. If the
protein is found to be not resistant to the protease, another
modification is introduced, and the confirmation is conducted again
in the same manner as above. The IL-6R.cndot.IL-6 fusion protein,
which has been made resistant to the protease secreted by the host
cells has been imparted, is produced by use of the same host cells,
and the confirmation test is conducted with the cell culture in the
same manner as above.
[0036] The IL-6R.cndot.IL-6 fusion protein of the present invention
can readily be produced with a gene for coding for it by a gene
recombination technique. The gene for coding for the IL-6R or IL-6
has already been isolated, and the base sequence is well known.
Therefore, the fusion protein of the present invention can be
produced by preparing the necessary gene sequences from the amino
acid sequences of the IL-6R and the IL-6, and linking them by use
of a restriction enzyme. Instead of employing the natural gene
sequence, a codon therein may be replaced by another codon which
codes for the same amino acid residue but has a different base
sequence in consideration of the codon condensation. This is
because the use of a specific codon can sometimes improves the
expression ratio or the translation ratio in expression of a
protein by gene recombination.
[0037] The host for production of the fusion protein of the present
invention by gene recombination is not limited specially, and
Escherichia coli, or an animal cells typified by CHO cells usually
employed in gene recombination operation may be used according to
literature (Yasukawa et al.: J.Biotech., 108, p.673, 1990). Of the
hosts, Pichia pastoris yeast employed in Examples is particularly
preferred since this yeast is capable of growing with methanol as
the only carbon source, and can be cultivated at a low cost in
comparison with the animal cells like CHO cells.
[0038] An example of the IL-6R.cndot.IL-6 fusion protein resistant
to the protease secreted by the yeast of Pichia pastoris species
which is a suitable host cells in genetic production of the
IL-6R.cndot.IL-6 fusion protein is the one derived from an IL-6R
with a modified IL-6 in which at least the sequence from the
N-terminal 28th alanine residue to the N-terminal 37th lysine
residue is deleted. The protease-resistant IL-6R.cndot.IL-6 fusion
protein of the present invention can be mass-produced by preparing
a gene for coding for the protease-resistant IL-6R.cndot.IL-6
fusion protein, preparing an expression vector having the gene
incorporated therein, transforming the host cells, and cultivating
the transformed host cells. The IL-6R.cndot.IL-6 fusion protein
made resistant to protease secreted by the host cells as described
above is not cleaved or hardly cleaved by the protease after it is
expressed in the host cells. Therefore, it can readily be produced
in a larger amount in comparison with the non-resistant
IL-6R.cndot.IL-6 fusion protein.
[0039] The above-described gene for producing the fusion protein of
the present invention prepared by gene recombination technique is
incorporated into an expression vector to introduce it into the
host (for transformation). Into the expression vector, an
expression-controlling gene, a gene for selecting the transformed
host, and so forth are incorporated in addition to the
aforementioned gene. The genes to be incorporated are selected
suitably depending on the host employed. For instance, when the
yeast of Pichia pastoris strain is used as the host, there are
introduced the upstream sequence and the downstream sequence of an
alcohol oxidase gene for introduction of the gene for coding for
the IL-6R.cndot.IL-6 fusion protein in chromosomal DNA, a histidine
synthesis gene as a selection indicator, and a promotor sequence of
an alcohol oxidase gene for expression control; and when E. coli is
used as the host, there are introduced an ampicillin-resistant gene
as a selection indicator, and a Lac promotor/operator sequence. A
commercial expression vector (e.g., pPIC9, an expression vector for
the Pichia pastoris strain yeast, produced by Invitrogen Co.) can
be used by introducing the gene of the present invention.
[0040] In the present invention, when the Pichia pastoris strain
yeast is transformed by an expression vector containing a gene for
coding for the IL-6R.cndot.IL-6 fusion protein for the production
of the fusion protein, are preferably incorporated, into the
expression vector, the inherent signal peptide of IL-6R and a
signal peptide of .alpha.-factor as the signal peptide of the
fusion protein, especially a signal peptide of the .alpha.-factor
for high expression.
[0041] The fusion protein can be produced by cultivating the
aforementioned transformed host under suitable conditions and
causing expression of the fusion protein in relation to the
expression-controlling gene in the expression vector as necessary.
In an example of the present invention, a jar fermenter is used
when employing preferably Pichia pastoris yeast as the host.
Specifically, a preliminarily prepared 20% glycerol-frozen yeast of
Pichia pastoris strain capable of expressing the IL-6R.cndot.IL-6
fusion protein is inoculated into a 100 mL of a culture contained
in a 500-mL shaking flask. The yeast is cultivated at 28-30.degree.
C. for 20 hours. The resulting 100-mL liquid culture is inoculated
into a 6-9 L of culture contained in a 16-L jar fermenter, and is
cultivated at 28-30.degree. C. with aeration and stirring. For
monitoring the state of the cultivated yeast, the OD600, the pH,
the dissolved oxygen concentration, stirring rate, and the
temperature are preferably monitored and controlled. The culture
medium is not limited, provided that a carbon source of natural
origin is contained. Specific compositions are shown in Examples. A
commercial jar fermenter may be used for the cultivation. During
the cultivation, methanol is added when the glycerol has been
consumed totally. The consumption of the glycerol is detected by
monitoring the dissolved oxygen. The methanol is preferably added
within five hours after the complete consumption. An excessive
amount of methanol is toxic to the yeast, whereas the deficiency of
methanol suppresses the function of the promoter sequence of the
alcohol oxidase gene. Therefore, the amount of the methanol is
preferably not less than 0.5% and not more than 5%
(weight/volume).
[0042] The fusion protein produced by the cultivation is collected
from the culture by a suitable method. In the case where
Escherichia coli is employed as the host, the expressed fusion
protein is accumulated as insoluble granules in the E. coli cells,
so that the E. coli mass is crushed and subjected to refolding or
purification under suitable conditions. In the case where the
Pichia pastoris yeast is employed as the host, the fusion protein
can be obtained by purification from the supernatant liquid of the
culture. The material solution for purification is not limited,
provided that it contains the fusion protein. An example of the
liquid is a liquid culture of Pichia pastoris yeast containing the
fusion protein. The solution may be treated as it is, but may be
treated after dilution with a buffer solution or pure water, or
after concentration by ultrafiltration or by use of ammonium
sulfate. The purification may be conducted by liquid
chromatography. Preferably three types of chromatography,
ion-exchange chromatography, hydrophobic chromatography, and gel
filtration chromatography may be employed in combination.
[0043] The supernatant liquid culture of Pichia pastoris yeast,
since it is voluminous, is preferably treated firstly by
ion-exchange chromatography. The ion-exchange chromatography
includes cation chromatography and anion chromatography, and either
may employed suitably in consideration of protein removal
efficiency. For example, in the cation chromatography, SP is used
as the cation exchange functional group; and in the anion
chromatography, DEAE is used and the anion exchange functional
group. In consideration of the flow rate of 100 mL/min or higher
and the nature of the sample solution not treated with a filtered
of 1 .mu.m or a smaller pore size, a preferred example is the
cation chromatography employing a fluidized adsorption bed, Stream
line SP C-50 column (produced by Amasham Pharmacia Co.). The
fraction containing the fusion protein obtained above is then
subjected to hydrophobic chromatography to obtain a fraction
containing the fusion protein of a higher purity. This fraction is
concentrated by cation chromatography to conduct efficiently the
subsequent gel filtration chromatography as shown later in
Examples.
[0044] The inventors of the present invention investigated
comprehensively the stem cell amplification effect of the
IL-6R.cndot.IL-6 fusion protein in which one of the amino acid
residues of the IL-6R and one of the amino acid residues of IL-6
are linked directly. As the results, it was found that, in
cultivation of CD34-positive cells on a methylcellulose plate in
the presence of the fusion protein and a stem cell factor (SCF),
the colony-forming ability is increased remarkably in comparison
with the reported one in cultivation in the presence of IL-6,
IL-6R, and SCF. The present invention is accomplished based on ex
vivo amplification of hematopoietic stem cells by the
IL-6R.cndot.IL-6 fusion protein in which one of the amino acid
residues of IL-6R and one of the amino acid residues of IL-6 are
linked directly. The present invention relates to an ex vivo
amplifying agent for the hematopoietic stem cells, and an ex vivo
amplifying method for the hematopoietic stem cells by employing the
IL-6R.cndot.IL-6 fusion protein.
[0045] The fusion protein of the present invention is effective by
itself to a certain extent as an ex vivo amplifying agent for
hematopoietic stem cells, and is valuable practically. The effect
is made remarkable by addition of any one of cytokines stimulating
tyrosine kinase such as SCF, and FLK2 ligand, particularly SCF.
Further, interleukin-3 (IL-3) or a platelet proliferation factor
(TPO) may be added thereto. The hematopoietic stem cells can be
obtained from umbilical blood, peripheral blood, or bone marrow by
CD34 selection as a fraction containing the hematopoietic stem
cells. The fusion protein of the present invention can be supplied
as a kit comprising vials containing separately the fusion protein
of the present invention and another cytokine, or may be supplied
as a mixture thereof contained in one vial. The ex vivo
amplification of the hematopoietic stem cell can be conducted by
cultivating a fraction containing the hematopoietic cells in a
serum-free culture containing the fusion protein of the present
invention, and SCF or the like at 37.degree. C. for 1-3 weeks in a
vessel like a plastic bag. The amplified hematopoietic stem cells
can be returned to a patient in the same manner as the conventional
peripheral blood stem cell transfusion.
[0046] The inventors of the present invention investigated
comprehensively the blood platelet proliferation effect of the
IL-6R.cndot.IL-6 fusion protein in which one of the amino acid
residues of the IL-6R and one of the amino acid residues of IL-6
are linked directly. Consequently, as shown later in Example, it
was found that, in the mouse dosed with the fusion protein,
proliferation of blood platelets is significantly promoted, and
that, in the mouse preliminarily dosed with a carcinostatic agent
and dosed with the fusion protein, the recovery of the platelets is
accelerated significantly. The present invention is based on the
newly found effects on proliferation or recovery of blood platelets
by the fusion protein in which one of the amino acid residues of
IL-6R and one of the amino acid residues of IL-6 are linked
directly. The present invention relates also to a blood
platelet-proliferating agent containing the fusion protein as the
main component, and a method for proliferating the platelets with
the agent. The blood platelet-proliferating agent of the present
invention is preferably dosed by parenteral administration such as
intravenous administration, intramuscular administration, and
percutaneous administration. The amount of the dose is decided
depending on the kind of the disease causing the blood platelet
deficiency, the state of the patient, and so forth, generally
ranging from 1 to 500 .mu.g/kg/day. The agent is dosed periodically
depending on the state of proliferation of the blood platelets. The
blood platelet-proliferating agent of the present invention can be
formulated by mixing a conventional vehicle or an activator such as
a physiological saline, a glucose solution, mannitol,
methylcellulose, gelatin, and human serum albumin. The blood
platelet-proliferating agent of the present invention may be
freeze-dried. The freeze-dried product is redissolved before use in
an isotonic solution such as a physiological saline, a glucose
solution, and a Ringer's solution.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1(a) illustrates an IL-6R.cndot.IL-6 fusion protein
formed by linking IL-6R with IL-6 through a linker sequence. FIG.
1(b) illustrates an IL-6R.cndot.IL-6 fusion protein formed by
linking IL-6R with IL-6 directly without a linker.
[0048] FIG. 2 shows the structure of the plasmid pBS6R6S prepared
in Example 1, in which amp denotes an ampicillin-tolerant gene, ori
denotes a transcription initiation site, IL-6 denotes an IL-6 gene,
and IL-6R denotes an IL-6R gene.
[0049] FIG. 3 shows the procedure of insertion of two kinds of
annealed oligonucleotides into the plasmid pBS6R6S shown in FIG. 2
by the method shown in Example 2.
[0050] FIG. 4 shows the structure of the plasmid pBS6R6L prepared
in Example 1, in which amp denotes an ampicill-intolerant gene, ori
denotes a transcription initiation site, IL-6 denotes an IL-6 gene,
and IL-6R denotes an IL-6R gene.
[0051] FIG. 5 shows the procedure of insertion of two kinds of
annealed oligonucleotides into the plasmid pBS6R6L shown in FIG. 4
by the method shown in Example 2.
[0052] FIG. 6 shows the structure of the plasmid pBS6R6L for
expressing a derivative 333A.DELTA.A prepared in Example 2. In FIG.
6, amp denotes an ampicillin-tolerant gene; ori, a transcription
initiation site; HIS4, a histidine-synthesizing gene; 3'AOXTT, a
terminator; IL-6, an IL-6 gene; IL-6R, an IL-6R gene; S, a gene for
coding for a signal sequence; and 5'AOX1, an upstream region of an
alcohol oxidase gene containing a promoter sequence.
[0053] FIG. 7 shows (A) the kind and number of the amino acid at
C-terminal of IL-6R region, (B) the amino acid sequence (the symbol
"-" means absence of the linker), (C) the kind and number of the
amino acid at N-terminal of IL-6R region, (D) the average
biological activity for the respective 216 culture medium
supernatant liquids derived in Example 4 and measured by the method
shown in example 5.
[0054] FIG. 8 shows the change caused in the cultivation as shown
in Example 6 as a function of the culture time. In FIG. 6, MeOH
conc. signifies a methanol concentration (unit: % (wt/vol)); EIA
signifies sandwich immunoassay, and Bio Assay signifies a
biological activity.
[0055] FIG. 9 shows the biological activity of FP6 as measured by
the method of Example 8.
[0056] FIG. 10 shows the result of SDS polyacrylamide gel
electrophoresis of FP6 having treated by endoglycosidase H for 0 to
180 minutes according to the method shown in Example 10. In FIG.
10, the lane of symbol H shows the simple endoglycosidase H, and
the lanes denoted by the numerals 0 to 180 show the results of PF6
treated with the endoglycosidase H for the time (minutes) denoted
by the numeral respectively. The bar marks denoted by the numerals
97.4 and 66.3 show respectively the position of detection of the
molecular weight marker (molecular weight of 97.4 kDa, or 66.3
kDa).
[0057] FIG. 11 shows the colony-forming activity of 500
CD34-positive cells by the method of Example 11 with (1)
combination of IL-6 (100 ng/mL) and IL-6R (100 ng/mL), (2)
combination of IL-6 (100 ng/mL) and IL-6R (200 ng/mL), (3) FP6 (300
ng/mL), and (4) FP-6 (600 ng/mL). In FIG. 11, G denotes a
granulocyte colony; M, a macrophage colony; GM, a
granulocyte/macrophage mixed colony; Blast, a blast colony; Mix, a
granulocyte/macrophage/erythroblast mixed colony; and BFU-E, an
erythroblast colony.
[0058] FIG. 12 shows average values of the number of blood
platelets of C57BL6 mouse not dosed with FP6 (Group 1), and dosed
with FP6 (Groups 2-5) in Example 12. In FIG. 12, the asterisk shows
a significant difference from the blood platelet number from that
of Group 1 (P<0.05).
[0059] FIG. 13 shows average values of the number of blood
platelets of C57BL6 mouse preliminarily dosed with 5FU, and not
dosed with FP6 (Group 1, solid squares) and dosed with FP6 (Groups
2, hollow squares) in Example 13. In FIG. 13, the asterisk shows a
significant difference from the blood platelet number from that of
Group 1 (P<0.05).
[0060] FIG. 14 shows relative average biological activities of (1)
nine strains of mut.sup.s capable of expression of fusion protein
112VAA, and (2) 11 strains of mut.sup.s capable of expression of
fusion protein 116EAA derived in Example 14 measured according to
the method of Example 5 as the relative values (%) to III-108
strain which was the most active of 11 strains of mut.sup.s in
expressing 333A.DELTA.A.
[0061] FIG. 15 shows the results of western blotting of culture
medium supernatant liquid containing (a) 20LAA, (b) 20LAD, (c)
116EAA, (d) 116EAD, (e) 112VAA, and (f) 112VAD according to the
method shown in Example 8.
BEST MODE FOR PRACTICING THE INVENTION
[0062] Examples are shown below for explaining the invention in
more detail. However, the present invention is not limited by the
Examples.
EXAMPLE 1
Preparation of Intermediate Plasmids
[0063] Intermediate plasmids pBS6RS and pBS6RL were prepared by
inserting an oligonucleotide coding for a linker for the purpose of
preparing a gene (CDNA) coding for an IL-6R.cndot.IL-6 fusion
protein linked through a linker as below.
[0064] Firstly, a cloning vector, pBluescript II KS(-) (produced by
Toyobo Co.), was cut by a restriction enzyme, KpnI, treated with a
Klenow fragment, and subjected to ligation reaction to obtain
plasmid pBS with the KpnI site deleted.
[0065] Then IL-6R gene (cDNA) was amplified by using a primer
p6RAB20L (SEQ ID NO:45) and a primer p6RF320S (SEQ ID NO:46), and
was cut by XhoI. This was inserted into pBS having preliminarily
been cut by XhoI and EcoRV to obtain pBS6R.
[0066] IL-6 gene (cDNA) was amplified by using a primer pIL6B2 (SEQ
ID NO:47) and a primer pIL6F (SEQ ID NO:48), and was cut by Bgl II
and NotI. This was inserted into a plasmid pBS6R having
preliminarily been cut by Bgl II and NotI to obtain pBS6R6S. FIG. 2
shows the structure of the obtained pBS6R6S.
[0067] Then, an oligomers 320S333Ab (SEQ ID NO:49) and 320S333Af
(SEQ ID NO:50) were annealed in a conventional manner. This was
inserted into the plasmid pBS6R6S having preliminarily been cut by
Bgl II and Kpn I to obtain pBS6R6L. FIG. 4 shows the structure of
the obtained pBS6R6L.
EXAMPLE 2
Preparation of Expression Plasmids
[0068] Into pBS6R6S having preliminarily been cut by Bgl II and
KpnI, Annealed Sequences 1-5 obtained by annealing two kinds of
oligonucleotides were respectively inserted as shown below to
obtain five kinds of plasmids having as an insert a gene for coding
for the IL-6R.cndot.IL-6 fusion protein.
[0069] Annealed Sequence 1 (323AG4SA): The oligonucleotide of SEQ
ID NO:1 is at the sense side, and the oligonucleotide of SEQ ID
NO:2 is at the antisense side.
[0070] Annealed Sequence 2 (323AG4S2A): The oligonucleotide of SEQ
ID NO:3 is at the sense side, and the oligonucleotide of SEQ ID
NO:4 is at the antisense side.
[0071] Annealed Sequence 3 (334LPA): The oligonucleotide of SEQ ID
NO:5 is at the sense side, and the oligonucleotide of SEQ ID NO:6
is at the antisense side.
[0072] Annealed Sequence 4 (333A.DELTA.A): The oligonucleotide of
SEQ ID NO:7 is at the sense side, and the oligonucleotide of SEQ ID
NO:8 is at the antisense side.
[0073] Annealed Sequence 5 (323A.DELTA.A): The oligonucleotide of
SEQ ID NO:9 is at the sense side, and the oligonucleotide of SEQ ID
NO:10 is at the antisense side.
[0074] Annealed Sequence 1 (323AG4SA) expresses a fusion protein
formed by linking the N-terminal 323th alanine of IL-6R and the
N-terminal 28th alanine of IL-6 through a linker GGGGS (SEQ ID
NO:65). Annealed Sequence 2 (323AG4S2A) expresses a fusion protein
formed by linking the N-terminal 323th alanine of IL-6R and the
N-terminal 28th alanine of IL-6 through a linker GGGGSGGGGS (SEQ ID
NO:66). Annealed Sequence 3 expresses a fusion protein formed by
linking the N-terminal 334th leucine of IL-6R and the N-terminal
28th alanine of IL-6 through a linker P. Annealed Sequence 4
(333A.DELTA.A) expresses a fusion protein of the present invention
formed by directly linking the N-terminal 333th alanine of IL-6R
and the N-terminal 28th alanine of IL-6 without a linker (SEQ ID
NO:67). Annealed Sequence 5 (323A.DELTA.A) expresses a fusion
protein of the present invention formed by directly linking the
N-terminal 323th alanine of IL-6R and the N-terminal 28th alanine
of IL-6 without a linker (SEQ ID NO:68).
[0075] Next, into pBS6R6L having preliminarily been cut by Eco47
III and KpnI, Annealed Sequences 6-22 obtained by annealing two
kinds of oligonucleotides are respectively inserted as shown below
to obtain 17 kinds of plasmids having a gene as an insert for
coding for the IL-6R.cndot.IL-6 fusion protein.
[0076] Annealed Sequence 6 (333AG4SA): The oligonucleotide of SEQ
ID NO:11 is at the sense side, and the oligonucleotide of SEQ ID
NO:12 is at the antisense side.
[0077] Annealed Sequence 7 (333AG4S2A): The oligonucleotide of SEQ
ID NO:13 is at the sense side, and the oligonucleotide of SEQ ID
NO:14 is at the antisense side.
[0078] Annealed Sequence 8 (343IG4S2A): The oligonucleotide of SEQ
ID NO:15 is at the sense side, and the oligonucleotide of SEQ ID
NO:16 is at the antisense side.
[0079] Annealed Sequence 9 (333AG4S3A): The oligonucleotide of SEQ
ID NO:17 is at the sense side, and the oligonucleotide of SEQ ID
NO:18 is at the antisense side.
[0080] Annealed Sequence 10 (333AG2A): The oligonucleotide of SEQ
ID NO:19 is at the sense side, and the oligonucleotide of SEQ ID
NO:20 is at the antisense side.
[0081] Annealed Sequence 11 (333AG4A): The oligonucleotide of SEQ
ID NO:21 is at the sense side, and the oligonucleotide of SEQ ID
NO:22 is at the antisense side.
[0082] Annealed Sequence 12 (343IG2A): The oligonucleotide of SEQ
ID NO:23 is at the sense side, and the oligonucleotide of SEQ ID
NO:24 is at the antisense side.
[0083] Annealed Sequence 13 (343IG4A): The oligonucleotide of SEQ
ID NO:25 is at the sense side, and the oligonucleotide of SEQ ID
NO:26 is at the antisense side.
[0084] Annealed Sequence 14 (361S.DELTA.A): The oligonucleotide of
SEQ ID NO:27 is at the sense side, and the oligonucleotide of SEQ
ID NO:28 is at the antisense side.
[0085] Annealed Sequence 15 (358D.DELTA.A): The oligonucleotide of
SEQ ID NO:29 is at the sense side, and the oligonucleotide of SEQ
ID NO:30 is at the antisense side.
[0086] Annealed Sequence 16 (352T.DELTA.A): The oligonucleotide of
SEQ ID NO:31 is at the sense side, and the oligonucleotide of SEQ
ID NO:32 is at the antisense side.
[0087] Annealed Sequence 17 (346R.DELTA.A): The oligonucleotide of
SEQ ID NO:33 is at the sense side, and the oligonucleotide of SEQ
ID NO:34 is at the antisense side.
[0088] Annealed Sequence 18 (343I.DELTA.A): The oligonucleotide of
SEQ ID NO:35 is at the sense side, and the oligonucleotide of SEQ
ID NO:36 is at the antisense side.
[0089] Annealed Sequence 19 (338K.DELTA.A): The oligonucleotide of
SEQ ID NO:37 is at the sense side, and the oligonucleotide of SEQ
ID NO:38 is at the antisense side.
[0090] Annealed Sequence 20 (335T.DELTA.A): The oligonucleotide of
SEQ ID NO:39 is at the sense side, and the oligonucleotide of SEQ
ID NO:40 is at the antisense side.
[0091] Annealed Sequence 21 (343I.DELTA.P): The oligonucleotide of
SEQ ID NO:41 is at the sense side, and the oligonucleotide of SEQ
ID NO:42 is at the antisense side.
[0092] Annealed Sequence 22 (338K.DELTA.P): The oligonucleotide of
SEQ ID NO:43 is at the sense side, and the oligonucleotide of SEQ
ID NO:44 is at the antisense side.
[0093] Annealed Sequence 6 (333AG4SA) expresses a fusion protein
formed by linking the N-terminal 333th alanine of IL-6R and the
N-terminal 28th alanine of IL-6 through a linker GGGGS (SEQ ID
NO:65). Annealed Sequence 7 (333AG4S2A) expresses a fusion protein
formed by linking the N-terminal 333th alanine of IL-6R and the
N-terminal 28th alanine of IL-6 through a linker GGGGSGGGGS (SEQ ID
NO:66). Annealed Sequence 8 (343IG4S2A) expresses a fusion protein
formed by linking the N-terminal 343th isoleucine of IL-6R and the
N-terminal 28th alanine of IL-6 through a linker GGGGSGGGGS (SEQ ID
NO:66). Annealed Sequence 9 (333AG4S3A) expresses a fusion protein
formed by linking the N-terminal 333th alanine of IL-6R and the
N-terminal 28th alanine of IL-6 through a linker GGGGSGGGGSGGGGS
(SEQ ID NO:61). Annealed Sequence 10 (333AG2A) expresses a fusion
protein formed by directly linking the N-terminal 333th alanine of
IL-6R and the N-terminal 28th alanine of IL-6 through a linker GG.
Annealed Sequence 11 (333AG4A) expresses a fusion protein formed by
linking the N-terminal 333th alanine of IL-6R and the N-terminal
28th alanine of IL-6 through a linker GGGG (SEQ ID NO:69). Annealed
Sequence 12 (343IG2A) expresses a fusion protein formed by linking
the N-terminal 343th isoleucine of IL-6R and the N-terminal 28th
alanine of IL-6 through a linker GG. Annealed Sequence 13 (343IG4A)
expresses a fusion protein formed by linking the N-terminal 343th
isoleucine of IL-6R and the N-terminal 28th alanine of IL-6 through
a linker GGGG (SEQ ID NO:69). Annealed Sequence 14 (361S.DELTA.A)
expresses a fusion protein of the present invention formed by
linking directly the N-terminal 361th serine of IL-6R and the
N-terminal 28th alanine of IL-6 without a linker (SEQ ID NO:70).
Annealed Sequence 15 (358D.DELTA.A) expresses a fusion protein of
the present invention formed by linking directly the N-terminal
358th aspartic acid of IL-6R and the N-terminal 28th alanine of
IL-6 without a linker (SEQ ID NO:71). Annealed Sequence 16
(352T.DELTA.A) expresses a fusion protein of the present invention
formed by directly linking the N-terminal 352th threonine of IL-6R
and the N-terminal 28th alanine of IL-6 without a linker (SEQ ID
NO:72). Annealed Sequence 17 (346R.DELTA.A) expresses a fusion
protein of the present invention formed by linking the N-terminal
346th arginine of IL-6R and the N-terminal 28th alanine of IL-6
without a linker (SEQ ID NO:73). Annealed Sequence 18
(343I.DELTA.A) expresses a fusion protein of the present invention
formed by linking directly the N-terminal 343th isoleucine of IL-6R
and the N-terminal 28th alanine of IL-6 without a linker(SEQ ID
NO:74). Annealed Sequence 19 (338K.DELTA.A) expresses a fusion
protein of the present invention formed by linking directly the
N-terminal 338th lysine of IL-6R and the N-terminal 28th alanine of
IL-6 without a linker (SEQ ID NO:75). Annealed Sequence 20
(335T.DELTA.A) expresses a fusion protein of the present invention
formed by directly linking the N-terminal 335th threonine of IL-6R
and the N-terminal 28th alanine of IL-6 without a linker (SEQ ID
NO:76). Annealed Sequence 21 (343I.DELTA.P) expresses a fusion
protein of the present invention formed by linking the N-terminal
343th isoleucine of IL-6R and the N-terminal 29th proline of IL-6
without a linker (SEQ ID NO:77). Annealed Sequence 22
(338K.DELTA.P) expresses a fusion protein of the present invention
formed by linking the N-terminal 338th lysine of IL-6R and the
N-terminal 29th proline of IL-6 without a linker (SEQ ID
NO:78).
[0094] The pBS6R6S itself has a gene for coding for the
IL-6R.cndot.IL-6 fusion protein as an insert. The fusion protein
(334LAV) expressed by this gene is a fusion protein of the present
invention formed by linking directly the N-terminal 334th leucine
of IL-6R and the N-terminal 30th valine of IL-6 without a linker
(SEQ ID NO:79).
[0095] Finally, the 23 kinds of plasmids obtained above and pBS6R6L
were cut respectively by XhoI and NotI to obtain genes for coding
for the IL-6R.cndot.IL-6 fusion protein. The genes were
respectively inserted into pPIC9 having been preliminarily cut by
XhoI and NotI to obtain 24 kinds of expression plasmids. FIG. 6
shows the structure of the expression plasmid 333A.DELTA.A as an
example.
EXAMPLE 3
Preparation of Transformants
[0096] From Pichia pastoris GS115 strain (Invitrongen Co.),
competent cells were prepared by use of an EasyComp Transformation
Kit (Invitrogen Co.), and thereto the respective expression
plasmids having been linearized by Bgl II were introduced. The
transformed cells were cultivated in a minimal nutrient culture
medium, and transformed cells having lost the histidine-requiring
property were selected.
[0097] The obtained transformants were inoculated onto MD plates
(1.34% (W/V) of YNB wo AA (Yeast Nitrogen Base Without Amino Acid),
0.00004% (W/V) of biotin, and 2% (W/V) of glucose), and MM plates
(1.34% (W/V) of YNB wo AA, 0.00004% (W/V) of biotin, and 0.5% (V/V)
of methanol). Thereby, the respective transformants were examined
for mut.sup.+ and mut.sup.s.
[0098] Table 1 shows the numbers of the obtained transformants and
the numbers of the obtained mut.sup.s strains for the respective
derivatives. For the respective derivatives, were obtained the
strains transformed by the expression plasmids, average 9.4 in
number of mut.sup.s strains (lowest: 4, highest: 17, total:
216).
1 TABLE 1 Number of obtained Number of obtained Derivative
transformants mut.sup.s strain 323AG4SA 60 13 333AG4SA 149 13
323AG4S2A 36 4 333AG4S2A 37 9 3431G4S2A 65 7 333AG4S3A 87 16 334LPA
33 13 333AG2A 70 10 333AG4A 49 8 3431G2A 60 7 3431G4A 100 14
361S.DELTA.A 23 6 358D.DELTA.A 100 7 352T.DELTA.A 106 17
346R.DELTA.A 54 7 343I.DELTA.A 27 10 343I.DELTA.P 52 5 338K.DELTA.A
93 6 338K.DELTA.P 111 11 334L.DELTA.V 27 5 335T.DELTA.A 92 9
333A.DELTA.A 81 11 323A.DELTA.A 56 8
EXAMPLE 4
Cultivation of Transformants
[0099] The mut.sup.s strains (216 kinds in total), and the
mut.sup.s strains which express a fusion protein formed by linking
the N-terminal 344th leucine of IL-6R and the N-terminal 28th
alanine of IL-6 through a linker SSELV (SEQ ID NO:62) (described in
Japanese Patent Application 10-2921) were respectively cultivated
at 30.degree. C. for 120 hours in test tubes containing 3 mL of
culture medium composed of a BMGY culture medium (1% (W/V) yeast
extract, 2% (W/V) peptone, 1.34% (W/V) of YNB wo AA (Yeast Nitrogen
Base Without Amino Acid), 0.4 mg/L of biotin, 100 mM potassium
phosphate (pH 6.0), and 1% (W/V) of glycerol). At 120 hours of
cultivation, a sample was taken out from the respective liquid
culture mediums. The samples were respectively centrifuged to
obtain a supernatant liquid.
EXAMPLE 5
Measurement of Biological Activity
[0100] The biological activity of the IL-6R.cndot.IL-6 fusion
proteins in the culture supernatant liquids derived in Example 4
was measured by a method employing BAF130 cells. The BAF130 is a
cell prepared by transforming a mouse cell BAF not expressing
inherently gp130 (Hatakeyama et al.: cell, 63, p.154, 1989) by
introducing a gene for coding for human gp130 protein to express
this protein. Therefore, this cell exhibits growth activity in the
presence of physiologically active IL-6R and IL-6.
[0101] A suspension of BAF130 was placed on 96-well plates in an
amount of 2.times.10.sup.4 cells per well. The 216 kinds of culture
supernatants were diluted to 1%, 0.25%. 0.061%, and 0.015%, and the
fractions of the diluted matters were respectively introduced to
the wells. Two days later, the light absorbance was measured at 405
nm with the reference wavelength of 600 nm by use of Cell Counting
Kit (produced by Wako Junyaku K. K.). For the standard, 1 .mu.g/mL
of IL-6 was diluted to several concentrations and was added with
100 ng/mL of soluble IL-6R in place of the culture supernatant. The
activity of the culture supernatant giving the same absorbance
dependency on the concentration as this is defined as 1 unit/mL.
FIG. 7 shows the average biological activities of the culture
supernatants of 4-17 kinds of mut.sup.s strains transformed
respectively by the same fusion protein expression vector for each
of the fusion proteins.
[0102] FIG. 7 shows that, in all of the 12 kinds of fusion proteins
having no linker sequence, at least one culture supernatant of the
mut.sup.s transformed by the same fusion protein expression vector
exhibited the activity. This means that the IL-6R.cndot.IL-6 fusion
protein of the present invention in which C-terminal of any one of
the 39 amino acid residues from N-terminal 323th alanine residue to
the N-terminal 361th serine residue is linked to the N-terminal
amino acid of IL-6 has a signal transmission property.
[0103] FIG. 7 shows also that the fusion proteins 323A.DELTA.A,
333A.DELTA.A, 334L.DELTA.V, 335T.DELTA.A, 338K.DELTA.P,
338K.DELTA.A, 343I.DELTA.P, and 343I.DELTA.A, which are
respectively an IL-6R.cndot.IL-6 fusion protein in which the amino
acid residue of the N-terminal of IL-6 is linked to C-terminal of
the 323th alanine residue, 333th alanine residue, 334th leucine
residue, 335th threonine residue, 338th lysine residue, or 343th
isoleucine, are more active than the known fusion protein
334LSSELVA (described in Japanese Patent Application No. 10-2921)
which is formed by linking the N-terminal 344th leucine of IL-6R
and the N-terminal 28th alanine of IL-6 through a linker SSELV.
[0104] FIG. 7 further shows that the fusion proteins 323A.DELTA.A,
333A.DELTA.A, 334L.DELTA.V, 335T.DELTA.A, 338K.DELTA.P,
338K.DELTA.A, 343I.DELTA.P, or 343I.DELTA.A, which are respectively
an IL-6RIL-6 fusion protein in which the amino acid residue of
N-terminal of IL-6 is linked to the C-terminal of the 323th alanine
residue, 333th alanine residue, 334th leucine residue, 335th
threonine residue, 338th lysine residue, or 343th isoleucine, are
nearly equally active or slightly less active than 323AG4SA,
333AG4SA, 323AG4S2A, 333AG4S2A, 343IG4S2A, and 333AG4S3A which are
respectively an IL-6R.cndot.IL-6 fusion protein formed by linking
with a linker of peptide composed of 5-15 amino acid residues
having a high freedom degree like a glycine residue or a serine
residue; or 334LPA, 333AG2A, 333AG4A, 343IG2A, and 343IG4A, which
are respectively an IL-6R.cndot.IL-6 fusion protein formed by
linking with a linker of peptide composed of 1-4 amino acids.
Therefore, of the IL-6R.cndot.IL-6 fusion proteins of the present
invention, particularly preferred are those in which the N-terminal
amino acid residue of IL-6 is linked to any one of the six amino
acid residues of the 323th alanine residue, the 333th alanine
residue, the 334th leucine residue, the 335th threonine residue,
the 338th lysine residue, and the 343 isoleucine residue.
EXAMPLE 6
Mass Cultivation of mut.sup.s Strains for Expressing Fusion
Protein
[0105] Of the eleven mut.sup.s strains expressing 333A.DELTA.A
described in Example 3, the most active III-108 strain in
expression in the method of Examples 4 and 5 was cultivated by use
of a 16-liter jar. A glycerol stock of III-108 strain was
inoculated into 100 mL of a culture medium BMGY (10 g/L of Bacto
Yeast Extract, 20 g/L of Bacto Peptone, 1.34 g/L of Yeast Nitrogen
Base without Amino Acid, 100 mM potassium phosphate (pH 6.0), 10
g/L of glycerol, and 0.4 mg/L of biotin), and cultivated at
30.degree. C. for 24 hours by a G-20 Shaking Cultivator
(manufactured by NBS Co.) at 200 rpm. The entire culture liquid was
inoculated into 8 liters of BMGY (10 g/L of Bacto Yeast Extract, 20
g/L of Bacto Peptone, 1.34 g/L of Yeast Nitrogen Base without Amino
Acid, 100 mM potassium phosphate (pH 6.0), 10 g/L of glycerol, and
0.4 mg/L of biotin), and cultivated at 28.degree. C. at stirring
rate of 350 rpm and aeration of 1 vvm by a 10-liter jar SF-116
(manufactured by NBS Co.). After 16 hours from the start of the
cultivation, was added 1.6 liters of a liquid mixture of 240 mL of
methanol, 50 g/L of Bacto Yeast Extract, and 100 g/L of Bacto
Peptone to initiate the introduction of expression of a fusion
protein. FIG. 8 shows the progress of the cultivation.
[0106] The methanol concentration in the liquid culture was
monitored by gas chromatography. The pH of the liquid culture was
measured by a pH meter. The OD600 value (index of the biomass) was
measured by means of a spectrophotometer for a 100-fold dilution of
the culture liquid with a physiological saline. The concentration
of the fusion protein was measured by sandwich enzyme immunoassay
by use of the anti-human IL-6R monochronal antibody MT-18 (Hirato
et al.: J.Immunol., vol.143, 2900 1989) as the solid antibody, the
anti-human IL-6 polychronal antibody (produced by Dienzyme Co.) as
the detecting antibody, and the purified fusion protein obtained in
Example 7 as the standard substance. The biological activity of the
fusion protein was measured by use of BAF 130 cells as shown in
Example 5.
EXAMPLE 7
Purification of Fusion Protein
[0107] The liquid culture picked up in Example 6 was centrifuged to
separate the biomass and the supernatant. The supernatant (11.6 L)
was diluted with a distilled water to obtain an electric
conductivity corresponding to about 50 mM NaCl concentration (66.05
L). Then the pH of the solution was adjusted to 4.5 by acetic
acid.
[0108] The above diluted solution was introduced to an fluidized
adsorption bed, Streamline SP C-50 column, (50 mm ID.times.100 cm,
gel volume 300 mL) having been equilibrated with a 20 mM acetate
buffer solution (pH 4.5) (produced by Amasham Pharmacia Co.) from
the column bottom upward (linear velocity: 300 cm/hour). After
completion of the introduction, the column was washed with the
equilbrating buffer solution introduced upward. Then the direction
of the liquid introduction was reversed, and an eluting buffer
solution (500 mM NaCl, 5% glycerol, 20 mM phosphate buffer solution
(pH 6.5)) was introduced downward from the column top (linear
velocity 150 cm/hour) to obtain an elution fraction (Streamline
elution fraction: 300 mL). The detection of the elution fraction
was conducted by measuring the light absorbance at 280 nm.
[0109] In the Streamline elution fraction, an ammonium sulfate
solution cooled to -20.degree. C. was dissolved to a concentration
of 2 M. The solution was introduced to a TSKgel Phenyl-5PW column
(21.5 mmID.times.15 cm, produced by Tosoh Corp.) having been
equilibrated with 2M ammonium sulfate solution and 20 mM phosphate
buffer solution (pH 6.5) (flow rate: 5 mL/min). After the
introduction, the equilibrating buffer solution was introduced
there to elute proteins which show weak hydrophobic interaction
with the adsorption group. Then the ammonium sulfate concentration
in the eluting buffer solution was gradually lowered, and the
fraction eluted at a 0.4M ammonium sulfate concentration was
collected as the elution fraction of fusion protein (Phenyl-5PW
elution fraction, 67 mL). The detection of the fusion protein was
conducted by the proliferation activity of BAF 130 cells as the
index as shown in Example 5.
[0110] The Phenyl-5PW elution fraction was desalted by dialysis
with 20 mM acetate buffer solution (pH 4.5) containing 5% glycerol.
The desalted solution was introduced to a TSKgel SP-5PW column (7.5
mmID.times.7.5 cm)(produced by Tosoh Corp.) (flow rate: 2 mL/min)
equilibrated with the same buffer solution. After the introduction,
the equilibrating buffer solution was allowed to flow to elute
proteins less interactive with the adsorption group. Then 20 mM
phosphate buffer solution (pH 6.5) containing 500 mM NaCl was
allowed to flow (1 mL/min) to obtain a fraction containing the
fusion protein at a high concentration (SP-5PW elution fraction, 10
mL). The detection of the fusion protein was conduced by absorbance
measurement at 280 nm.
[0111] The SP-5PW elution fraction was added portionwise (5 mL
every minute) to a TSKgel G3000SW column (21.5 mmID.times.30 cm)
having been equilibrated with a 20 mM phosphate buffer solution
containing 100 mM NaCl (pH 6.5). Thereby the fusion protein was
collected by measuring the absorbance at 280 nm.
EXAMPLE 8
Measurement of Activity of Fusion Protein
[0112] The fusion protein 333A.DELTA.A of the present invention
described in Example 5 and purified by the method described in
Example 7 (hereinafter referred to as FP6) was used in the
experiment below.
[0113] The biological activity of FP6 was measured at various
concentrations (0.625 to 40 ng/mL) according to the method of
measurement of biological activity shown in Example 5 employing
BAF130 cells. FIG. 9 shows obviously the concentration dependency
of FP6.
[0114] Not shown in the drawing, the concentration dependency curve
of FP6 derived by diluting the 1 .mu.g/mL FP6 solution to various
concentrations (1- to 1024-fold dilution) nearly coincides with the
concentration dependency curve of IL-6 derived by diluting the 5
.mu.g/mL solution to various concentrations (1- to 1024-fold
dilution). On the other hand, as shown in Example 5, the activity
of the culture supernatant giving the same concentration dependency
curve as the diluted solution of IL-6 in various concentrations
together with 100 ng/mL of soluble IL-6R is defined as one unit/mL.
Therefore, 1 .mu.g of FP6 was shown to correspond to 5 units.
EXAMPLE 9
Analysis of Amino Acid Sequence of Fusion Protein
[0115] The fusion protein 333A.DELTA.A of the present invention
described in Example 5 and purified by the method described in
Example 7 (hereinafter referred to as FP6) was used in the
experiment below.
[0116] FP-6 was further purified by reversed phase chromatography
with Phenyl-5PW RP column. The elution was conducted with a
gradient from 20% acetonitrile/0.1% aqueous trifluoroacetic acid
solution to 60% acetonitrile/0.1% aqueous trifluoroacetic acid
solution. The obtained fraction was dried up under a reduced
pressure. The solid was re-dissolved in aqueous 20%
acetonitrile/0.1% trifluoroacetic acid solution, and was analyzed
by protein sequencer 477A (manufactured by Applied Biosystem
Co.).
[0117] Consequently, leucine, alanine, proline, and arginine were
detected in this order from the N-terminal, which is consistent
with the N-terminal sequence coded by the gene.
EXAMPLE 10
Measurement of Molecular Weight of Sugar Chain Portion of Fusion
Protein
[0118] The fusion protein 333A.DELTA.A of the present invention
described in Example 5 and purified by the method described in
Example 7 (hereinafter referred to as FP6) was used in the
experiment below.
[0119] A 150 .mu.L portion of a solution of 0.1% SDS, and 100 mM
sodium chloride in 20 mM phosphate buffer solution (pH 6.0)
containing 0.1 mg/mL of FP6 was boiled for five minutes, and was
cooled by water. A portion of 10 pL of the solution was mixed with
a portion of 1 .mu.L of endoglycosidase H of 1 unit/mL (produced by
Sigma Co.). The mixtures prepared thus were allowed to react at
25.degree. C., for 30 seconds to 3 hours. The reaction was stopped
by addition of 10 .mu.L of 500 mM glycine/hydrochloric acid buffer
solution (pH 2.5). Separately, a reference sample of the
endoglycosidase H of reaction time of zero minute was provided by
mixing successively 10 .mu.L of a 500 mM glycine/hydrochloric acid
buffer (pH 2.5), 10 .mu.L of a preliminarily cooled FP6 solution,
and 1 .mu.L of endoglycosidase H.
[0120] To the solution in which the reaction has been stopped, 3
.mu.L of 72% glycerol and 10% 2-mercaptoethanol colored with
bromophenyl blue. This solution was subjected to SDS polyacrylamide
gel electrophoresis. After the electrophoresis, the protein in the
gel was detected by staining with Coomassie Brilliant Blue
R250.
[0121] As shown clearly in FIG. 10, the FP6 not having reacted with
the endoglycosidase H was detected as one bond at the position
ranging from 76 to 93 kDa. This means that the molecular weight had
a nonuniformity range of 17 kDa. The FP6 having completely digested
by the endoglycosidase H was detected at the position of 57 kDa.
Not shown in FIG. 9, the FP6 which was denatured with SDS and was
reacted with a ten-fold amount of Triton X-100, and N-glycosidase
for 18 hours, was detected a the position of 57 kDa. This shows
that the molecular weight of the sugar chain bonded by Nglycoside
linkage of FP6 ranges from 19 to 36 kDa.
EXAMPLE 11
Effect of Fusion-Protein on Proliferation of Stem Cells
[0122] The fusion protein 333A.DELTA.A of the present invention
described in Example 5 and purified by the method described in
Example 7 (hereinafter referred to as FP6) was used in the
experiment below.
[0123] CD34-positive cells were isolated and purified from 20 mL of
human umbilical blood according to the method disclosed in Japanese
Patent Application No. 9-325847. The obtained 500 cells were
cultivated with 1.2% methylcellulose (Shin-Etsu Kagaku K. K.), 30%
bovine serum albumin (Hyclone Laboratories Inc.), 1% bovine serum
albumin (hereinafter referred to as BSA) (Sigma Co.), 0.05 mM
2-mercaptoethanol (Sigma Co.), and SCF (stem cell factor) (100
ng/mL) under any of the conditions of (1) combination of IL-6 (100
ng/mL) and IL-6R (100 ng/mL), (2) combination of IL-6 (100 ng/mL)
and IL-6R (200 ng/mL), (3) FP6 (300 ng/mL), and (4) FP6 (600
ng/mL), with dispensation of 1 mL fractions of .alpha.-MEM (Flow
Co.) on a 35-mm plastic cultivation plate (Nunc Co.) for suspension
cultivation at 37.degree. C., 5% CO.sub.2, and humidity 100%.
[0124] Two weeks later, the colonies were identified by observation
with an inverted microscope. FIG. 11 show the results. The colonies
were classified into six kinds: a granulocyte colony (G), a
macrophage colony (M), granulocyte-macrophage mixed colony (GM), a
blast colony (Blast), a
granulocyte.cndot.macrophage.cndot.erythroblast mixed colony (Mix),
and erythroblast colony (BFU-E).
[0125] As shown in FIG. 11, under the known Conditions (1) and (2),
a colony-forming activity was nearly the same as the reported one
(Sui et al.: Proc.Natl.Acad.Sci. USA, 92, p.2589, 1995). On the
other hand, under Condition (3) (FP6 (300 ng/mL)) of the present
invention, the colonyforming activity was much higher than that of
the same concentration of the combination of IL-6 (100 ng/mL) and
IL-R (200 ng/mL). Although not shown in the drawing, the sizes of
the colonies formed under Condition (3) or (4) of the present
invention were remarkably larger than that of the known Condition
(1) or (2). This shows that the fusion protein of the present
invention has ex vivo proliferation effect for hematopoietic stem
cells higher than the known combination of IL-6 with IL-6R.
EXAMPLE 12
Blood Platelet Proliferation Effect of Fusion Protein for Mouse
[0126] The fusion protein 333A.DELTA.A of the present invention
described in Example 5 and purified by the method described in
Example 7 (hereinafter referred to as FP6) was used in the
experiment below.
[0127] Five groups of C57BL6 mouse (male, 8 weeks old), each group
having five mice, were employed. The first group mice as an
FP6-nondosed group (control group) were dosed with 300 .mu.L of PBS
and 1% bovine serum albumin (BSA); the second group mice were dosed
with 300 .mu.L of PBS containing 0.5 .mu.g of FP6, and 1% bovine
serum albumin (BSA); the third group mice were dosed with 300 .mu.L
of PBS containing 1 .mu.g of FP6, and 1% bovine serum albumin
(BSA); the fourth group mice were dosed with 300 .mu.L of PBS
containing 2 .mu.g of FP6, and 1% bovine serum albumin (BSA); and
the fifth group mice were dosed with 300 .mu.L of PBS containing 5
.mu.g of FP6, and 1% bovine serum albumin (BSA). The dose was given
to each of the mice intraperitoneally every twelve hours, namely
twice a day, for five days. On the sixth day, the mice were
exsanguinated from the descending vein. The number of blood
platelets was counted by means of a hemocytometer CC-180A
(manufactured by Toa Iyou Densi K. K.).
[0128] As shown in FIG. 12, the groups dosed with 1 .mu.g or more
of FP6 (third to fifth groups) showed significant proliferation of
blood platelets in comparison with the FP6-nondosed group (first
group). On the other hand, it is reported that the dose of 0.5
.mu.g of IL-6 (molecular weight: about 21 kDa) having twice number
of molecules than 1 .mu.g of FP6 (molecular weight: about 84 kDa)
under the same conditions did not cause significant proliferation
of blood platelets (Ishibashi et al.: Blood, 74, p.1241, 1989).
This means the higher effect of blood platelet proliferation in
vivo of the fusion protein of the present invention in comparison
with IL-6.
EXAMPLE 13
Blood Platelet Recovery Effect of Fusion Protein for 5FU-Dosed
Mouse
[0129] The fusion protein 333A.DELTA.A of the present invention
described in Example 5 and purified by the method described in
Example 7 (hereinafter referred to as FP6) was used in the
experiment below.
[0130] Two groups of C57BL6 mice (male, 8 weeks old), each group
having 15 mice, were employed. The first-group mice as an
FP6-nondosed group (control group) were dosed with 300 .mu.L of PBS
and 1% bovine serum albumin (BSA); and the second group mice were
dosed with 300 .mu.L of PBS containing 5 .mu.g of FP6, and 1%
bovine serum albumin. All of the mice were dosed with 150 mg/kg of
5FU into the tail vein. The above medical agent was dosed to each
of the mouse intraperitoneally every twelve hours, namely twice a
day, for five days from the second day to the sixth day. Every day
from the seventh day to the ninth day, the five mice of each of the
groups were exsanguinated from the descending artery. The number of
blood platelets was counted by means of a hemocytometer CC-180A
(manufactured by Toa Iyoh Denshi K. K.).
[0131] FIG. 13 shows clearly that the group dosed with FP6 (Second
Group) recovered the blood platelets significantly at eighth day
and ninth day in comparison with the group not dosed with FP6
(First Group). This means that the fusion protein of the present
invention has an effect of recovery in vivo of blood platelet
production function having lowered by dose of 5FU.
EXAMPLE 14
Expression of Immunoglobulin-Like Domain-Deficient Type Fusion
Protein
[0132] An expression plasmid for coding for an IL-6 derivative
112VL (233 amino acids from the N-terminal 112th valine to the
N-terminal 344th leucine) , and another expression plasmid for
coding for an IL-6 derivative 116EL (229 amino acids from the
N-terminal 116th glutamic acid to the N-terminal 344th leucine)
were constructed as below.
[0133] An IL-6R gene (cDNA) was amplified with a primer p6RAB112V
(SEQ ID NO:51) and a primer p344F (SEQ ID NO:52), and was cut by
Xhol and XbaI. The cut product was inserted into pPIC9 having been
cut preliminarily by Xhol and Avr II to obtain pPIC9-112VL.
[0134] An IL-6R gene (cDNA) was amplified with a primer p6RAB116E
(SEQ ID NO:53) and a primer p344F (SEQ ID NO:54), and was cut by
Xhol and XbaI. The cut product was inserted into pPIC9 having been
cut preliminarily by Xhol and Avr II to obtain pPIC9-116EL.
[0135] An expression plasmid pPIC9-112VAA coding for an
immunoglobulin-like domain-deficient type fusion protein (a fusion
protein of the present invention having as the N-terminal 112th
valine of IL-6R, and the N-terminal 333th alanine of IL-6R and the
N-terminal 28th alanine of IR-6 being directly bonded without a
linker), and an expression plasmid pPIC9-116EEA coding for the
fusion protein 116EAA (a fusion protein of the present invention
having as the N-terminal the N-terminal 116th glutamic acid of
IL-6R, and the N-terminal 333th alanine of IL-6R and the N-terminal
28th alanine of IR-6 being directly bonded without a linker) were
constructed as below.
[0136] A fragment obtained by cutting pPIC9-112VL by XhoI and PmaCI
was inserted to a 333A.DELTA.A-expression plasmid pPIC9-333A A
having been preliminarily cut by XhoI and PmaCI to obtain
pPIC9-112VAA.
[0137] A fragment obtained by cutting pPIC9-116EL by XhoI and PmaCI
was inserted to a 333A.DELTA.A-expression plasmid
pPIC9-333A.DELTA.A having been preliminarily cut by XhoI and PmaCI
to obtain pPIC9-116EAA.
[0138] Transformants were prepared with the above pPIC9-112VAA, and
pPIC9-116EAA according to the method shown in Example 3. As the
results, 9 mut5 strains for expressing 112VAA and 11 mut.sup.s
strains for expressing 116EAA were established. These were
cultivated according to the method shown in Example 4, and the
biological activity of the liquid supernatant was measured
according to the method shown in Example 5.
[0139] The immunoglobulin-like domain-deficient fusion proteins
112VAA and 116EAA had the activities nearly equal to the activity
of fusion protein 333A.DELTA.A having the immunoglobulin-like
domain as shown clearly in FIG. 14.
EXAMPLE 15
Determination of the Protease Cutting Site
[0140] To 1.9 mL of the Streamline elution fraction derived in
Example 7, were added 0.1 mL of 2% SDS, 64% glycerol, and 0.25%
bromophenol blue. The mixture was boiled. This was subjected to SDS
polyacrylamide gel electrophoresis, and was blotted electrically in
10% methanol/10 mM CAPS buffer solution (pH 11) onto a film of
polyvinylidene difluoride (PVDF). The blots were detected by
staining with Coomassie Brilliant Blue G-250. The detected protein
portions were cut and recovered respectively, and were tested by
Protein Sequencer 477A (manufactured by Applied Biosystem Co.).
Consequently, the protein of a molecular weight 18 kD was found to
be a portion of the fusion protein having aspartic acid
residue/valine residue/alanine residue/alanine residue/proline
residue/histidine residue, which was produced by cutting of the
peptide linkage by a protease of Pichia yeast between the
N-terminal 37th lysine residue and the N-terminal 38th aspartic
acid residue of IL-6 portion.
EXAMPLE 16
Expression of Protease-Resistant Fusion Protein (1)
[0141] From pBS6R6L obtained as in Example 1, IL-6 gene (cDNA) was
amplified by using a primer pKN6B38D (SEQ ID NO:55) and a primer
pIL6F2 (SEQ ID NO:56), and was cut by NruI and NotI. This was
inserted to pBS6R6L having been preliminarily cut by Eco47III and
NotI to obtain a plasmid pBS6R6L-38D.
[0142] The pBS6R6L-38D was cut by XhoI and NotI to obtain a gene
for coding for IL-6R.cndot.IL-6 fusion protein, and this was
inserted in pPIC9 having been preliminarily cut by XhoI and NotI to
obtain an expression plasmid pPIC9-20LAD for coding for a
protease-resistant fusion protein 20LAD.
[0143] IL-6R gene was amplified by using a primer p6RAB112V (SEQ ID
NO:57) and a primer p344F (SEQ ID NO:58), and was cut by XhoI and
XbaI. This was inserted to pPIC9 having been preliminarily cut by
XhoI and AvrII to obtain a pPIC9-112VL. The pPIC9-112VL was cut by
XhoI and PmaCI. The obtained fraction was inserted in pPIC9-20LAA
having been preliminarily cut by XhoI and PmaCI. Thereby, an
expression plasmid pPIC9-112VAA was prepared which codes for the
immunoglobulin-like domain-deficient fusion protein 112VAA (fusion
protein having the N-terminal 112th valine of IL-6R as the
N-terminal, and directly linked between the N-terminal 333th
alanine of. IL-6R and the N-terminal 28th alanine of IL-6 without
interposition of a linker).
[0144] Separately, IL-6R gene was amplified by using a primer
p6RABl16E (SEQ ID NO:59) and a primer p344F (SEQ ID NO:58), and was
cut by XhoI and XbaI. This was inserted to pPIC9 having been
preliminarily cut by XhoI and AvrII to obtain a pPIC9-116EL. The
pPIC9-116EL was cut by XhoI and PmaCI. The obtained fraction was
inserted in pPIC9-20LAA having been preliminarily cut by XhoI and
PmaCI. Thereby, an expression plasmid pPIC9-116EAA was prepared
which codes for the immunoglobulin-like domain-deficient fusion
protein 116EAA (fusion protein having the N-terminal 116th glutamic
acid of IL-6R as the N-terminal, and directly linked between the
N-terminal 333th alanine of IL-6R and the N-terminal 28th alanine
of IL-6 without interposition of a linker).
[0145] The pPIC9-20LAD was cut by XhoI and PmaCI to obtain a gene
for coding for IL-6R. This was inserted in pPIC9-112VAA having been
preliminarily cut by XhoI and PmaCI to obtain a protease-resistant
fusion protein 112VAD of the present invention (fusion protein
having the N-terminal 112th valine of IL-6R as the N-terminal, and
directly linked between the N-terminal 333th alanine of IL-6R and
the N-terminal 38th aspartic acid of IL-6 without interposition of
a linker). Separately, the above cut pPIC9-20LAD was inserted in
pPIC9-116EAA having been preliminarily cut by XhoI and PmaCI to
obtain a protease-resistant fusion protein 116EAD of the present
invention (fusion protein having the N-terminal 116th glutamic acid
of IL-6R as the N-terminal, and directly bonded between the
N-terminal 333th alanine of IL-6R and the N-terminal 38th aspartic
acid of IL-6 without interposition of a linker).
EXAMPLE 17
Expression of Protease-Resistant Fusion Protein (2)
[0146] The five expression plasmids described in Example 16
(pPIC9-20LAD, pPIC9-112VAA, pPIC9-116EAA, pPIC9-112VAD, and
pPIC9-116EAD) were respectively introduced to Pichia yeast
according to the method described in Example 3. Consequently, there
were obtained 10 strains of mut.sup.s transformed by pPIC9-20LAD, 7
strains of mut.sup.s transformed by pPIC9-112VAA, 10 strains of
mut.sup.s transformed by pPIC9-116EAA, 1 strain of mut.sup.s
transformed by pPIC9-112VAD, and 6 strains of mut.sup.s transformed
by pPIC9-116EAD. These strains were cultivated according to the
method described in Example 4, and the supernatant liquids were
collected. Their shown in Example 5. Consequently, biologically
active were 10 strains out of 10 mut.sup.s strains transformed by
pPIC9-20LAD, 7 strains out of 7 mut.sup.s strains transformed by
pPIC9-112VAA, 7 strains of 10 mut.sup.s strains transformed by
pPIC9-116EAA, 1 strain of 1 mut.sup.s strain transformed by
pPIC9-112VAD, and 6 strains out of 6 mut.sup.s strains transformed
by pPIC9-116EAD. This means that the deletion of the sequence of
the N-terminal 28th alanine to the N-terminal 37th lysine of IL-6
does not affect the biological activity.
EXAMPLE 18
Evaluation of Resistance to Protease
[0147] The transformant strains which express respectively
333A.DELTA.A (hereinafter referred to as "20LAA") described in
Example 3, and the protease-resistant fusion protein 20LAD of the
present invention, the fusion protein 112VAA, the
protease-resistant fusion protein 112VAD of the present invention,
the fusion protein 116EAA, and the protease-resistant fusion
protein 116EAD of the present invention described in Example 17
were cultivated by the method as show below. The cultivation was
conducted in a test tube with 3 mL of a BMGY culture medium (1%
(W/V) yeast extract, 2% (W/V) peptone, 1.34% (W/V) YNB wo AA,
0.00004% (W/V) biotin, 100 mM potassium phosphate (pH 6.0), and 1%
(W/V) glycerol) at 30.degree. C. for 48 hours. The culture was
centrifuged to collect the biomass. The biomass pellet was
suspended in 2 mL of a BMMY culture medium (1% (W/V) yeast extract,
2% (W/V) peptone, 1.34% (W/V) YNB wo AA, 0.00004% (W/V) biotin, 100
mM potassium phosphate (pH 6.0), and 0.5% (W/V) methanol), and
cultivated at 30.degree. C. for 24 hours. At the time of the
cultivation for 24 hours, a sample was taken from each of the
liquid cultures. The sample was centrifuged to recover the culture
supernatant, and the supernatant was subjected to SDS
polyacrylamide gel electrophoresis. After the electrophoresis, the
proteins in the gel was transferred to a PVDF film, and tested by
western blotting with rabbit anti-IL-6 polyclonal antibody
(produced by Dienzyme Co.). FIG. 15 shows the results.
[0148] As shown in FIG. 15, the band of the molecular weight 18 kD
presumably caused by cleavage at the C-terminal of the N-terminal
37th lysine of IL-6 region was detected clearly for 20LAA, 116EAA,
and 112VAA, whereas the band was not detected or hardly detected
for the protease-resistant fusion proteins 20LAD, 116EAD, and
112VAD of the present invention. This means that the modification
by deletion of the sequence of 10 amino acids from the 28th alanine
to the 37th lysine gave the protease resistance.
Industrial Applicability
[0149] The IL-6R.cndot.IL-6 fusion protein of the present invention
having no linker sequence is promising in increasing medical
efficacy and lowering remarkably antigenicity. Therefore, this
fusion protein is important as a novel remedy in the hematopoiesis
field, and is expected in development as an ex vivo amplifier of
hematopoietic stem cells and the proliferating agent for blood
platelets.
[0150] The fusion protein made resistant to the protease secreted
by the host cells can be readily mass-produced more efficiently by
the reason that the fusion protein dose not cut by the protease in
a production process by genetic transformation of host cells, and
other reasons. The final production yield can be raised because the
fusion protein is not cleaved in a purification process in the
presence of the protease. The purification process can be
simplified since deactivation of the protease before the
purification may be omitted advantageously. Furthermore, the
purified product can be more uniform, since contamination of the
final purified product by cleaved molecules is not caused.
[0151] In addition to the above effects, the substance which is
resistant to the protease is expected to retain the biological
activity even in the environment in which protease may be present.
Sequence CWU 1
1
60 1 39 DNA Artificial Sequence Annealed Sequence 1 gatcccctcc
agctggcggt ggtggatccg ccccggtac 39 2 31 DNA Artificial Sequence
Annealed Sequence 2 cggggcggat ccaccaccgc cagctggagg g 31 3 54 DNA
Artificial Sequence Annealed Sequence 3 gatcccctcc agctggtggc
ggtggctcgg gcggtggtgg gtcggccccg gtac 54 4 46 DNA Artificial
Sequence Annealed Sequence 4 cggggccgac ccaccaccgc ccgagccacc
gccaccagct ggaggg 46 5 60 DNA Artificial Sequence Annealed Sequence
5 gatcccctcc agctgagaac gaggtgtcca cccccatgca ggcacttcca gccccggtac
60 6 52 DNA Artificial Sequence Annealed Sequence 6 cggggctgga
agtgcctgca tgggggtgga cacctcgttc tcagctggag gg 52 7 54 DNA
Artificial Sequence Annealed Sequence 7 gatcccctcc agctgagaac
gaggtgtcca cccccatgca ggcagccccg gtac 54 8 46 DNA Artificial
Sequence Annealed Sequence 8 cggggctgcc tgcatggggg tggacacctc
gttctcagct ggaggg 46 9 24 DNA Artificial Sequence Annealed Sequence
9 gatcccctcc agctgccccg gtac 24 10 16 DNA Artificial Sequence
Annealed Sequence 10 cggggcagct ggaggg 16 11 26 DNA Artificial
Sequence Annealed Sequence 11 aggtggcggt ggatccgccc cggtac 26 12 22
DNA Artificial Sequence Annealed Sequence 12 cggggcggat ccaccgccac
ct 22 13 41 DNA Artificial Sequence Annealed Sequence 13 aggtggcggt
ggctcgggcg gtggtgggtc ggccccggta c 41 14 37 DNA Artificial Sequence
Annealed Sequence 14 cggggccgac ccaccaccgc ccgagccacc gccacct 37 15
71 DNA Artificial Sequence Annealed Sequence 15 acttactact
aataaagacg atgataatat tggtggcggt ggctcgggcg gtggtgggtc 60
ggccccggta c 71 16 67 DNA Artificial Sequence Annealed Sequence 16
cggggccgac ccaccaccgc ccgagccacc gccaccaata ttatcatcgt ctttattagt
60 agtaagt 67 17 56 DNA Artificial Sequence Annealed Sequence 17
aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc ggatctgccc cggtac 56 18
52 DNA Artificial Sequence Annealed Sequence 18 cggggcagat
ccgccgccac ccgacccacc accgcccgag ccaccgccac ct 52 19 17 DNA
Artificial Sequence Annealed Sequence 19 aggtggcgcc ccggtac 17 20
13 DNA Artificial Sequence Annealed Sequence 20 cggggcgcca cct 13
21 23 DNA Artificial Sequence Annealed Sequence 21 aggtggcggt
ggcgccccgg tac 23 22 19 DNA Artificial Sequence Annealed Sequence
22 cggggcgcca ccgccacct 19 23 47 DNA Artificial Sequence Annealed
Sequence 23 acttactact aataaagacg atgataatat tggtggcgcc ccggtac 47
24 43 DNA Artificial Sequence Annealed Sequence 24 cggggcgcca
ccaatattat catcgtcttt attagtagta agt 43 25 53 DNA Artificial
Sequence Annealed Sequence 25 acttactact aataaagacg atgataatat
tggtggcggt ggcgccccgg tac 53 26 49 DNA Artificial Sequence Annealed
Sequence 26 cggggcgcca ccgccaccaa tattatcatc gtctttatta gtagtaagt
49 27 95 DNA Artificial Sequence Annealed Sequence 27 acttactact
aataaagacg atgataatat tctcttcaga gattctgcaa atgcgacaag 60
cctcccagtg caagattctt cttcagcccc ggtac 95 28 91 DNA Artificial
Sequence Annealed Sequence 28 cggggctgaa gaagaatctt gcactgggag
gcttgtcgca tttgcagaat ctctgaagag 60 aatattatca tcgtctttat
tagtagtaag t 91 29 86 DNA Artificial Sequence Annealed Sequence 29
acttactact aataaagacg atgataatat tctcttcaga gattctgcaa atgcgacaag
60 cctcccagtg caagatgccc cggtac 86 30 82 DNA Artificial Sequence
Annealed Sequence 30 cggggcatct tgcactggga ggcttgtcgc atttgcagaa
tctctgaaga gaatattatc 60 atcgtcttta ttagtagtaa gt 82 31 68 DNA
Artificial Sequence Annealed Sequence 31 acttactact aataaagacg
atgataatat tctcttcaga gattctgcaa atgcgacagc 60 cccggtac 68 32 64
DNA Artificial Sequence Annealed Sequence 32 cggggctgtc gcatttgcag
aatctctgaa gagaatatta tcatcgtctt tattagtagt 60 aagt 64 33 50 DNA
Artificial Sequence Annealed Sequence 33 acttactact aataaagacg
atgataatat tctcttcaga gccccggatc 50 34 46 DNA Artificial Sequence
Annealed Sequence 34 cggggctctg aagagaatat tatcatcgtc tttattagta
gtaagt 46 35 41 DNA Artificial Sequence Annealed Sequence 35
acttactact aataaagacg atgataatat tgccccggta c 41 36 37 DNA
Artificial Sequence Annealed Sequence 36 cggggcaata ttatcatcgt
ctttattagt agtaagt 37 37 26 DNA Artificial Sequence Annealed
Sequence 37 acttactact aataaagccc cggtac 26 38 22 DNA Artificial
Sequence Annealed Sequence 38 cggggcttta ttagtagtaa gt 22 39 17 DNA
Artificial Sequence Annealed Sequence 39 acttactgcc ccggtac 17 40
13 DNA Artificial Sequence Annealed Sequence 40 cggggcagta agt 13
41 38 DNA Artificial Sequence Annealed Sequence 41 acttactact
aataaagacg atgataatat tccggtac 38 42 34 DNA Artificial Sequence
Annealed Sequence 42 cggaatatta tcatcgtctt tattagtagt aagt 34 43 23
DNA Artificial Sequence Annealed Sequence 43 acttactact aataaaccgg
tac 23 44 19 DNA Artificial Sequence Annealed Sequence 44
cggtttatta gtagtaagt 19 45 32 DNA Artificial Sequence Primer 45
ctcgagaaga ggctggcccc aaggcgctgc cc 32 46 33 DNA Artificial
Sequence Primer 46 agatctggat tctgtccaag gcgtgcccat ggc 33 47 28
DNA Artificial Sequence Primer 47 agatctggta cccccaggag aagattcc 28
48 29 DNA Artificial Sequence Primer 48 atgcggccgc tacatttgcc
gaagagccc 29 49 51 DNA Artificial Sequence Oligomer 49 gatcccctcc
agctgagaac gaggtgtcca cccccatgca agcgctggta c 51 50 43 DNA
Artificial Sequence Oilgomer 50 cagcgcttgc atgggggtgg acacctcgtt
ctcagctgga ggg 43 51 33 DNA Artificial Sequence Primer 51
ctcgagaaga gggttccccc cgaggagccc cag 33 52 22 DNA Artificial
Sequence Primer 52 tctctagaga atattatcat cg 22 53 29 DNA Artificial
Sequence Primer 53 ctcgagaaga gggagcccca gctctcctg 29 54 22 DNA
Artificial Sequence Primer 54 tctctagaga atattatcat cg 22 55 24 DNA
Artificial Sequence Primer 55 tctcgcgatg tagccgcccc acac 24 56 29
DNA Artificial Sequence Primer 56 atgcggccgc tacatttgcc gaagagccc
29 57 33 DNA Artificial Sequence Primer 57 ctcgagaaga gggttccccc
cgaggagccc cag 33 58 22 DNA Artificial Sequence Primer 58
tctctagaga atattatcat cg 22 59 29 DNA Artificial Sequence Primer 59
ctcgagaaga gggagcccca gctctcctg 29 60 13 PRT Artificial Sequence
Linker 60 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Glu 1 5
10
* * * * *