JP2001008690A - IL-6 receptor / IL-6 direct fusion protein - Google Patents

Abstract

(57) [Problem] To provide an IL-6R / IL-6 fusion protein or the like in which IL-6R and IL-6 are directly bound without a linker. An IL-6 receptor / IL-6 fusion protein characterized in that one of the amino acid residues constituting the IL-6 receptor and one of the amino acid residues constituting the IL-6 are directly bound. .

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an interleukin-6 receptor (hereinafter abbreviated as IL-6R) / interleukin-6 (hereinafter referred to as I-6) directly linked without a linker sequence.
L-6) fusion protein, a gene encoding the fusion protein, a host transformed with an expression vector containing the gene, a method of culturing the host, and purifying the fusion protein from a culture of the host The present invention relates to a method, a novel ex vivo amplifying agent for hematopoietic stem cells comprising the fusion protein, and a novel agent for increasing platelets containing the fusion protein as a main component.

[0002]

2. Description of the Related Art IL-6, IL-11, and Ciliary neurot belong to IL-6 type cytokines.
ropic factor, Leukemia inh
ifactory factor, oncostatin
Both -M and Cardiotropin-1 are known to transmit signals via at least one receptor complex containing the signaling protein gp130. Taking IL-6 as an example, IL-6 is IL-6R
And the resulting IL-6.IL-6R complex binds to gp130.

[0003] The hematopoietic system is one of the biological defense systems in which IL-6 plays an important role. IL-6 in hematopoietic cells
The expression pattern of R is different between human and mouse. Human undifferentiated hematopoietic progenitor cells capable of forming granulocyte-macrophage colonies, erythroblast colonies, megakaryocyte colonies, and mixed colonies on methylcellulose medium do not express a sufficient number of IL-6R (Tajima et al.,
J. Exp. Med. 184, 1996). Therefore, human undifferentiated hematopoietic progenitor cells hardly react to IL-6, but strongly react to the IL-6 / IL-6R complex. On the other hand, undifferentiated hematopoietic progenitor cells of mice express a sufficient number of IL-6R. Furthermore, Nakahata and the present inventors have determined that human megakaryocyte progenitor cells have a sufficient number of ILs.
It was found that -6R was not expressed (1997 Patent Application No. 325847). These findings indicate that when IL-6 is administered to mice, a significant increase in platelet count is observed,
Consistent with the limited effect of administering IL-6 to humans.

It has been reported that the binding constant between IL-6 and soluble IL-6R is 5 × 10 ⁻⁹ M (Yasuk).
awa et al. Biochem. 108 volumes, 673 pages,
1990). This is 200 ng / ml (1 × 10
^-8 M) IL-6 (molecular weight 20,000) and 500 ng / ml
When (1 × 10 ⁻⁸ M) soluble IL-6R (molecular weight: 50,000) is mixed, it means that half of the molecules exist alone. In fact, to fully act on cells that do not express IL-6R, soluble IL-6 of 1000 ng / ml or more is required.
R is required.

[0005] When genetic engineering is used, two types of proteins that are naturally present as separate proteins can be expressed as a fusion protein consisting of a single polypeptide chain. When two kinds of proteins having the property of binding to each other are expressed as a fusion protein, if the two kinds of proteins can take their original structures (structures capable of expressing a biological activity) in a fused state, both of them can be expressed. It is conceivable that the bond becomes stronger and dissociation is less likely to occur than in the case where no fusion is performed.

In order for two kinds of proteins to take an original structure in a fusion protein, it is necessary that steric hindrance does not occur between the two kinds of proteins. Furthermore, it is necessary that the two types of proteins having the original structure fused have a degree of freedom such that they can bind to each other. For this reason, conventionally, it is usual to connect 5 to 20 amino acid residues having a high degree of freedom, such as glycine residues and serine residues, as a linker, and to fuse the protein via the linker. By indirectly fusing through an inherently unrelated linker that is not included in the proteins to be fused, two types of proteins do not cause steric hindrance and can bind to each other. The degree of freedom can be given.

For example, when the V region of the H chain and the V region of the L chain of an antibody having the property of binding to each other are expressed as a fusion protein, GGGGSGGGGGSGGGGS (G is a glycine residue, S is a serine residue) is a linker sequence. (Houston et al., Proc. Nat).
l. Acad. Sci. USA, 85, 5879, 1
988).

[0008] IL-6R and I having the property of binding to each other
As for L-6, a fusion protein in which both are linked via a linker (FIG. 1 (a) is an illustration) has recently been reported. One is RGGG represented by SEQ ID NO: 60 on the C-terminal side of the alanine residue at position 323 of IL-6R.
GSGGGGSVE is a fusion protein which is not contained in IL-6 and IL-6R and which is not originally contained in IL-6 and IL-6R.
isher et al., Nature Biotech, 15,
142, 1997). In the second, a linker called EFM (E is a glutamic acid residue, F is a phenylalanine residue, and M is a methionine residue) is bonded to the C-terminal of the 356th valine residue of IL-6R, and further to the C-terminal side. It is a fusion protein bound to IL-6 (Chebat
h et al., Eur. CytokineNetw. , 8,35
9, p. 1997). In addition, the present applicants also reported that IL-6
SSELV at the C-terminal of the 344th leucine residue of R
(L is a leucine residue, V is a valic acid residue) and a fusion protein in which IL-6 is further bonded to the C-terminal side of the linker (1998 Patent Application No. 2921).
issue).

In general, it has been pointed out that when a heterologous protein is produced by genetic recombination, the expressed heterologous protein is cleaved by the action of a protease naturally expressed by the host. Therefore, the IL-6R / IL-6 fusion protein may be cleaved by the host protease after being expressed in the host. In particular, it is known that yeast of the species Pichia pastoris expresses various proteases, but this is not mentioned in the above-mentioned report. If the IL-6R / IL-6 fusion protein is cleaved by the cleavage action of a protease expressed by the host, by providing an IL-6R / IL-6 fusion protein resistant to such cleavage action, Larger amount of IL-6R-IL- than before
6 fusion proteins can be obtained from cultures of yeasts of the species Pichia pastoris and other hosts.

[0010]

Since the fusion protein is considered to be able to maintain the binding state of the two proteins firmly, these two proteins bind together, such as IL-6R and IL-6. It is considered to be particularly effective when a protein forming an IL-6 signal transduction system is administered to a human body or the like as a pharmaceutical in this state.

When a fusion protein is developed as a drug for continuous administration into the body, the linker is a sequence that is not contained in the protein to be bound and has a sequence independent of these proteins, and has a unique three-dimensional structure. The problem is that there is a high possibility that the above-mentioned immune response will be induced. Therefore, it is generally preferred that the linker be as short as possible, and most preferably that no linker is present.

However, as described above, fusion proteins include:
It is necessary that steric hindrance and the like do not occur between the two types of proteins, and that the two types of proteins having the fused original structure have enough freedom to bind to each other. For example, IL-6R and IL-
When direct fusion of 6 does not cause steric hindrance of both,
And in order to have a degree of freedom that allows both to bind to each other, the order of the proteins to be fused, which C-terminal amino acid residue to which amino acid residue of the N-terminal protein, etc. There are many decisions that need to be made.

[0013] As for the fusion protein of IL-6R and IL-6, only an example using the above-mentioned three types of linkers has been reported, and only a protein in which both are linked via a linker is known. FIG. 1A is an illustration of a fusion protein in which both are bound via a linker. On the other hand, there has been no report on the case where both are bound without the intervention of a linker.

Accordingly, the present invention provides an IL-6R / IL-6 fusion protein in which IL-6R and IL-6 are directly bound without a linker, as shown in FIG. 1 (b). It is intended to do so. The present invention also relates to an IL-IL having resistance to the cleavage action of a protease expressed by a host, particularly a yeast of the species Pichia pastoris.
Another object is to provide a 6R-IL-6 fusion protein and a gene encoding the fusion protein.

[0015]

Means for Solving the Problems In order to achieve the above object, the present inventors have conducted intensive studies on IL-6R / IL-6 fusion protein, and found that IL-6R is located on the N-terminal side without a linker. The fusion protein in which IL-6 was located on the C-terminal side was completed. That is, the present invention relates to IL-
An IL-6R / IL-6 fusion protein characterized in that one of the amino acid residues constituting 6R and one of the amino residues constituting IL-6 are directly bonded. In addition, the present inventors have found that the C-terminal of the lysine residue at the N-terminal 37 of IL-6
A protease resistant IL-6R / IL-6 fusion protein characterized in that the terminal side is subjected to a protease cleavage action, a mutation is inserted into the protease cleavage site in the primary structure, and the protease has resistance to protease. Among them, on the C-terminal side of IL-6R, 10 to 10 amino acids from the 28th alanine residue to the 37th lysine residue are found.
IL-6 to which IL-6 from which amino acid residues have been deleted is bound.
The R.IL-6 fusion protein and the gene encoding it have been completed.

The present invention also encodes an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino residues constituting IL-6 are directly bound. Is a gene.

Further, the present invention encodes an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino residues constituting IL-6 are directly bound. Pichia pastoris (Pichia pas) transformed with an expression vector containing the gene
toris).

Further, the present invention encodes an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino acid residues constituting IL-6 are directly bound. A yeast of the species Pichia pastoris transformed with the expression vector containing the gene is cultured, and the IL-6R-IL-6 fusion protein is collected from the culture as a secretory protein. I
This is a method for producing an L-6 fusion protein.

Further, the present invention encodes an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino acid residues constituting IL-6 are directly bound. An IL comprising culturing a yeast of the species Pichia pastoris transformed with an expression vector containing a gene in a medium containing no carbon and containing a carbon source derived from a natural product, and culturing with the addition of methanol on the way.
This is a method for producing a -6R-IL-6 fusion protein.

The present invention further includes an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino acid residues constituting IL-6 are directly bound. The solution is subjected to ion exchange chromatography,
It is subjected to three types of chromatography, hydrophobic chromatography and gel filtration chromatography, and IL-6R.
Collecting an IL-6 fusion protein;
This is a method for producing a 6R-IL-6 fusion protein.

Further, the present invention includes an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino acid residues constituting IL-6 are directly bound. Ex vivo hematopoietic stem cells characterized by the following:
It is an amplifying agent.

The present invention is based on an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino acid residues constituting IL-6 are directly bound. Is a platelet-increasing agent. Hereinafter, the present invention will be described in detail.

The fusion protein of the present invention has an I-terminal
L-6R is located, IL-6 is located at the C-terminal side, and both proteins are directly linked without a linker. As described above, when the purpose is only to impart resistance to the protease,
The two proteins may be linked via a polypeptide linker, but are preferably directly linked without a polypeptide linker in consideration of, for example, reducing antigenicity during administration. When a linker is used, for example, a known linker sequence (Fisher et al., Na
cure Biotech, 15, 142, 1997
Year; Chebath et al., Eur. Cytokine N
etw. , 8, 359, 1997).
A linker of serine residue-serine residue-glutamic acid residue-leucine residue-varic acid residue is bonded to the C-terminal of the 344th leucine residue of IL-6R, and IL-6 is further added to the C-terminal side thereof. Binding can be exemplified. IL-6 constituting IL-6R / IL-6 fusion protein of the present invention
R is a membrane protein composed of a total of 468 amino acid residues, and is composed of a signal region, an extracellular region, a transmembrane region and an intracellular region (Yamasaki et al., Science,
241, 825, 1988). Human IL-6R
In the case of, the signal region extends from the vicinity of the N-terminal first methionine residue to the vicinity of the 19th alanine residue, the extracellular region extends from the vicinity of the 20th leucine residue to the vicinity of the 358th aspartic acid residue, and the transmembrane region. Is thought to be from the vicinity of the 359th serine residue to the vicinity of the 386th leucine residue, and the intracellular region is approximately from the vicinity of the 387th arginine residue to the vicinity of the 468th arginine residue. The extracellular region is divided into an immunoglobulin-like region and a cytokine receptor region. The immunoglobulin-like region extends from near the 20th leucine residue to around the 111th aspartic acid residue, and the cytokine receptor region lies near the 112th valine residue. To the vicinity of the alanine residue at position 323.

[0024] In IL-6R, it is known that a cytokine receptor region is essential for binding to IL-6, and an immunoglobulin-like region is unnecessary. The cytokine receptor region is a structure in which two short barrel-like structures composed of seven β sheets are connected (Yawata et al., EMBO J., 1
2, p. 1705, 1993).

In the present invention, not only the full-length IL-6R but also the whole extracellular region or the cytokine receptor region, that is, a partial IL-6R can be used. This is because it is the cytokine receptor region that binds to IL-6 to form its signal transduction system, and the extracellular region is a region containing this region.

According to the findings of the present inventors, specifically, I
As the N-terminus of L-6R, a leucine residue at position 20 at the N-terminal, a valine residue at position 112 at the N-terminal, and
The second glutamic acid residue can be exemplified.

According to the findings of the present inventors, specifically, as the C-terminus of IL-6R, 39 amino acid residues from the alanine residue at position 323 to the serine residue at position 361 of the N-terminus were determined. Any one of them, particularly preferably 32
3rd alanine residue, 333rd alanine residue, 3
For example, it is possible to use any one of the six amino acid residues of the 34th leucine residue, the 335th threonine residue, the 338th lysine residue, and the 343rd isoleucine residue. The C-terminal side of this partial IL-6R is linked to the N-terminal of IL-6. Note that I
The N-terminus of L-6R can be appropriately deleted in consideration of the effect of the fusion protein on signal transduction.

IL-6, which constitutes the IL-6 receptor / IL-6 fusion protein, is a secretory protein having a total length of 212 amino acid residues and consisting of four α helices (Hir
Nano et al., Nature, 324, 731, 1986.
Year). It is known that all four α helices are required for IL-6 to be active.
Therefore, as the IL-6 used in the present invention, four α
There is no particular limitation as long as it has all the helices. That is, let alone full-length IL-6, for example, N
Partial I in which some amino acid residues at the terminal and C-terminal have been deleted
It may be L-6. More specifically, secreted IL-
Illustrative examples of IL-6 from the N-terminal 28th alanine residue or the 29th proline residue to the 212th methionine residue known as the structure of No. 6 can be given. In addition, examples of expression of IL-6 (for example, Yasuka
wa et al., Biotech. Lett. , 12,419
P. 1990) and examples of expression of IL-6R / IL-6 fusion proteins (Fisher et al., Nature Biotec).
h. , 15, 142, 1997) to determine what sequence to use as partial IL-6.

In the fusion protein of the present invention in which a mutation has been inserted into the protease cleavage site in the primary structure to impart resistance to the protease, the cleavage site of the protease is different for each type. Mutation is introduced, that is, which part is deleted, which part is added with an amino acid residue which is not originally present, or which part is replaced with an amino acid residue is appropriately determined to be less likely to be cleaved by a protease. I do. In the introduction of a mutation by substitution, the protein molecule does not take the original three-dimensional structure by the substitution, so that the protein molecule is not or hardly subjected to the cleavage action of the protease. In this case, substitution of the target amino acid residue with glycine or serine, which is an amino acid residue having a small molecular size, can be exemplified as a preferable substitution. In introducing a mutation by deletion, cleavage of the protease is performed by removing the target amino acid residue so that the protease cannot recognize the cleavage action site, or by preventing the protein molecule from taking an original three-dimensional structure. Be unaffected or hardly affected.

The mutation can be introduced by genetic engineering using IL-6R.
-It may be performed on the gene encoding the IL-6 fusion protein. The amino acid residue may be deleted, the amino acid residue may be inserted, or the amino acid residue may be substituted. However, the operation is relatively easy, and the antigenicity due to the substitution of an amino acid residue which is not originally present may be obtained. It is particularly preferable that one or more consecutive amino acid residues near the cleavage site of the protease are deleted and deleted because the influence of a change or the like is considered to be small.

A more specific selection criterion is the I of the present invention.
The purpose of the present invention is to impart resistance to a protease secreted by a host cell used in producing an L-6R-IL-6 fusion protein by genetic engineering. That is, as shown in the examples below, IL-6 to be produced by genetic engineering is used.
In the process of expressing the R.IL-6 fusion protein in the selected host cells and partially purifying it by a method using ordinary liquid chromatography or the like, a peak which is considered to be a degradation product due to a protease is collected, or a normal SDS- A band considered to be a degradation product of the protease is collected by a method using PAGE, and the amino acid sequence at the N-terminus is analyzed, whereby any of the IL-6R / IL-6 fusion proteins is secreted by the protease secreted by the selected host cell. It is possible to know whether the part is under cutting action.

When a protease cleavage site in the primary structure of the IL-6R / IL-6 fusion protein is detected as described above, amino acid residues near the cleavage site are deleted, and the protease is cleaved. By making it less or less susceptible, it becomes possible to obtain an IL-6R / IL-6 fusion protein having resistance to protease.

In addition to the above, the protease-resistant IL-6R / IL-6 of the present invention is also used for the purpose of maintaining the biological activity of the IL-6R / IL-6 fusion protein for a long time in the presence of a protease, such as a pharmaceutical. The fusion protein is effective. In order to produce such an IL-6R / IL-6 fusion protein, a protease which coexists in an environment in which the IL-6R / IL-6 fusion protein should exert a biological activity is obtained, and the protease is used for mutagenesis. IL-6R · I
It is only necessary to detect which site of the L-6 fusion protein is cleaved and to mutate the site as described above.

The IL-6 into which the mutation has been introduced as described above
Regarding the R.IL-6 fusion protein, it is preferable to confirm that the biological activity is maintained after the introduction of the mutation. For this purpose, the mutated IL-6R-IL-
For example, a 6 fusion protein is prepared and its biological activity is subjected to confirmation using, for example, a BAF130 cell line as described in Examples below. If no biological activity is observed in the IL-6R / IL-6 fusion protein into which the mutation has been introduced by this confirmation, the procedure of introducing another mutation and performing the same confirmation may be repeated.

Subsequently, the mutated IL-6R · I
It is preferable to confirm that the L-6 fusion protein is protease resistant. For this purpose, a mutation-introduced IL-6R / IL-6 fusion protein was allowed to coexist with a protease for a certain period of time, followed by SDS-PA
What is necessary is just to provide to the analysis which combined GE and western blotting. As a result, if no resistance to the protease is observed, the procedure of introducing another mutation and performing the same confirmation may be repeated. In the case of an IL-6R / IL-6 fusion protein imparted with resistance to a protease secreted by the host cell, after preparing the IL-6R / IL-6 fusion protein using the host cell, the above-described confirmation operation is performed on the cell culture solution. Good.

The IL-6R / IL-6 fusion protein of the present invention can be easily prepared by performing a gene recombination operation using a gene encoding the same. IL-
The gene encoding 6R or IL-6 has already been isolated, and its nucleotide sequence is well known. Therefore, when preparing the fusion protein of the present invention, a necessary gene sequence is prepared from the amino acid sequences of IL-6R and IL-6 constituting the fusion protein, and these are ligated using a restriction enzyme. good. Here, in addition to the use of a natural gene sequence, any codon may be replaced with one which encodes the same amino acid residue but has a different base sequence in consideration of codon condensation. This is because when a protein is expressed in a host by genetic recombination, use of a specific codon may improve the expression rate and the translation rate.

There are no particular restrictions on the host used when the fusion protein of the present invention is produced by genetic recombination, and Escherichia coli and CHO cells used in ordinary genetic recombination procedures can be used with reference to the conventional reports. Animal cells represented by, for example, (Yasukawa et al., J. Bio
tech. , 108, 673, 1990). Among them, the yeast of the species Pichia pastoris shown in the present example is a yeast that can grow using methanol as a sole carbon source, and can be cultured at a lower cost than animal cells such as CHO cells. It can be exemplified as a preferred host.

A yeast of the species Pichia pastoris, which is a suitable host cell for producing an IL-6R / IL-6 fusion protein by genetic engineering, (Pichia pastoris)
IL-6RI which is resistant to proteases secreted by
Examples of the L-6 fusion protein include a fusion protein of IL-6R and IL-6 in which at least the alanine residue at the N-terminal 28th position to the lysine residue at the N-terminal 37th position are deleted. IL-6R having protease resistance
A gene encoding an IL-6 fusion protein is prepared, an expression vector is prepared by incorporating the gene, and the host cell is transformed. By culturing the transformed host cell, an IL having the protease resistance of the present invention can be obtained. -6R ・ IL
The -6 fusion protein can be produced in large quantities. As described above, the IL-6R / IL-6 fusion protein imparted with resistance to the protease secreted by the host cell is not cleaved by the protease after being expressed in the host cell, or is hardly cleaved by the protease. I do not
As compared with the L-6R-IL-6 fusion protein, a larger amount of protein can be obtained.

The above-described gene for producing the fusion protein of the present invention by genetic recombination is used by incorporating it into an expression vector when introducing (transforming) it into a host. The expression vector incorporates, in addition to the gene, an expression control gene, a gene serving as an indicator for selecting a transformed host, and the like. Such a gene may be appropriately selected and used in relation to the host to be used. . For example, if yeast of the species Pichia pastoris is used as a host, upstream and downstream sequences of an alcohol oxidase gene for introducing a gene encoding an IL-6R / IL-6 fusion protein into its chromosomal DNA, The histidine synthesis gene and the promoter sequence of the alcohol oxidase gene for expression control are used as an indicator of ampicillin resistance gene and Lac promoter / operator sequence for expression control, which are selection indicators if Escherichia coli is used as a host. Etc. can be exemplified. The gene of the present invention can be introduced into a commercially available expression vector (for example, pPIC9, an expression vector for Pichia pastoris yeast, manufactured by Invitrogen) and used.

In the present invention, preferably, when a yeast of the species Pichia pastoris is transformed with an expression vector containing a gene encoding an IL-6R / IL-6 fusion protein to prepare a fusion protein, the expression vector is It is preferable to incorporate an intrinsic signal peptide of IL-6R or a signal peptide of α-factor as a signal peptide of the fusion protein. In particular, it is preferable to use a signal peptide of α-factor because high expression can be realized.

The above-mentioned transformed host is cultured under suitable conditions, and the fusion protein can be produced by inducing the expression of the fusion protein as required in relation to the expression control gene in the expression vector. . In the present invention, when a yeast of Pichia pastoris is preferably used as a host, a method using a jar fermenter can be exemplified. More specifically, IL-6 prepared beforehand was placed in a 500 mL shake flask charged with 100 mL of medium.
A 20% glycerol frozen strain of Pichia pastoris yeast expressing the R.IL-6 fusion protein was inoculated with 28
Incubate with shaking at ３０30 ° C. for 20 hours. Next, 100 mL of the above culture solution is inoculated into a 16 L capacity jar fermenter charged with 6 to 9 L of medium, and aeration and stirring culture is started at 28 to 30 ° C. In order to monitor the state of the yeast during the culture, it is preferable to monitor and control the OD600, pH, dissolved oxygen concentration, stirring speed, and temperature. The type of the medium is not particularly limited as long as it contains a carbon source derived from a natural product, but the specific composition may be referred to the examples. As a jar fermenter for performing the culture, a commercially available device can be used. After the start of the culture, methanol may be added at the stage when the glycerol in the culture solution is depleted. Glycerol depletion can be determined by monitoring dissolved oxygen. It is desirable to add methanol within 5 hours after depletion. If the amount of methanol added is too large, it is toxic to yeast, but if the amount is too small, considering that the promoter sequence of the alcohol oxidase gene does not function sufficiently,
0.5% or more and 5% or less (mass / volume) are preferable.

The fusion protein produced by culturing can be obtained from a medium or the like by an appropriate method. When Escherichia coli is used as a host, the expressed fusion protein accumulates as an insoluble mass in Escherichia coli. Therefore, after the cells are disrupted, refolding and purification operations may be performed under appropriate conditions. When a yeast of the Pichia pastoris species is preferably used as a host, the fusion protein can be purified and obtained from the culture supernatant. The purification raw material is not particularly limited as long as it is a solution containing the fusion protein, and examples thereof include a culture solution of a yeast of Pichia pastoris containing the fusion protein. The solution may be used as it is,
Or dilute them with buffer or pure water,
It may be used after being concentrated with an ultrafiltration membrane or ammonium sulfate. As a purification operation, a liquid chromatography operation can be exemplified. Preferably, a combination of three types of chromatography, such as ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography, can be exemplified.

Since the culture supernatant of yeast of the species Pichia pastoris is large in volume, it is preferable to first apply it to ion exchange chromatography. Ion exchange chromatography is divided into cation exchange chromatography and anion exchange chromatography, and can be appropriately selected in consideration of protein removal efficiency. The former can be exemplified by SP as a cation exchange group, and the latter can be exemplified by DEAE as an anion exchange group. Here, the flow rate can be increased to 100 ml or more per minute and 1 μm
Considering that it is possible to add a sample that has not passed through a filter having a size of not more than m, as shown in the examples below, the adsorption fluidized bed S
A preferred example is a streamline SP C-50 column (manufactured by Amersham Pharmacia). The fraction containing the fusion protein obtained by the above method is then subjected to hydrophobic chromatography, whereby a fraction having a higher purity of the fusion protein can be obtained. This fraction
As shown in the examples below, gel filtration chromatography can be performed efficiently by concentrating by, for example, cation exchange chromatography.

The present inventors have proposed a stem cell of an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino acid residues constituting IL-6 are directly bound. As a result of intensive studies on the amplification effect, the fusion protein and stem cell factor (SC
F) When CD34-positive cells were cultured on methylcellulose plates in the presence of IL-6, I, which had been reported previously,
It was discovered that the colony forming ability was significantly higher than when cultured in the presence of L-6R and SCF. The present invention relates to an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino acid residues constituting IL-6 are directly bound. Based on the ex vivo expansion of hematopoietic stem cells, the ex vivo expansion agent of hematopoietic stem cells containing the fusion protein as a main component or the ex vivo expansion of hematopoietic stem cells.
This is a vivo amplification method.

The ex vivo amplifying agent for hematopoietic stem cells of the present invention is of practical value since it exhibits some effects even when the fusion protein of the present invention alone is used. However, any of cytokines such as SCF and FLK2 ligand that stimulate tyrosine kinases can be used. The effect becomes remarkable when one of them is added, particularly preferably SCF. Furthermore, interleukin-3 (IL-
3) or platelet growth factor (TPO) may be added. Hematopoietic stem cells can be obtained from cord blood, peripheral blood, or bone marrow by CD34 selection as a fraction containing hematopoietic stem cells. The fusion protein of the present invention can be provided as a kit containing individual containers containing other cytokines,
Alternatively, they can be provided as a mixture in a single container. Ex vivo expansion of hematopoietic stem cells is performed by, for example, culturing a fraction containing hematopoietic stem cells in a serum-free medium supplemented with the fusion protein of the present invention and, for example, SCF at 37 ° C. for 1 to 3 weeks in a container such as a plastic bag. do it. When returning the expanded hematopoietic stem cells to the patient, the same method as in the current peripheral blood stem cell transplantation may be used.

The present inventors have proposed a platelet of an IL-6R / IL-6 fusion protein in which one of the amino acid residues constituting IL-6R and one of the amino acid residues constituting IL-6 are directly bound. As a result of intensive studies on the effect of increasing the number of platelets, as shown in Examples below, platelets were significantly increased when the fusion protein was administered to mice, and platelets were increased when the fusion protein was administered to mice previously administered with an anticancer agent. He found that recovery was significantly faster. The present invention relates to one of these newly found amino acid residues constituting IL-6R and IL-6R.
IL-6R / IL-6 fusion protein to which one of the amino acid residues constituting -6 is directly bound, based on the increase or recovery of platelets, and containing the fusion protein as a main component. Platelet-increasing agents or platelet-increasing methods. The platelet-increasing agent of the present invention is preferably administered by parenteral administration, for example, intravenous administration, intramuscular administration, transdermal administration and the like. The dose is appropriately selected depending on the type of the disease showing the platelet deficiency symptom, the condition of the patient, and the like. Generally, the dose is in the range of 1 to 500 μg / kg / day. Good. The platelet-increasing agent of the present invention can be formulated by mixing with a commonly used excipient such as physiological saline, a glucose solution, an activator such as mannitol, methylcellulose, gelatin, or human serum albumin. In addition, the platelet increasing agent of the present invention can be a freeze-dried product. In the case of freeze-drying, it may be redissolved with an isotonic solution such as physiological saline, glucose solution, Ringer's solution or the like immediately before use.

[0047]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1 Preparation of Intermediate Plasmid Insertion of oligonucleotides encoding various linkers resulted in the fusion of IL-
The gene encoding the 6R-IL-6 fusion protein (cDN
In order to be able to produce A), the intermediate plasmid pBS6R
6S and pBS6R6L were produced by the following method.

First, the cloning vector pBl
usscript II KS (-) (Toyobo Co., Ltd.)
Was digested with the restriction enzyme KpnI and treated with Klenow fragment, followed by a ligation reaction to obtain KpnI.
A plasmid pBS having a lost site was prepared.

Next, a primer p6RAB20L (SEQ ID NO: 45) and a primer p6RF320S (SEQ ID NO: 4)
Amplifying the IL-6R gene (cDNA) using
Cleavage with XhoI. This is previously converted to XhoI and EcoR
Plasmid pBS6R was obtained by insertion into pBS cut with V.

Next, the IL-6 gene (cDNA) was amplified with the primers pIL6B2 (SEQ ID NO: 47) and pIL6F (SEQ ID NO: 48), and the Bgl I
Cut with I and NotI. This was inserted into a plasmid pBS6R which had been cut with Bgl II and Not I in advance, to obtain pBS6R6S. Obtained pBS6R
FIG. 2 shows the structure of 6S.

Next, the oligomers 320S333Ab (SEQ ID NO: 49) and 320S333Af (SEQ ID NO: 50) were annealed by a conventional method. This is previously converted to Bgl I
PBS6R6L was obtained by insertion into plasmid pBS6R6S cut with I and KpnI. FIG. 4 shows the structure of the obtained pBS6R6L.

Example 2 Preparation of Expression Plasmid pBS6R6S previously cut with Bgl II and KpnI
Into each of the annealing sequences 1 to 5 obtained by annealing the two types of oligonucleotides, respectively,
Five types of plasmids having as their inserts genes encoding IL-6R / IL-6 fusion proteins were prepared.

Annealed sequence 1 (323AG4SA): The oligonucleotide of SEQ ID NO: 1 on the sense side, and the oligonucleotide of SEQ ID NO: 2 on the antisense side.

Annealed sequence 2 (323AG4S2A);
The oligonucleotide of SEQ ID NO: 3 is on the sense side, and the oligonucleotide of SEQ ID NO: 4 is on the antisense side.

Annealed sequence 3 (334 LPA); the sense side is an oligonucleotide of SEQ ID NO: 5, the antisense side; an oligonucleotide of SEQ ID NO: 6.

Annealed sequence 4 (333AΔA): Sense side: oligonucleotide of SEQ ID NO: 7, antisense side: Oligonucleotide of SEQ ID NO: 8

Annealed sequence 5 (323AΔA); oligonucleotide on the sense side of SEQ ID NO: 9, antisense side; oligonucleotide on SEQ ID NO: 10

The annealing sequence 1 (323AG4S
A) shows that N-terminal alanine 323 of IL-6R and I
This is for expressing a fusion protein in which alanine at the N-terminal 28th position of L-6 is fused via a linker called GGGGS, and has an annealing sequence 2 (323AG4S2A).
Is the N-terminal alanine at position 323 of IL-6R and IL-
Alanine at the N-terminal 28th position of GGGGSGGGGS
Is used to express a fusion protein fused through a linker called IL-6R. The annealed sequence 3 binds the N-terminal leucine 334 of IL-6R and the N-terminal alanine 28 of IL-6 via a linker P. This is for expressing the fused fusion protein, and is used for annealing sequence 4 (33
3AΔA) is for expressing a fusion protein of the present invention in which the alanine at the N-terminal 333 of IL-6R and the alanine at the N-terminal 28 of IL-6 are directly fused without using a linker. Sequence 5 (323AΔ
A) shows that N-terminal alanine 323 of IL-6R and I
This is for expressing the fusion protein of the present invention in which alanine at the N-terminal 28th position of L-6 is directly fused without using a linker.

Next, Eco47 III and KpnI
Into pBS6R6L cut in step 2.
Annealed sequences 6 to 22 obtained by annealing various kinds of oligonucleotides were inserted, respectively, to prepare a plasmid 17 having as its insert a gene encoding an IL-6R / IL-6 fusion protein.

Annealed sequence 6 (333AG4SA); oligonucleotide on the sense side of SEQ ID NO: 11, antisense side; oligonucleotide on SEQ ID NO: 12.

Annealed sequence 7 (333AG4S2A);
The oligonucleotide of SEQ ID NO: 13 is on the sense side, and the oligonucleotide of SEQ ID NO: 14 is on the antisense side.

Annealed sequence 8 (343IG4S2A);
The oligonucleotide of SEQ ID NO: 15 is on the sense side, and the oligonucleotide of SEQ ID NO: 16 is on the antisense side.

Annealed sequence 9 (333AG4S3A);
The oligonucleotide of SEQ ID NO: 17 is on the sense side, and the oligonucleotide of SEQ ID NO: 18 is on the antisense side.

Annealed sequence 10 (333AG2A); oligonucleotide of SEQ ID NO: 19 on the sense side, oligonucleotide of SEQ ID NO: 20 on the antisense side.

Annealed sequence 11 (333AG4A): The oligonucleotide of SEQ ID NO: 21 on the sense side, and the oligonucleotide of SEQ ID NO: 22 on the antisense side.

Annealed sequence 12 (343IG2A): Oligonucleotide of SEQ ID NO: 23 on the sense side, oligonucleotide of SEQ ID NO: 24 on the antisense side.

Annealed sequence 13 (343IG4A); 1
The oligonucleotide of SEQ ID NO: 25 is on the sense side, and the oligonucleotide of SEQ ID NO: 26 is on the antisense side.

Annealed sequence 14 (361SΔA): Oligonucleotide of SEQ ID NO: 27 on the sense side, oligonucleotide of SEQ ID NO: 28 on the antisense side

Annealed sequence 15 (358DΔA): Oligonucleotide of SEQ ID NO: 29 on the sense side, oligonucleotide of SEQ ID NO: 30 on the antisense side.

Annealed sequence 16 (352TΔA): The oligonucleotide of SEQ ID NO: 31 on the sense side, and the oligonucleotide of SEQ ID NO: 32 on the antisense side.

Annealed sequence 17 (346RΔA); oligonucleotide of SEQ ID NO: 33 on the sense side, oligonucleotide of SEQ ID NO: 34 on the antisense side

Annealed sequence 18 (343IΔA): The oligonucleotide of SEQ ID NO: 35 on the sense side, and the oligonucleotide of SEQ ID NO: 36 on the antisense side.

Annealed sequence 19 (338KΔA): The oligonucleotide of SEQ ID NO: 37 on the sense side, and the oligonucleotide of SEQ ID NO: 38 on the antisense side.

Annealed sequence 20 (335TΔA): The oligonucleotide of SEQ ID NO: 39 on the sense side, and the oligonucleotide of SEQ ID NO: 40 on the antisense side.

Annealed sequence 21 (343IΔP): The oligonucleotide of SEQ ID NO: 41 on the sense side and the oligonucleotide of SEQ ID NO: 42 on the antisense side.

Annealed sequence 22 (338KΔP): The oligonucleotide of SEQ ID NO: 43 on the sense side, and the oligonucleotide of SEQ ID NO: 44 on the antisense side.

The annealing sequence 6 (333AG4S
A) is alanine at the N-terminal 333th position of IL-6R and IL
-6 is for expressing a fusion protein in which the N-terminal 28th alanine of -6 is fused via a linker called GGGGS, and annealed sequence 7 (333AG4S2A)
Alanine at position 333 of the N-terminal of L-6R and N-terminal of IL-6
This is for expressing a fusion protein in which alanine at the 28th terminal is fused via a linker called GGGGSGGGGS. Annealed sequence 8 (343IG4S2A) has an isoleucine at the 343rd N-terminal of IL-6R and IL-
Alanine at the N-terminal 28th position of GGGGSGGGGS
For expressing a fusion protein fused via a linker called annealed sequence 9 (333AG4S3
A) is alanine at the N-terminal 333th position of IL-6R and IL
The N-terminal 28th alanine of -6 was replaced with GGGGSGGGG
This is for expressing a fusion protein fused via a linker called SGGGGS, and contains an annealed sequence 10 (3
33AG2A) is for expressing a fusion protein in which alanine at position 333 at the N-terminus of IL-6R and alanine at position 28 at the N-terminus of IL-6 are fused via a linker called GG. Annealed sequence 11 (333AG4A) Is for expressing a fusion protein in which the alanine at the N-terminal 333 of IL-6R and the alanine at the N-terminal 28 of IL-6 are fused via a linker called GGGG. The annealed sequence 12 (343IG2A) -6R
Is used to express a fusion protein obtained by fusing the N-terminal isoleucine at position 343 of A. and the alanine at position 28 of the N-terminal of IL-6 via a linker called GG. The annealed sequence 13 (343IG4A) has the N-terminal of IL-6R. End 34
This is for expressing a fusion protein in which the third isoleucine and alanine at the N-terminal 28th position of IL-6 are fused via a linker called GGGG.
(361SΔA) is for expressing a fusion protein of the present invention in which the N-terminal serine at position 361 of IL-6R and the alanine at position 28 of the N-terminal of IL-6 are directly fused without using a linker. Array 15 (358DΔ
A) is for expressing the fusion protein of the present invention in which aspartic acid at the N-terminal 358 of IL-6R and alanine at the N-terminal 28 of IL-6 are directly fused without using a linker. Sequence 16 (352TΔA)
Is the threonine at the N-terminal 352 of IL-6R and IL
This is for expressing the fusion protein of the present invention in which the N-terminal 28th alanine of -6 is directly fused without using a linker, and the annealing sequence 17 (346RΔA) contains IL-
This is for expressing a fusion protein of the present invention in which arginine at the N-terminal 346 of 6R and alanine at the N-terminal 28 of IL-6 are directly fused without using a linker, and the annealing sequence 18 (343IΔA) is This is for expressing a fusion protein of the present invention in which the isoleucine at the N-terminal 343 of IL-6R and the alanine at the N-terminal 28 of IL-6 are directly fused without using a linker, and annealed sequence 19 (338KΔA) Is the N-terminal 338 of IL-6R
This is for expressing the fusion protein of the present invention in which the lysine at position 28 and the alanine at position 28 of the N-terminus of IL-6 are directly fused without using a linker.
TΔA) is for expressing the fusion protein of the present invention in which threonine at position 335 of the N-terminus of IL-6R and alanine at position 28 of the N-terminus of IL-6 are directly fused without using a linker. 21 (343IΔP)
Is the isoleucine at the N-terminal position 343 of IL-6R and I
This is for expressing the fusion protein of the present invention in which the N-terminal proline at the N-terminal of L-6 is directly fused without using a linker, and annealed sequence 22 (338KΔP)
Is the N-terminal lysine 338 of IL-6R and IL-6
This is for expressing the fusion protein of the present invention in which the proline at the N-terminus of No. 29 is directly fused without using a linker.

PBS6R6L itself is IL-6R · IL-
6 has a gene encoding the fusion protein in the insert, and the fusion protein expressed by the gene (334LΔ
V) shows the N-terminal leucine at position 334 of IL-6R and I
It is a fusion protein of the present invention obtained by directly fusing the N-terminal valine at position 30 of L-6 without using a linker.

Finally, the plasmid 2 prepared by the above method was used.
XhoI and NotI were used for the three species and pBS6R6L, respectively.
To obtain a gene encoding the IL-6R / IL-6 fusion protein, and inserted into pPIC9 previously cut with XhoI and NotI to prepare 24 types of expression plasmids. As an example of these expression plasmids, 333AΔ
The structure of the expression plasmid for A is shown in FIG.

Example 3 Preparation of Transformant EasyComp Trans from Pichia pastoris strain GS115 (Invitrogen)
Competent cells were prepared using a formation kit (Invitrogen), and each expression plasmid linearized with Bgl II was introduced. Transformants were cultured in a minimal nutrient medium, and those that lost histidine auxotrophy were selected.

Next, the obtained transformant was placed on an MD plate (1.34% (W / V) YNB wo AA (Yeas).
t Nitrogen Base Without A
mino Acid), 0.00004% (W / V) biotin, 2% (W / V) glucose, and MM plate (1.34% (W / V) YNB wo AA, 0.00
004% (W / V) biotin and 0.5% (V / V) methanol).
It was checked whether it was t ⁺ or mut ^s .

[0083] for each derivative, a count of the number of transformants obtained and mut ^s strains shown in Table 1. For each strain transformed with each expression plasmid, average 9.4 pieces of mut ^s strains (minimum four, maximum 17 kinds, total 216 species) were acquired.

[0084]

[Table 1]

[0085] Example 4 mut ^s strains obtained in culture example 3 transformants (total 216 species) and I
Leucine at the N-terminal 344 of L-6R and N of IL-6
End 28th methanol alanine using each tube (described in 1998 patent application No. 2921) mut ^s bacteria expressing a fusion protein via a linker that SSELV 5% (V / V) Medium containing 1% (W / V) yeast extract, 2% (W / V) peptone, 1.34% (W / V) YNB wo AA (Yea
st Nitrogen BaseWithout A
mino Acid), 0.4 mg / l biotin, 10
The cells were cultured in 3 ml of 0 mM potassium phosphate (pH 6.0) and 1% (w / v) glycerol at 30 ° C. for 120 hours. A 120-hour culture solution was sampled from each culture solution, and the supernatant was obtained by centrifugation.

Example 5 Measurement of biological activity IL-6R · IL in the culture supernatant obtained in Example 4
The biological activity of the -6 fusion protein was measured by a biological activity evaluation method using BAF130 cells. BAF130 is a mouse cell B that does not originally express human gp130 protein.
AF (Hatakeyama et al., Cell, 63, 15)
(See page 4, 1989), a gene encoding the human gp130 protein was introduced into the cells, and the cells were transformed to express the protein. Therefore, a biologically active IL
It shows proliferative activity in the presence of -6R and IL-6.

The suspension of BAF130 was added to a 96-well plate at 2 × 10 ⁴ cells / well, and 216 kinds of culture supernatants were added at 1%, 0.25%, and 0.061%, respectively.
%, 0.015%. Two days later, Cel
l Counting Kit (Wako Pure Chemical Industries, Ltd.)
Of the reference wavelength of 600 nm.
An absorbance of 5 nm was obtained. 1 μg /
of IL-6 to various concentrations, 100
The activity of the culture supernatant showing the same concentration dependency as the absorbance when added together with soluble ng / ml of IL-6R was 1 un
It / ml. FIG. 7 shows that each fusion protein was transformed with the same fusion protein expression vector.
It showed an average value of the biological activity of ut ^s bacteria 4-17 kinds of the culture supernatant.

FIG. 7 shows that at least one mut ^s transformed with the same fusion protein expression vector was used for all 12 types of fusion proteins having no linker sequence.
It can be seen that the activity was observed in the culture supernatant of the bacteria. This corresponds to the N-terminal amino acid of IL-6 at the C-terminal of any one of the 39 amino acid residues from the alanine residue at position 323 to the serine residue at position 361 of the N-6 terminal of IL-6R. The IL-6R • of the present invention to which the residue is bound.
2 shows that the IL-6 fusion protein has IL-6 signaling activity.

Further, from FIG. 7, the alanine residue at position 323, the alanine residue at position 333, the leucine residue at position 334, the threonine residue at position 335, the lysine residue at position 338, or the isoleucine residue at position 343 323AΔA which is an IL-6R / IL-6 fusion protein in which the N-terminal amino acid residue of IL-6 is bound to any one of the C-terminals of
333AΔA, 334LΔV, 335TΔA, 338
KΔP, 338KΔA, 343IΔP or 343IΔA
Is the same as the previously reported leucine at the N-terminal 344 of IL-6R and the alanine at the N-terminal 28 of IL-6.
334LSSELVA which is a fusion protein fused via a linker called SELV (1998 Patent Application No. 292)
No. 1).

Further, from FIG. 7, the alanine residue at position 323, the alanine residue at position 333, the leucine residue at position 334, the threonine residue at position 335, the lysine residue at position 338, or the isoleucine residue at position 343 323AΔA which is an IL-6R / IL-6 fusion protein in which the N-terminal amino acid residue of IL-6 is bound to any one of the C-terminals of
333AΔA, 334LΔV, 335TΔA, 338
KΔP, 338KΔA, 343IΔP or 343IΔA
Are 323AG4SA, 333AG4SA, which are IL-6R / IL-6 fusion proteins fused via a linker consisting of a peptide consisting of 5 to 15 amino acid residues having a high degree of freedom such as glycine residues and serine residues. , 3
23AG4S2A, 333AG4S2A, 343IG4
334L which is an IL-6R / IL-6 fusion protein fused via S2A, 333AG4S3A or a linker consisting of a peptide having 1 to 4 amino acid residues connected together
PA, 333AG2A, 333AG4A, 343IG2
A and 343IG4A have almost the same or slightly lower activity. Thus, the 323rd alanine residue, the 333th alanine residue, 334
The leucine residue at position 335, the threonine residue at position 335,
An IL-6 of the present invention in which the N-terminal amino acid residue of IL-6 is bound to the C-terminal of any one of the six amino acid residues of the lysine residue at position 38 and the isoleucine residue at position 343; It turns out that the 6R-IL-6 fusion protein is particularly preferred.

[0091] mut ^s expressing Example 6 fusion protein
Mut ^s strains 1 expressing 333AΔA according to mass culture in Example 3 of bacteria
Of the one strain, the strain III-108, which had the highest expression level in the method described in Examples 4 and 5, was cultured in a 16-liter jar. The glycerol stock of strain III-108 was added to 100 ml of BMGY (Bac
to Yeast Extract 10g / l, Ba
cto Peptone 20g / l, Yeast N
itrogen Base with out Amin
o Acid 1.34 g / l, 100 mM potassium phosphate buffer pH 6.0, glycerol 10 g / l,
Biotin (0.4 mg / l) was inoculated into a medium, and pre-cultured in a G-20 shaking incubator (NBS) at 30 ° C. and 200 rpm for 24 hours. 8 L of this culture solution
BMGY (Bacto Yeast Extract)
10g / l, Bacto Peptone 20g /
1, Yeast Nitrogen Base wit
hout Amino Acid 1.34 g / l, 1
A 00 mM potassium phosphate buffer (pH 6.0, glycerol 10 g / l, biotin 0.4 mg / l) was inoculated into a medium, and 16 liter jar SF-116 (manufactured by NBS) at a temperature of 28 ° C., a stirring speed of 350 rpm, and aeration. Culture was performed under the condition of an amount of 1 vvm. 16 hours after the start of the culture, 240 ml of methanol, Bacto Yeast
Extract 50 g / l and Bacto Pep
1.6 l of a 100 g / l mixed solution was added to initiate induction of fusion protein expression. The figure shows the progress of the main culture.

The concentration of methanol in the culture solution was measured by gas chromatography. The pH in the culture was measured with a pH meter. OD600 in culture solution (index of bacterial cell density)
Was measured with a spectrophotometer after diluting the culture solution 100-fold with physiological saline. The concentration of the fusion protein in the culture was
L-6R monoclonal antibody MT-18 (Hirata
J. et al. Immunol. 143, 2900, 19
1989) as a solid phase antibody, an anti-human IL-6 polyclonal antibody (Genzyme) as a detection antibody, and a purified fusion protein obtained by the method shown in Example 7 as a standard substance. It was measured by immunoassay. The biological activity of the fusion protein was measured by the method using BAF130 cells described in Example 5.

Example 7 Purification of Fusion Protein The culture solution selected in Example 6 was separated into cells and supernatant by centrifugation. The obtained supernatant (11.6 L) was diluted with distilled water until the conductivity became about 50 mM in terms of NaCl concentration (66.05 L). Next, the pH was adjusted to 4.5 using acetic acid.

Adsorbed fluidized bed Streamline SP C-5 equilibrated with 20 mM acetate buffer (pH 4.5)
0 column (50 mm ID x 100 cm, gel amount 300 m
l) The above-mentioned diluted preparation was added to (Amersham Pharmacia) by sending it upward from the bottom of the column (linear velocity: 300 cm / h). After the addition was completed, the equilibration buffer was further fed upward to wash. Next, the direction of the solution was changed, and the elution buffer solution (500 mM NaC
l, 5% glycerol, 20 mM phosphate buffer (pH
6.5)) (150 cm / h linear velocity) and eluted fraction (Streamline eluted fraction, 300 m
L) was obtained. The eluted fraction was detected by measuring the absorbance at 280 nm.

In the Streamline elution fraction, ammonium sulfate cooled to -20 ° C. was dissolved to a concentration of 2 M, and TSKgel Phe was equilibrated with 2 M ammonium sulfate and 20 mM phosphate buffer (pH 6.5).
nyl-5PW column (21.5mmID × 15cm)
(Made by Tosoh Corporation) (flow rate 5 ml / min). After the addition was completed, the equilibration buffer was further sent to elute proteins having weak hydrophobic interaction with the adsorbed groups. Then, the concentration of ammonium sulfate in the buffer was gradually reduced, and the fraction eluted at a concentration of 0.4 M ammonium sulfate was collected as a fusion protein elution fraction (Phenyl-5PW elution fraction, 67 ml). The detection of the fusion protein was carried out by using B
The proliferation activity of AF130 cells was used as an index.

The phenyl-5PW eluted fraction was separated from a 20 mM acetate buffer (pH: 5%) containing 5% glycerol.
It was dialyzed in 4.5) and desalted. The desalted solution was equilibrated with the same buffer and a TSKgel SP-5PW column (7.5
mmID × 7.5 cm) (manufactured by Tosoh Corporation) (flow rate 2 ml / min). After the addition was completed, the equilibration buffer was further fed to elute proteins having weak interaction with the adsorbed groups. Next, a 20 mM phosphate buffer solution (pH 6.5) containing 500 mM NaCl was fed (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 performed by measuring the absorbance at 280 nm.

The SP-5PW elution fraction was added to 100
20 mM phosphate buffer (pH 6.
The mixture was added several times to a TSKgel G3000SW column (21.5 mm ID × 30 cm) (manufactured by Tosoh) equilibrated in 5) (5 ml per minute), and the absorbance at 280 nm was measured to separate the fusion protein.

Example 8 Measurement of Activity of Fusion Protein The following experiment was performed using the fusion protein 333AΔA of the present invention described in Example 5 purified by the method described in Example 7 (hereinafter abbreviated as FP6). .

Various concentrations of FP6 (0.625 to 40 ng)
/ Ml) was measured by the biological activity evaluation method using BAF130 cells described in Example 5. As is clear from FIG. 9, FP6 showed concentration dependency.

Although not shown in the figure, 1 μg / ml F
A concentration-dependent curve obtained by diluting P6 to various concentrations (1 to 1024 times) and IL-6 at 5 μg / ml at various concentrations (1 to 1
(024 times), and the concentration-dependent curves obtained by dilution were almost the same. On the other hand, as described in Example 5, 1 μg / m
of IL-6 to various concentrations, each with 100 n
The activity of the culture supernatant showing the same concentration-dependent curve as that obtained when added together with g / ml of soluble IL-6R was defined as 1 unit / ml. Therefore, 1 μg
Of FP6 was shown to correspond to 5 units.

Example 9 Analysis of Amino Acid Sequence of Fusion Protein The following experiment was performed using the fusion protein 333AΔA of the present invention described in Example 5 purified by the method described in Example 7 (hereinafter abbreviated as FP6). went.

FP6 was further purified by reverse phase chromatography on a Phenyl-5PW RP column.
Elution was performed with a gradient from an aqueous solution of 20% acetonitrile and 0.1% trifluoroacetic acid to an aqueous solution of 60% acetonitrile and 0.1% trifluoroacetic acid. After the obtained fraction was dried under reduced pressure, 20% acetonitrile,
The sample was redissolved in a 0.1% trifluoroacetic acid aqueous solution, and analyzed with a protein sequencer 477A (manufactured by Applied Biosystems).

As a result of the analysis, leucine, alanine, proline, and arginine were detected in this order from the N-terminal, and it was confirmed that the sequence was consistent with the N-terminal sequence encoded by the gene.

Example 10 Analysis of Molecular Weight of Sugar Chain Portion of Fusion Protein The fusion protein 333AΔA of the present invention described in Example 5 was purified by the method described in Example 7 (hereinafter abbreviated as FP6). Was conducted.

150 μm containing 0.1 mg / ml FP6
l 0.1% SDS, 100 mM sodium chloride, 20
mM phosphate buffer (pH 6.0) was boiled for 5 minutes, and after cooling with water, 10 μl was mixed with 1 μl of 1 unit / ml endoglycosidase H (manufactured by Sigma), and the mixture was added at 25 ° C. for 3 hours.
The reaction was performed from 0 seconds to 3 hours. The reaction was stopped by adding 10 μl of 500 mM glycine hydrochloride buffer (pH 2.5). In addition, 500 mM glycine hydrochloride buffer (pH 2.
5) 10 μl, 10 μl of a pre-cooled FP6 solution, and 1 μl of endoglycosidase H were sequentially mixed,
A sample having an endoglycosidase H reaction time of 0 minutes was used.

3 μl of 72% glycerol and 10% 2-mercaptoethanol colored with bromophenyl blue were added to each of the above solutions after the reaction was stopped. This was subjected to SDS polyacrylamide gel electrophoresis. Proteins in the gel after electrophoresis were detected by staining with Coomassie Brilliant Blue R250.

As is clear from FIG. 10, FP6 not reacted with endoglycosidase H was detected as one band at the position of 76 to 93 kDa. This means that FP6
Indicates that the sugar chain site has a heterogeneity of 17 kDa. FP6 completely digested with endoglycosidase H was detected at a position of 57 kDa. Furthermore,
Although not shown in the figure, after denaturing FP6 with SDS, 10-fold amount of Triton X-100 was added, N-glycosidase F was added, and the reaction was carried out for 18 hours.
It was detected at a position of 7 kDa. This means that FP6 N
The molecular weight of the sugar chains linked by glycosidic bonds is 19-36k
Da.

Example 11 Stem Cell Amplifying Effect of Fusion Protein The following experiment was carried out using the fusion protein 333AΔA of the present invention described in Example 5 purified by the method described in Example 7 (hereinafter abbreviated as FP6). Was.

From 20 ml of human cord blood, Japanese Patent Application No. 9-325
CD34-positive cells were purified and separated according to the method described in No. 847. 500 cells obtained, 1.2% methylcellulose (Shin-Etsu Chemical), 30% fetal bovine serum albumin (H
yclone Laboratories In
c. ), 1% bovine serum albumin (hereinafter abbreviated as BSA) (Sigma), 0.05 mM 2-mercaptoethanol (Sigma), SCF (stem cell factor)
(100 ng / ml) and any of the following four conditions (IL-6 (100 ng / ml) and IL-6R (10
0 ng / ml), IL-6 (100 ng / ml)
/ Ml) and IL-6R (200 ng / ml), FP6 (300 ng / ml), FP6 (600
ng / ml)) with 1 ml of α-MEM (Flow)
The mixture was dispensed into a 5 mm plastic culture dish for suspension culture (Nunc), and cultured at 37 ° C., 5% CO _{2 and} 100% humidity.

After two weeks, colonies were identified by observation under an inverted microscope. The results are shown in FIG. The colonies are granulocyte colony (G), macrophage colony (M), mixed colony of granulocyte / macrophage (GM), blast colony (Blast), mixed colony of granulocyte / macrophage / erythroblast (Mix), erythroblast Spherical colony (BFU
-E).

As is clear from FIG. 11, if the condition has already been reported, the existing report (Sui et al.,
Proc. Natl. Acad. Sci. USA, 9
2, p. 2589, 1995). On the other hand, under the conditions of the present invention (FP6 (300 ng / ml)), the same concentration of IL-
6 and IL-6R (IL-6 (10
0ng / ml) and IL-6R (200ng / ml). Furthermore, although not shown in the figure, the size of the mixed colonies under or formed under the conditions of the present invention was significantly larger than under the conditions already reported. These facts indicate that the fusion protein of the present invention has a better ex vivo expansion effect of hematopoietic stem cells than the previously reported combination of IL-6 and IL-6R.

Example 12 Platelet-Increasing Effect of Fusion Protein on Mice Using the fusion protein 333AΔA of the present invention described in Example 5 purified by the method described in Example 7 (hereinafter abbreviated as FP6), An experiment was performed.

C57BL6 mice (male, 8 weeks old) were
A group of 5 animals, a total of 5 groups, the first group was a group to which 300 μl of PBS, 1% bovine serum albumin (BSA) was administered, and the second group was 0.5 μg of FP6 as an FP6 non-administration group (control group).
Of PBS, 1% bovine serum albumin (B
SA) administration group, the third group containing 1 μg of FP6 3
A group to which 00 μl of PBS and 1% bovine serum albumin (BSA) are administered, and a fourth group to which 300 μl containing 2 μg of FP6
, PBS, 1% bovine serum albumin (BSA), group 5 was treated with 300 μl of PB containing 5 μg of FP6
S, a group to which 1% bovine serum albumin (BSA) was administered. Each group was intraperitoneally administered twice a day every 12 hours for 5 consecutive days. On the sixth day, whole blood was collected from the descending vein, and the platelet count was counted using a hemocytometer CC-180A (manufactured by Toa Medical Electronics Co., Ltd.).

As is evident from FIG. 12, a significant increase in platelets was observed in the group to which 1 μg or more of FP6 was administered (Group 3-5) as compared to the group to which FP6 was not administered (Group 1). On the other hand, when 0.5 μg of IL-6 (molecular weight of about 21 kDa), which is twice as many as 1 μg of FP6 (molecular weight of about 84 kDa), was administered under the same conditions, no significant increase in platelets was reported. (Ishibashi et al.,
Blood, 74, 1241, 1989). This indicates that the fusion protein of the present invention has a better platelet-increasing effect in vivo than IL-6.

Example 13 Platelet Recovery Effect of Fusion Protein on 5FU-Administered Mice The fusion protein 333AΔA of the present invention described in Example 5 was purified by the method described in Example 7 (hereinafter abbreviated as FP6). Was conducted.

C57BL6 mice (male, 8 weeks old) were
A group of 15 animals, a total of 2 groups, the first group was a group to which 300 μl of PBS and 1% bovine serum albumin (BSA) were administered as an FP6 non-administration group (control group), and the second group was 300 μl containing 5 μg of FP6. PBS, 1% bovine serum albumin (BS
A) was a group to be administered. In all groups, 150 mg / kg of 5FU was administered via the tail vein, and the drug was intraperitoneally administered twice a day every 12 hours for 5 consecutive days from the second day to the sixth day. For three consecutive days from the 7th day to the 9th day, all blood was collected from the descending artery of 5 mice in each group, and the platelet count was measured using a hemocytometer CC-180A (manufactured by Toa Medical Electronics Co., Ltd.).
Was counted.

As is clear from FIG. 13, significant platelet recovery was observed on the 8th and 9th days in the group to which FP6 was administered (Group 2), as compared with the group to which FP6 was not administered (Group 1). .
This indicates that the fusion protein of the present invention has an in vivo recovery effect on the decrease in platelet production function due to 5FU administration.

Example 14 Expression of fusion protein lacking immunoglobulin-like region IL-6R derivative 112VL (N-terminal 11 of IL-6R
Expression plasmid pPIC encoding 233 amino acids from the second valine to the N-terminal 344th leucine)
9-112VL and IL-6R derivative 116EL (I
An expression plasmid pPIC9-116EL encoding L-6R (229 amino acids from the N-terminal 116th glutamic acid to the N-terminal 344th leucine) was constructed by the following method.

Using the primer p6RAB112V (SEQ ID NO: 51) and the primer p344F (SEQ ID NO: 52), the IL-6R gene (cDNA) was amplified and cut with XhoI and XbaI. This is previously converted to XhoI and AvrI
By insertion into pPIC9 cut with I, pPI
C9-112VL was obtained.

Next, primer p6RAB116E (SEQ ID NO: 53) and primer p344F (SEQ ID NO: 54)
To amplify the IL-6R gene (cDNA) using Xh
Cleavage with oI and XbaI. XhoI and Av
By inserting into pPIC9 cut with rII,
pPIC9-116EL was obtained.

Next, an immunoglobulin-like region deleted fusion protein 112VAA (N-terminal is N-terminal 11 of IL-6R)
An expression plasmid pPIC9-112VAA encoding the fusion protein of the present invention in which the valine at the N-terminal 333 of IL-6R and the alanine at the N-terminal 28 of IL-6 are directly fused without using a linker. And the fusion protein 116EAA (N-terminal is glutamic acid at position 116 of the N-terminal of IL-6R, and N-terminal 3 of IL-6R.
An expression plasmid pPIC9-116 encoding the fusion protein of the present invention in which alanine at position 33 and alanine at position 28 at the N-terminus of IL-6 are directly fused without using a linker.
EAA was constructed in the following manner.

PPIC9-12-1VL was converted to XhoI and Pm
The fragment obtained by cutting with aCI was previously digested with XhoI and Pma.
333AΔA expression plasmid pPIC cut with CI
9-333AΔA, pPIC9-112VAA
Was prepared.

Next, pPIC9-116EL was converted to XhoI
And a fragment obtained by digesting with PmaCI, the expression plasmid p of 333AΔA previously digested with XhoI and PmaCI.
Inserted into PIC9-333AΔA, pPIC9-116
EAA was prepared.

PPIC9-112VAA, pPIC9-
As a result of producing a transformant by the method shown in Example 3 using 116EAA, mut ^s fungus 9 expressing 112VAA
It was established mut ^s strains 11 strains expressing the stock and 116EAA. Further, this was cultured by the method shown in Example 4, and the biological activity in the supernatant was measured by the method shown in Example 5.

As apparent from FIG. 14, the fusion proteins 112VAA and 116E lacking the immunoglobulin-like region were deleted.
AA is a fusion protein 3 having an immunoglobulin-like region.
It had almost the same activity as 33AΔA.

Example 15 Determination of Protease Cleavage Site A stream of 2% SDS, 64% glycerol and 0.25% bromophenol blue was added to 1.9 ml of the streamline elution fraction obtained as in Example 7 in an amount of 0.1 m.
After adding l, the mixture was boiled. After subjecting this to SDS polyacrylamide gel electrophoresis, 10% methanol, 10%
Electroblotting was performed on a polyvinylidene difluoride (PVDF) membrane in a mM CAPS buffer (pH 11). This is Komaji Brilliant Blue G-25
After staining with 0 and detection, several of the detected protein portions were cut and recovered, respectively, and applied to a protein sequencer 477A (manufactured by Applied Biosystems). As a result, a protein having a molecular weight of 18 kD was converted into an aspartic acid residue-valine residue-alanine residue-alanine residue-
Proline residue-histidine residue, a part of the fusion protein, in which the peptide bond between the N-terminal 37th lysine residue and the 38th aspartic acid residue of the IL-6 portion is cleaved by a Pichia yeast-derived protease. It was caused by

Example 16 Expression of Protease-Resistant Fusion Protein (1) From pBS6R6L obtained as in Example 1, using the primer pKN6B38D (SEQ ID NO: 55) and the primer pIL6F2 (SEQ ID NO: 56), the IL-6 gene was used. (CDNA) was amplified and cut with NruI and NotI. This was inserted into pBS6R6L, which had been cut with Eco47III and NotI beforehand, to obtain plasmid p.
BS6R6L-38D was obtained.

PBS6R6L-38D was ligated with XhoI and No.
An expression plasmid pPIC9 encoding the protease-resistant fusion protein 20LAD of the present invention is obtained by obtaining the gene encoding the IL-6R / IL-6 fusion protein by cutting with tI, inserting the gene into pPIC9 previously cut with XhoI and NotI. -20 LAD was produced.

Using the primer p6RAB112V (SEQ ID NO: 57) and the primer p344F (SEQ ID NO: 58), the IL-6R gene was amplified and cut with XhoI and XbaI. This was previously cut with XhoI and AvrII.
By inserting into PIC9, pPIC9-112V
L was obtained. pPIC9-112VL was converted to XhoI and Pma
The fragment obtained by cutting with CI was previously digested with XhoI and PmaC
I was inserted into pPIC9-20LAA digested with I, and the immunoglobulin-like region deleted fusion protein 112VAA (N-terminal is valine at the N-terminal 112th position of IL-6R; IL-
An expression plasmid pPIC9- encoding a fusion protein in which the N-terminal alanine of 6R is directly fused with the N-terminal alanine of IL-6 without a linker.
112 VAA was produced.

Next, primer p6RAB116E (SEQ ID NO: 59) and primer p344F (SEQ ID NO: 58)
To amplify the IL-6R gene using XhoI and Xba
I cut. By inserting this into pPIC9 previously cut with XhoI and AvrII, pPIC9-
116EL was obtained. pPIC9-116EL was replaced with XhoI
And the fragment obtained by digestion with PmaCI were inserted into pPIC9-20LAA previously digested with XhoI and PmaCI, and the fusion protein 116E lacking the immunoglobulin-like region was deleted.
AA (a fusion protein in which the N-terminal is glutamic acid at the N-terminal 116 of IL-6R and the alanine at the N-terminal 333 of IL-6R is directly fused to the alanine at the N-terminal 28 of IL-6 without a linker) Expression plasmid pPIC
9-116 EAA was produced.

Further, pPIC9-20LAD was replaced with XhoI.
To obtain a gene encoding IL-6R by digestion with pmaCI and pPI previously digested with XhoI and PmaCI.
By inserting into C9-112VAA, the protease-resistant fusion protein 112VAD of the present invention (the N-terminal is the valine at position 112 of the N-terminal of IL-6R, and the IL-6R is
Alanine at position 333 of N-terminal and N-terminal 38 of IL-6
Expression plasmid pPIC9 encoding a fusion protein obtained by directly fusing the aspartic acid without a linker
-112VAD was inserted into pPIC9-116EAA which had been cut with XhoI and PmaCI to obtain the protease-resistant fusion protein 116EAD of the present invention.
(N-terminal is glutamic acid at the N-terminal 116th of IL-6R, alanine at the N-terminal 333 of IL-6R and IL
Expression plasmids pPIC9-116EAD encoding fusion proteins obtained by directly fusing the N-terminal 38th aspartic acid of -6 without a linker) were prepared.

Example 17 Expression of Protease-Resistant Fusion Protein (2) Five types of expression plasmids (pPIC) described in Example 16
9-20 LAD, pPIC9-12VAA, pPIC
9-116EAA, pPIC9-12VAD, pPI
C9-116 EAD) was introduced into Pichia yeast by the method described in Example 3. As a result, pPIC9-20
10 shares mut ^s strains transformed with LAD, pP
Seven mut ^s strains transformed with IC9-112VAA, ten mut ^s strains transformed with pPIC9-116EAA, one mut ^s strain transformed with pPIC9-12-VAD, one pPIC9-11
The mut ^s strains transformed were obtained 6 strains by 6EAD. This was cultured by the method described in Example 4 to obtain a supernatant, and the biological activity was measured by the method described in Example 5, and as a result,
mut ^s transformed by pPIC9-20LAD
10 strains of 10 strains in strain, 7 out of the 7 strains in mut ^s strains transformed by PPIC9-112VAA, pPIC
7 out of 10 mut ^s strains transformed with 9-116EAA, 1 out of 1 mut ^s strain transformed with pPIC9-112VAD, pPIC 9-1 1
In the mut ^s strain transformed by 6EAD, 6 out of 6 strains
The strain had biological activity. This result indicates that N-6 of IL-6
This shows that the biological activity is not affected by deletion of the alanine at the terminal position 28 to the lysine position 37.

Example 18 Evaluation of Protease Resistance 333AΔA (hereinafter referred to as 20LAA) described in Example 3, 20LAD protease-resistant fusion protein of the present invention described in Example 17 and fusion protein 112VA described in Example 17
A, Protease-resistant fusion protein 112V of the present invention
Transformants expressing AD, fusion protein 116EAA, and the protease-resistant fusion protein 116EAD of the present invention were cultured by the following methods. Using a test tube, BMGY medium (1% (W / V) yeast extract, 2% (W / V)
V) Peptone, 1.34% (W / V) YNB wo A
A, 0.00004% (W / V) biotin, 100 mM
After culturing with 3 ml of potassium phosphate (pH 6.0) and 3% of 1% (W / V) glycerol at 30 ° C. for 48 hours, the cells were collected by centrifugation, and the cell pellet was transferred to a BMMY medium (1% (W / V)).
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), 2% of 0.5% (V / V) methanol) and cultured at 30 ° C. for 24 hours. The culture solution at 24 hours was sampled from each culture solution, centrifuged, and the culture supernatant was recovered and subjected to SDS polyacrylamide gel electrophoresis.
After the electrophoresis, the protein in the gel was transferred to a PVDF membrane, and subjected to Western blotting using a rabbit anti-IL-6 polyclonal antibody (Genzyme). FIG. 15 shows the results.

As is clear from FIG. 15, 20 LAA,
In 116EAA and 112VAA, each IL-6
A band having a molecular weight of 18 kD, which was thought to have been generated by cleavage at the C-terminal of lysine at position N-terminal 37, was clearly detected, but the protease-resistant fusion protein 20 of the present invention was not detected.
LAD, 116 EAD, 112 VAD did not detect or hardly detected. from this result,
It turns out that protease resistance was able to be imparted by introducing a mutation that deleted 10 amino acids from the N-terminal 28th to 37th amino acids of IL-6.

[0135]

According to the present invention, the IL-6R / IL-6 fusion protein having no linker sequence provided by the present invention is higher than the IL-6R / IL-6 fusion protein having a linker sequence reported hitherto. A significant decrease in efficacy and antigenicity is expected. This indicates that the fusion protein has great significance as a novel therapeutic agent in the hematopoietic domain,
It is expected to be developed as a vivo amplifying agent or a platelet increasing agent.

[0136] In the step of imparting resistance to the prosthesis secreted by the host cell, the step of producing the host cell by genetic engineering involves the step of transforming the host cell into a conventional one because it is not cleaved by the protease. It is possible to produce a larger amount in comparison. Further, even if the protease is subjected to a purification step in the coexistence state, it is not cleaved, so that the yield can be ultimately increased, and furthermore, the operation of inactivating the protease prior to the operation can be omitted. Therefore, there is also an effect that the purification operation can be simplified. Furthermore, since a molecule cleaved by the protease is not mixed in the final purified product, a more uniform purified product can be obtained.

In addition to the above effects, those having a resistance to a protease which is expected to coexist in an environment where the biological activity is to be exerted can be expected to exert the effect of sustaining the biological activity.

[0138]

[Sequence List] SEQUENCE LISTING <110> Tosoh Corporation <120> IL-6 receptor / IL-6 direct fusion protein <130> PA210-9867 <150> JP10-190597 <151> 1998-07-06 <150> JP11- 21788 <151> 1999-01-29 <150> JP11-123411 <151> 1999-04-30 <160> 60 <210> 1 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 1 gatcccctcc agctggcggt ggtggatccg ccccggtac 39 <210> 2 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 2 cggggcggat ccaccaccgc cagctggagg g 31 <210> 3 < 211> 54 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 3 gatcccctcc agctggtggc ggtggctcgg gcggtggtgg gtcggccccg gtac 54 <210> 4 <211> 46 <212> DNA <213> Artificial Sequence <220 > <223> Annealed sequence <400> 4 cggggccgac ccaccaccgc ccgagccacc gccaccagct ggaggg 46 <210> 5 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 5 gatcccctcc agctgagaac gaggtgtcca cccccatgcca cccccatgcca gccccggtac 60 <210> 6 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 6 cggggctgga agtgcctgca tgggggtgga cacctcgttc tcagctggag gg 52 <210> 7 <211> 54 <212> DNA <213> Artificial Sequence <220> < 223> Annealed sequence <400> 7 gatcccctcc agctgagaac gaggtgtcca cccccatgca ggcagccccg gtac 54 <210> 8 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 8 cggggctgcc tgcatggg gg tggacacctctt <210> 9 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 9 gatcccctcc agctgccccg gtac 24 <210> 10 <211> 16 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 10 cggggcagct ggaggg 16 <210> 11 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 11 aggtggcggt ggatccgccc cggtac 26 <210 > 12 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 12 cggggcggat ccaccgccac ct 22 <210> 13 <211> 41 <212> DNA <213> Artificial Sequence <220 > <223> Ani <400> 41 aggtggcggt ggctcgggcg gtggtgggtc ggccccggta c 41 <210> 14 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 37 cggggccgac ccaccaccgc ccgagccacc gccacct 37 <210> 15 <211> 71 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 15 acttactact aataaagacg atgataatat tggtggcggt ggctcgggcg gtggtgggtc 60 ggccccggta c 71 <210> 16 <211> 67 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 16 cggggccgac ccaccaccgc ccgagccacc gccaccaata ttatcatcgt ctttattagt 60 agtaagt 67 <210> 17 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400 > 17 aggtggcggt ggctcgggcg gtggtgggtc gggtggcggc ggatctgccc cggtac 56 <210> 18 <211> 52 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 18 cggggcagat ccgccgccac ccgaccgacc accc accc 211> 17 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 19 aggtggcgcc ccggtac 17 <210> 20 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 20 cggggcgcca cct 13 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223 > Annealed sequence <400> 21 aggtggcggt ggcgccccgg tac 23 <210> 22 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 22 cggggcgcca ccgccacct 19 <210> 23 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 23 acttactact aataaagacg atgataatat tggtggcgcc ccggtac 47 <210> 24 <211> 43 <212> DNA <213> Artificial Sequence <220> <223 > Annealed sequence <400> 24 cggggcgcca ccaatattat catcgtcttt attagtagta agt 43 <210> 25 <211> 53 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 25 acttactact aataaagacg atgataatat tggtggcggt ggcgcccccc tac 53 210> 26 <211> 49 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 26 cggggcgcca ccgccaccaa tattatcatc gtctttatta gtagtaagt 49 <210> 27 <211> 95 <212> DNA <213> Artificial Sequence < 220> <223> Annealed sequence <400> 27 acttactact aataaagacg atgataatat tctcttcaga gattctgcaa atgcgacaag 60 cctcccagtg caagattctt cttcagcccc ggtac 95 <210> 28 <211> 91 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence > 28 cggggctgaa gaagaatctt gcactgggag gcttgtcgca tttgcagaat ctctgaagag 60 aatattatca tcgtctttat tagtagtaag t 91 <210> 29 <211> 86 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 29 acttactgacta cagatagacatatagatatacatagacata gatag cctcccagtg caagatgccc cggtac 86 <210> 30 <211> 82 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 30 cggggcatct tgcactggga ggcttgtcgc atttgcagaa tctctgaaga gaatattatc 60 atcgtctta ttagtaga 31 > 68 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 31 acttactact aataaagacg atgataatat tctcttcaga gattctgcaa atgcgacagc 60 cccggtac 68 <210> 32 <211> 64 <212> DNA <213> Artificial Sequence < 220> <223> Annealed sequence <4 00> 32 cggggctgtc gcatttgcag aatctctgaa gagaatatta tcatcgtctt tattagtagt 60 aagt 64 <210> 33 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Annealing sequence <400> 33 acttactact aataaagacg atgataatat tctcttcaga g210cc 34 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 34 cggggctctg aagagaatat tatcatcgtc tttattagta gtaagt 46 <210> 35 <211> 41 <212> DNA <213> Artificial Sequence < 220> <223> Annealed sequence <400> 35 acttactact aataaagacg atgataatat tgccccggta c 41 <210> 36 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 36 cggggcaata ttatcatcgt ctttattagt agtaagt 37 <210> 37 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 37 acttactact aataaagccc cggtac 26 <210> 38 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 38 cggggcttta ttagtagtaa gt 22 <210> 39 <211> 17 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <4 00> 39 acttactgcc ccggtac 17 <210> 40 <211> 13 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 40 cggggcagta agt 13 <210> 41 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 41 acttactact aataaagacg atgataatat tccggtac 38 <210> 42 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 42 cggaatatta tcatcgtctt tattagtagt aagt 34 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Annealing sequence <400> 43 acttactact aataaaccgg tac 23 <210> 44 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Annealed sequence <400> 44 cggtttatta gtagtaagt 19 <210> 45 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 45 ctcgagaaga ggctggcccc aaggcgctgc cc 32 <210> 46 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 46 agatctggat tctgtccaag gcgtgcccat ggc 33 <210> 47 <211> 28 <212> DNA < 213> Artificial Sequence <220> <223> Primer <400> 47 agatctggta cccccaggag aagattcc 28 <210> 48 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 48 atgcggccgc tacatttgcc gaagagccc 29 <210> 49 <211> 51 <212 > DNA <213> Artificial Sequence <220> <223> Oligomer <400> 49 gatcccctcc agctgagaac gaggtgtcca cccccatgca agcgctggta c 51 <210> 50 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> Oligomer < 400> 50 cagcgcttgc atgggggtgg acacctcgtt ctcagctgga ggg 43 <210> 51 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 51 ctcgagaaga gggttccccc cgaggagccc cag 33 <210> 52 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 52 tctctagaga atattatcat cg 22 <210> 53 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer <400 > 53 ctcgagaaga gggagcccca gctctcctg 29 <210> 54 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Primer <400> 54 tctctagaga atattatcat cg 22 <210> 55 <211> 24 <212> DNA <213> Artificia l Sequence <223> Primer <400> 55 tctcgcgatg tagccgcccc acac 24 <210> 56 <211> 29 <212> DNA <213> Artificial Sequence <223> Primer <400> 56 atgcggccgc tacatttgcc gaagagccc 29 <210> 57 <211> 33 <212> DNA <213> Artificial Sequence <223> Primer <400> 57 ctcgagaaga gggttccccc cgaggagccc cag 33 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <223> Primer <400> 58 tctctagag aatattatca tcg 23 <210> 59 <211> 29 <212> DNA <213> Artificial Sequence <223> Primer <400> 59 ctcgagaaga gggagcccca gctctcctg 29 <210> 60 <211> 13 <212> <213> Artificial Sequence <223> Linker <400> 60 Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Val Glu 13

[Brief description of the drawings]

FIG. 1. (a) IL-6 via linker sequence.
(B) an IL-6R / IL-6 fusion protein of the present invention in which IL-6R and IL-6 are directly fused without the use of a linker, and an IL-6R / IL-6 fusion protein in which R and IL-6 are fused. It is a figure showing an illustration.

FIG. 2 shows the plasmid pBS prepared in Example 1.
It is a figure which shows the structure of 6R6S. amp is the ampicillin resistance gene, ori is the transcription initiation site, IL-6 is I
L-6 gene and IL-6R indicate the IL-6R gene, respectively.

FIG. 3 shows the plasmid pBS6R6 shown in FIG.
FIG. 9 is a diagram showing a procedure for inserting a product obtained by annealing two kinds of oligonucleotides in S in accordance with the method described in Example 2.

FIG. 4 shows the plasmid pBS prepared in Example 1.
It is a figure which shows the structure of 6R6L. amp is the ampicillin resistance gene, ori is the transcription initiation site, IL-6 is I
L-6 gene and IL-6R indicate the IL-6R gene, respectively.

FIG. 5 shows the plasmid pBS6R6 shown in FIG.
FIG. 7 is a diagram showing a procedure for inserting into L two oligonucleotides annealed by the method shown in Example 2;

FIG. 6 shows the derivative 333AΔ prepared in Example 2.
FIG. 2 shows the structure of the expression plasmid for A. In the figure, am
p is the ampicillin resistance gene, ori is the transcription initiation site, HIS4 is the histidine synthesis gene, 3'AOXT
T is a terminator, IL-6 is an IL-6 gene,
IL-6R indicates an IL-6 receptor gene, S indicates a gene encoding a signal sequence, and 5′AOX1 indicates an upstream region of an alcohol oxidase gene including a promoter sequence.

FIG. 7 shows (A) each derivative of each fusion protein.
The type and number of the C-terminal amino acid residue of the IL-6R region,
(B) the amino acid sequence of the linker (“-” indicates no linker), (C) the type and number of the amino acid residue at the N-terminal of the IL-6 region, and (D) the amino acid residue obtained in Example 4. 21
FIG. 7 is a view showing average biological activity values obtained by the method shown in Example 5 for each of the six types of culture supernatants.

FIG. 8 is a diagram showing a change over time when culturing is performed by the method described in Example 6. In the figure, MeOH con
c. Is the methanol concentration (unit is% (mass / volume)),
EIA will launch sandwich immunoassays, Bio As
“say” indicates a biological activity.

FIG. 9 is a diagram showing the biological activity of FP6 determined by the method described in Example 8.

FIG. 10 shows SDS of FP6 treated with endoglycosidase H for 0 to 180 minutes by the method described in Example 10.
It is a figure which shows the result when used for a polyacrylamide gel electrophoresis. In the figure, the lane indicated by H indicates only endoglycosidase H, and the lanes numbered from 0 to 180 indicate FP6 treated with endoglycosidase H for the time (minutes) of each number. 97.4 and 66. in the figure.
The bars with numerical values of 3 indicate the positions where the molecular weight marker proteins (molecular weights 97.4 kDa and 66.3 kDa) were detected, respectively.

FIG. 11 is a diagram showing a method of IL shown in FIG.
-6 (100 ng / ml) and IL-6R (100 ng / ml)
ml), IL-6 (100 ng / ml)
And IL-6R (200 ng / ml), F
P6 (300 ng / ml), FP6 (600 ng / m
FIG. 1 (b) is a drawing showing the colony forming ability of 500 CD34-positive cells in 1). In the figure, G represents a granulocyte colony,
M indicates a macrophage colony, GM indicates a granulocyte / macrophage mixed colony, Blast indicates a blast cell colony, Mix indicates a mixed granulocyte / macrophage / erythroblast colony, and BFU-E indicates an erythroblast colony. .

FIG. 12 is a diagram showing a C57B according to the method shown in Example 12.
When FP6 was not administered to L6 mice (first
FIG. 11 is a diagram showing the average values of the platelet counts when administered (group 2) and when administered (group 2-5). In the figure, an asterisk indicates a significant difference (P <0.05) from the platelet count of the mice of the first group.

FIG. 13 is a diagram showing 5F in advance according to the method shown in Example 13;
The mean value of the platelet count on days 7-9 when FP6 was not administered (Group 1, black squares) and when FP6 was administered (Group 2, white squares) to C57BL6 mice to which U was administered FIG. In the figure, the asterisk indicates a significant difference (P <0.05) from the platelet count of the mice in the first group on the same day.

Figure 14, mut ^s fungus 9 expressing obtained () fusion protein 112VAA the method shown in Example 14
Mut ^s expressing the shares and () fusion protein 116EAA
To bacteria 11, the biological activity value average value obtained in the method shown in Example 5, mut ^s bacteria 11 expressing 333AΔA
III-108, the strain with the highest expression level among the strains
It is a figure shown as a relative value (%) with respect to the strain.

FIG. 15 shows (a) 20LAA, (b) 20L
AD, (c) 116 EAA, (d) 116 EAD,
FIG. 7 shows the results of Western blotting of the culture supernatant containing (e) 112VAA and (f) 112 VAD by the method described in Example 8.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 14/715 C07K 19/00 19/00 C12N 1/19 C12N 1/19 C12P 21/02 C C12P 21 / 02 A61K 37/02 // (C12N 1/19 C12R 1:84) (C12P 21/02 C12R 1:84) (72) Inventor Shigeo Tsuchiya 2-7-3, Tachibanadai, Aoba-ku, Yokohama-shi, Kanagawa B317 (72) Inventor Teruhiko Ide 6-4-13 Minamino, Hachioji-shi, Tokyo F-term (reference) 4B024 AA01 BA26 BA63 CA04 DA12 EA04 GA11 GA19 HA01 4B064 AG03 AG20 BA15 BH09 CA06 CA19 CC24 CD06 CE06 CE07 CE11 DA01 4B065 AA72X AB01 BD02 BB06 BD15 CA24 CA44 4C084 AA02 AA06 AA07 BA41 BA44 CA53 DA18 DA46 MA17 MA44 MA63 MA66 NA06 NA07 ZA512 ZA532 ZB212 4H045 AA10 AA20 AA30 BA41 BA53 DA02 DA51 EA24 FA73 FA74 G A10 GA15 GA22 GA23 GA25 HA06

Claims (16)

[Claims] 1. An IL-6 receptor-IL-6 fusion, wherein one of the amino acid residues constituting IL-6 receptor and one of the amino acid residues constituting IL-6 are directly bound. protein. 2. An IL-6 receptor having a C-terminal amino acid residue at any one of 39 amino acid residues from the alanine residue at the N-terminal 323 to the serine residue at the N-terminal 361 of the IL-6 receptor. 6. The IL-6 receptor according to claim 1, wherein 6 N-terminal amino acid residues are bound.
IL-6 fusion protein. 3. A gene encoding an IL-6 receptor-IL-6 fusion protein in which one of the amino acid residues constituting the IL-6 receptor and one of the amino acid residues constituting the IL-6 are directly bound. . 4. An IL-6 receptor having a C-terminal amino acid residue at the C-terminal side of any one of 39 amino acid residues from the alanine residue at the N-terminal 323 to the serine residue at the N-terminal 361 of the IL-6 receptor. The gene according to claim 3, which encodes an IL-6 receptor / IL-6 fusion protein to which 6 N-terminal amino acid residues are bound. 5. A gene encoding an IL-6 receptor / IL-6 fusion protein in which one of the amino acid residues constituting IL-6 receptor and one of the amino acid residues constituting IL-6 are directly bound. Pichia pastoris (Pichia pastoris) transformed with an expression vector containing
astoris). 6. An IL-6 receptor having an IL-terminal C-terminal amino acid residue of any one of 39 amino acid residues from the alanine residue at the N-terminal 323 to the serine residue at the N-terminal 361 of the IL-6 receptor. 6. The Pichia pastoris according to claim 5, which is transformed with an expression vector containing a gene encoding an IL-6 receptor-IL-6 fusion protein to which an N-terminal amino acid residue of 6 is bound.
storis) species of yeast. 7. A gene encoding an IL-6 receptor / IL-6 fusion protein in which one of the amino acid residues constituting the IL-6 receptor and one of the amino acid residues constituting the IL-6 are directly bound. Pichia pastoris (Pichia pastoris) transformed with an expression vector containing
astoris) and cultivating yeast of the species
Collecting the L-6 receptor / IL-6 fusion protein as a secretory protein;
A method for producing an IL-6 fusion protein. 8. An IL-6 receptor having a C-terminal C-terminal to any one of the 39 amino acid residues from the alanine residue at the N-terminal 323 to the serine residue at the N-terminal 361 of the N-6 terminal. 6. The Pichia pastoris according to claim 5, which is transformed with an expression vector containing a gene encoding an IL-6 receptor-IL-6 fusion protein to which an N-terminal amino acid residue of 6 is bound.
cultivation of yeasts of the S. storis species and IL
IL-6 receptor I, characterized in that the IL-6 receptor IL-6 fusion protein is collected as a secretory protein
A method for producing an L-6 fusion protein. 9. A gene encoding an IL-6 receptor / IL-6 fusion protein in which one of the amino acid residues constituting IL-6 receptor and one of the amino acid residues constituting IL-6 are directly bound. Pichia pastoris (Pichia pastoris) transformed with an expression vector containing
A method for producing an IL-6 receptor / IL-6 fusion protein, which comprises culturing yeast of the species asteris) without a methanol and containing a carbon source derived from a natural product and adding methanol in the middle. . 10. An IL-6 receptor having a C-terminal amino acid residue at any one of 39 amino acid residues from the alanine residue at the N-terminal 323 to the serine residue at the N-terminal 361 of the IL-6 receptor. Pichia pastoris transformed with an expression vector containing a gene encoding the IL-6 receptor-IL-6 fusion protein to which the N-terminal 6 amino acid residues are bound
is) a type of yeast which is cultured in a medium not containing methanol but containing a carbon source derived from a natural product, and culturing with the addition of methanol on the way.
A method for producing a fusion protein. 11. A solution containing an IL-6 receptor / IL-6 fusion protein in which one of the amino acid residues constituting IL-6 receptor and one of the amino acid residues constituting IL-6 are directly bound. Is subjected to three types of chromatography, ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography, to obtain IL-6.
A method for producing an IL-6 receptor / IL-6 fusion protein, comprising collecting a receptor / IL-6 fusion protein. 12. An IL-6 receptor having an IL-terminal C-terminal to any one of the 39 amino acid residues from the alanine residue at the N-terminal 323 to the serine residue at the N-terminal 361 of the IL-6 receptor. The solution containing the IL-6 receptor / IL-6 fusion protein to which the N-terminal 6 amino acid residue is bound is subjected to three types of chromatography: ion exchange chromatography, hydrophobic chromatography, and gel filtration chromatography. , IL-6 receptor
Collecting an IL-6 fusion protein;
Method for producing 6 receptor / IL-6 fusion protein. 13. An IL-6 receptor / IL-6 fusion protein in which one of the amino acid residues constituting IL-6 receptor and one of the amino acid residues constituting IL-6 are directly bound. Characteristic hematopoietic stem cell ex vi
vo amplifying agent. 14. An IL-6 receptor having an IL-terminal C-terminal amino acid residue of any one of 39 amino acid residues from the alanine residue at the N-terminal 323 to the serine residue at the N-terminal 361 of the IL-6 receptor. An ex vivo amplifying agent for hematopoietic stem cells, comprising an IL-6 receptor-IL-6 fusion protein to which 6 N-terminal amino acid residues are bound. 15. An IL-6 receptor / IL-6 fusion protein, in which one of the amino acid residues constituting the IL-6 receptor and one of the amino acid residues constituting IL-6 are directly bound, as a main component. Platelet-increasing agents including. 16. An IL-6 receptor having a C-terminal amino acid residue at any one of 39 amino acid residues from the N-terminal alanine residue 323 to the N-terminal 361 serine residue on the C-terminal side of the IL-6 receptor. A platelet-increasing agent comprising, as a main component, an IL-6 receptor / IL-6 fusion protein to which 6 N-terminal amino acid residues are bound.