JP2839837B2 - DNA encoding the ligand-binding domain protein of granulocyte colony-stimulating factor receptor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial applications]

The present invention relates to a DNA encoding a ligand binding domain protein, ie, a G-CSF binding domain protein, in the extracytoplasmic domain of a granulocyte colony stimulating factor receptor (hereinafter referred to as G-CSF receptor). An expression vector capable of replicating in Escherichia coli;
The present invention relates to a method for producing a ligand binding region protein of a CSF receptor and a derivative thereof, and a protein produced in such a manner.

[0002]

BACKGROUND ART Granulocyte colony stimulating factor (G-CSF) is a member of a colony stimulating factor involved in blood cell proliferation and differentiation and plays an important role in neutrophilic granulocyte proliferation and differentiation. . This factor controls neutrophil concentration in the blood,
It is also known to be deeply involved in the activation of mature neutrophils. For example, G-CSF acts on a neutrophil precursor cell through a receptor (G-CSF receptor) or the like,
It stimulates its proliferation or differentiation to give mainly neutrophilic granulocytes. It has also been suggested that G-CSF acts as a regulator of neutrophils and has clinical utility in chemotherapy and bone marrow transplantation for cancer patients. On the other hand, it has been found that it may stimulate the growth of cancer cells such as myeloid leukemia cells. The cells on which G-CSF acts are restricted to neutrophil precursors and mature neutrophils, and various myeloid leukemia cells, whereas G-CSF
Receptors are also present on non-blood cells, such as human endothelial cells and placenta, and their ligands (ie, G-CSF)
Elucidation of the mechanism of the complex formation reaction between G-CSF and G-CSF receptor is effective for the study, treatment or prevention of related diseases and abnormalities. For example, it has been suggested that cancer cells such as myeloid leukemia cells proliferate by G-CSF, but it is possible to block it with a substance that antagonizes the G-CSF receptor. Already, mouse and human G-
The DNA encoding the CSF receptor has been cloned,
The sequence has been revealed [Fukunaga et al., Cell, 61 , 3]
41-350 (1990); Proc. Natl. Acad, Sc
i. USA, 87 , 8702-8706 (1990);
WO 91/14776 (released October 3, 1991)]. Fukunaga et al. Also report successful expression of the G-CSF receptor (Cell, supra; Proc. N.
atl. Acad, Sci. USA, supra; EMBO J .; , 1
0 , 2855-2865 (1991)).
The entire sequence of NA was expressed using animal cells. The expression level was extremely low, and G-
It was not optimal for the production of CSF receptor, and structural analysis and study of the reaction mechanism could not be performed sufficiently.

[0003]

SUMMARY OF THE INVENTION In order to achieve the above various objects, the present inventors do not need to use all molecules of the G-CSF receptor. Focusing on the usefulness of a region that plays an important role, that is, a protein in the ligand binding region, studies have been made to establish an effective method for producing such a protein. The amino acid sequence of the ligand binding region protein useful for the purpose of the present invention is known from the above-mentioned documents for humans and mice. Specifically, Fukunaga et al. Cloned cDNA (complementary DNA) encoding the G-CSF receptor, and this receptor consists of an immunoglobulin-like region, a cytokine receptor complementary region (hereinafter abbreviated as CRH region), and a fibronectin type III region. (Cell, supra; Pro
c. Natl. Acad, Sci. USA, supra).

[0004] The CRH region is interleukin 2-
7, erythropoietin, growth hormone, GM-CSF
In addition, cytokines such as interferon α, β, γ
From about 200 amino acids found in the extracytoplasmic region of the receptor
Region of complementarity, the ligand binding site of these receptors
It is believed that. This suggests that these receptors are
It is called the Itokine receptor family [Bazan
(Bazan), Proc. Natl. Acad. Sci. USA,8
7, 6934-6938 (1990)]. Fact G-CS
This CRH region is also essential for the F receptor,
The CRH region of the scale is the ligand binding of this family,
And one unit necessary for signal transmission
Have been. This CRH region is further added to the amino-terminal domain.
(Hereinafter abbreviated as BN domain) and C-terminal domain
Consists of two parts, and when the BN domain is deleted,
Activity is completely lost. This means that ligand binding
Says that the BN domain plays the most important role
Suggests that activity is retained only in the BN domain
It is not yet clear whether it is
-In the case of the leukin 6 receptor, the C-terminal domain is also required.
It is considered important. The above results are the results of the present invention.
BN domain or the B
A protein containing an analog having an activity equivalent to that of the N domain
Suggests usefulness, but only in the BN domain
There is no example of expression, and the binding activity is
It was unknown whether it had sex. Solve these issues
To do so, a large amount of G-CSF receptor binding region protein
Need to be manufactured.

A protein having such an activity (eg, Riga
Use of Escherichia coli for the production of
When it comes, its high production, ease of handling, and
It is extremely convenient and convenient due to the advantages of creating mutants.
Profound. Conventionally, ligand binding site of cytokine receptor
Expression of (CRH) in E. coli is considered to be very difficult
Was. The reason for this is that many binding systems
Contains Foldin easily, including in residues and proline residues
It was mentioned that not to do. Exceptionally human growth
Although the expression was successful in the case of the lemon receptor,
One of the reasons for this is that the human growth hormone receptor
Low cysteine residue content compared to other receptors
It is thought that there is. For example, the same cytokine receptor
Erythropoietin [Harr et al.
is), J. Biol. Chem. ,267, 15205-15
209 (1992)] and interferon gamma [Fontla
Kiunts et al. Biol. Chem. ,26
5, 13268-13275 (1990)].
Most of the products expressed in E. coli are
Has been solubilized. Difficulty in expressing these receptors
The reason is that these receptors have many complex domains
May be established by the combination of G-CSF receiving
As described above, CRH region, immunoglobulin
Complicated Structure Consisting of Chromosome-like Region and Fibronectin-like Region
Is shown. In the case of the above human growth hormone receptor,
The extraplasmic reticulum region consists only of the CRH region.
Another cause of successful growth hormone receptor expression
I think. However, this growth hormone receptor
The CRH region of the body also consists of the BN domain and the C-terminal region
And it cannot be asserted that it has a simple configuration. Present
In addition, the aforementioned interferon γ and erythropoietin
Its extracytoplasmic domain consists almost exclusively of the CRH region.
However, when expressed, most of them are insolubilized.

[0006] Incidentally, signal transmission requires the entire CRH region including the C-terminal region (Fukunaga et al., Supra, EMB).
O. J. , 10 , 2855-2865 (1991), as far as ligand binding is concerned, appears to be active even with the BN domain alone. That is, if only a BN domain of about 100 amino acid residues can be folded and expressed while maintaining almost the same structure as in nature, various cytokines including G-CSF receptor, which has been conventionally difficult to express, can be used. It is possible to remove a large number of functional domains of the receptor. Then, the ligand binding activity of the expression product is examined, and the degree of contribution of this portion to ligand binding can be clarified. In this way, it is possible to obtain a large amount of a protein having a small molecular weight ligand-binding activity. Such small molecular weight ligand binding proteins can be obtained by X-ray or NMR.
This is remarkably advantageous in the analysis of higher-order structures. For the above-mentioned reasons, the production of the ligand-binding domain protein of the G-CSF receptor disclosed in the present invention by the DNA recombination method has been difficult in the past to produce the ligand-binding domain protein of another receptor. Also open.

[0007]

DISCLOSURE OF THE INVENTION The present invention provides a DNA useful for producing a ligand-binding region protein of a G-CSF receptor by a recombinant method, an expression vector containing the DNA, and an expression vector transformed with the expression vector. It is intended to provide a method for producing a G-CSF receptor binding domain protein using an E. coli host cell and the transformant. As described later, the G-CSF receptor ligand-binding domain protein produced by the method of the present invention exhibited a specific G-CSF binding activity almost similar to that of the natural G-CSF receptor. Therefore,
The protein is useful for the study and treatment of diseases related to the interaction between the G-CSF receptor and the ligand, and when administered clinically, through antagonism of the G-CSF receptor in vivo, It is also useful for treating or preventing G-CSF-dependent diseases and abnormalities, for example, leukemia caused by abnormal granulocyte proliferation. Furthermore, the ligand binding region protein of the present invention has the following advantages because it is a small molecular weight protein consisting of about 100 amino acids as a BN domain. That is, when administered to the body, the possibility of producing an antibody is low, and the range of application is wide, such as binding to an appropriate carrier protein depending on the purpose of treatment. Moreover, X-ray and NMR
It is also advantageous for the analysis of higher-order structures by.

The G-CSF receptor binding domain protein of the present invention can be obtained by screening a peptide-based G-CSF agonist or antagonist using a peptide library or the like, or a non-peptide G-CSF agonist or antagonist. Enables screening of
The research on the binding between G-CSF and the G-CSF receptor (such as the analysis of its tertiary structure) has been drastically developed.
It enables the synthesis of SF agonists and antagonists. At present, G-CSF is used as a therapeutic agent for restoring leukopenia in cancer patients who have undergone radiation therapy or anticancer drug treatment, based on their induction of differentiation of myeloid leukemia cells and hyperactivity of mature granulocytes. Although expected to be useful, the G-CSF agonists or antagonists described above are useful in the treatment of patients with leukopenia.
It is expected that the same or better effect can be exhibited.

For purposes of the present invention, the terms used herein are defined below. In the present invention, the term G-CSF receptor means any naturally occurring G-CSF receptor, including humans, including humans, as well as variants of these natural G-CSF receptors produced by genetic engineering. Shall be considered. The ligand-binding domain protein of the G-CSF receptor refers to a protein constituting a region involved in binding to G-CSF in the G-CSF receptor. The protein has an amino acid sequence constituting a CRH region of a G-CSF receptor, especially a BN domain. For purposes of the invention, not only ligand binding domain proteins having a natural amino acid sequence, but also derivatives having similar activity obtained by altering one or more amino acids by methods known to those skilled in the art are useful. Therefore, in this specification, the term "ligand binding region protein" includes not only a protein having a natural amino acid sequence but also a derivative thereof (a mutant in the above sense).

BN, mBN (mouse-derived BN), hBN
(Human-derived BN) means the ligand-binding region of the G-CSF receptor as defined above, and includes mBN and hBN
Refers to the region from mouse and human, respectively. Further, in this specification, unless otherwise specified, proteins in the region and derivatives thereof are also represented. The DNA encoding the ligand binding region protein of the G-CSF receptor is G-CSF receptor.
-A DNA encoding an amino acid sequence constituting a region involved in binding to G-CSF in the CSF receptor.
The DNA contains the CRH region of the G-CSF receptor,
DNA encoding amino acid sequence constituting N domain
It contains. Similar to the definition of the protein described above, the DNA includes not only a DNA encoding a natural ligand binding region protein, but also a DNA encoding a derivative of the protein obtained by a method known to those skilled in the art. DN
A may be either a complementary DNA or a synthetic DNA.

[0011] The CRH region of the G-CSF receptor refers to a region complementary to a cytokine receptor, and the amino acid sequence of this region is mouse [SEQ ID NO: 1 in the sequence listing; Fukunaga et al., Cell, 61 ,
341-350 (1990)] and human [SEQ ID NO: 2 in the Sequence Listing; Fukunaga et al., Proc. Natl. Acad, Sci. USA,
87 , 8702-8706 (1990)]. In this specification, the expression of the ligand binding domain protein is shown using the complementary DNA of the region corresponding to the BN portion of the mouse or human G-CSF receptor, but it may be a synthetic DNA. As will be easily understood by those skilled in the art, even DNAs having different codons are included in the scope of the present invention and are not limited to the description of the Examples, as long as they encode the amino acid sequence of the protein of the present invention.

The complementary DNA encoding the amino acid sequence of SEQ ID NO: 1 has been incorporated into plasmid pBLJ17 [Fukunaga et al., Cell, 61 , 341-350 (1990)], and FERM BP-3312 (deposit date). Escherichia coli K12, deposited under the Ministry of International Trade and Industry, National Institute of Advanced Industrial Science and Technology,
Available from The complementary DNA encoding the amino acid sequence of SEQ ID NO: 2 is a plasmid pH
Q3 [Fukunaga et al., Proc. Natl. Acad. Sci. USA,
87 , 8702-8706 (1990)] and deposited with the Institute of Biotechnology and Industrial Technology, the Ministry of International Trade and Industry under the FERM BP-3314 (deposit date: June 28, 1990). , Escherichia coli pHQ3
Available from

As described above, the BN domain of the G-CSF receptor is the amino-terminal region when the CRH region of the G-CSF receptor is divided into two, and is the genomic DNA (genomic DNA) of the G-CSF receptor. Exons 5 and 6 of DNA)
[Seta, J. et al. Immunol. , 148 , 259-2
66 (1992)]. This is, specifically, in the sequence disclosed by Fukunaga et al.
-In the CSF receptor, refers to the portion from tyrosine 97 to valine 201 (in SEQ ID NO: 1,
Corresponding to tyrosine 1 and valine 105, respectively). Similarly, in the human G-CSF receptor, the region extends from tyrosine at position 98 to valine at position 202 (corresponding to tyrosine at position 1 and valine at position 105 in SEQ ID NO: 2, respectively).

However, the start and end sites of the BN domain are not necessarily strict for the present invention,
It does not significantly affect the three-dimensional structure of the entire BN domain. Provided that the function is retained, N
The terminus and / or C-terminus may be shifted in any direction of several residues, or the terminus and / or C-terminus may be modified by adding several residues of amino acids. Normal,
Such a slight difference in the primary structure does not significantly affect the three-dimensional structure of the entire BN domain, and it is considered that the function is maintained. In the examples described later, in the case of mice, a DNA fragment encoding the BN domain of the mouse G-CSF receptor was used. This DNA fragment starts from glycine-serine, passes through the tyrosine at position 97 in the original sequence (SEQ ID NO: 1), and encodes an amino acid sequence obtained by adding lysine at position 201 to valine at position 201 at the C-terminus (sequence). Number 3). In the case of human, a DNA fragment encoding the BN domain of the human G-CSF receptor was used.
This DNA fragment also encodes an amino acid sequence starting from glycine-serine at the N-terminus, passing through the 98th tyrosine of the original sequence (SEQ ID NO: 2), and adding a lysine to the 202nd valine at the C-terminus (sequence). No. 11).

Further, the DNA encoding the ligand-binding region protein of the present invention is modified by deletion, insertion or substitution of one or more nucleotides by a method well known to those skilled in the art, so that the DNA is encoded. Amino acids and peptides that constitute the ligand binding domain protein to be modified, having the same or more desirable biological properties as the natural ligand binding domain protein, derived from humans,
Alternatively, improved ligand binding proteins derived from other mammals can be produced. Desirable properties vary depending on individual purposes, but include high G-CSF binding activity, activity of inhibiting G-CSF and G-CSF receptor binding, and the like. Therefore, DNA modified by such a method is also included in the scope of the present invention. The present invention also provides G
A method for producing a CSF receptor binding domain protein. The production of such a protein is based on the DNA of the present invention.
Into a suitable expression vector, transforming a host cell with the expression vector, and culturing the transformant.

For expression of the G-CSF receptor of the present invention, for example, prokaryotic microorganisms such as Escherichia coli, eukaryotic microorganisms such as S. cerevisiae, and mammalian cells are used. Tissue culture cells include birds, or mammalian cells such as murine, rat and monkey cells. The selection and use of an appropriate host cell-vector system and the like are known to those skilled in the art.
D encoding the ligand binding domain protein of the CSF receptor
A system suitable for expression of NA can be arbitrarily selected.
However, for the purposes of the present invention, E. coli is preferred for the above reasons. DN encoding mBN or hBN
In order to express A in an E. coli host,
This DNA must be ligated to the E. coli gene expression system. There are many books on the expression system of Escherichia coli, and operation may be performed according to them. That is, a genetic engineering method using Escherichia coli is described in a literature known in the art [Maniatis et al. (1982), Molecular
Cloning: A Laboratory Manual, Cold Spring H
arbor Laboratory], and many other recently published laboratory books. Basic methods such as protein purification are also described in many books such as Biochemistry Laboratory Course (Nippon Biochemistry, Tokyo Kagaku Dojin). Although the method of the present invention can be carried out using these known methods,
The following is a brief description.

The mBN domain or hBN domain is
It can be taken out by the polymerase chain reaction method (hereinafter abbreviated as PCR method). Next, these BN domain genes are inserted into an expression plasmid derived from Escherichia coli. Many gene expression systems derived from Escherichia coli are derived from DNA fragments having promoter activity and translation ability of genes that function in their own cells. Such things include tryptophan gene, lactose gene, alkaline phosphatase gene, and furthermore,
There are tac gene which is a hybrid of tryptophan gene and lactose gene [Nakahara, Matsubara (198
6), edited by The Biochemical Society of Japan, “Genetic Engineering Research Method II”, 1-1
72, Tokyo Chemical Doujinshi]. Usually, the gene is expressed by ligating these DNA fragments before the gene of interest. Usually, there are two types of expression, intracellular expression and secretory expression, and an appropriate expression system exists for each type.

When the target substance is a protein which is originally secreted extracellularly, the gene of the signal sequence of the secretory protein derived from Escherichia coli is ligated to the N-terminal of the gene of the target substance, and the expression product is converted into the periplasm. Expression systems are often constructed to be secreted. In order to express the target substance as a fusion protein with another protein, a method of linking the gene of the target substance immediately after the gene encoding the latter is generally used. The protein to be fused is selected from those that are well expressed in Escherichia coli. Proteins that can be fused with the ligand binding region protein of the present invention include β
-Galactosidase, β-lactamase and maltose binding protein (MBP). Since these proteins are very well expressed under the control of expression in Escherichia coli, their expression as fusion proteins is often very good. Methods for separating a target protein from a fusion protein are also known. For example, when the target substance does not contain methionine, cyanogen bromide treatment is performed. In other cases, for example, a recognition sequence for the restriction protease factor Xa is inserted into the junction of each gene of the fusion protein,
For example, a technique that enables excision by restriction protease factor Xa is used.

Hereinafter, the present invention will be described specifically. In order to obtain DNA encoding a mouse-derived G-CSF receptor ligand-binding domain protein, as described in Example 1 below, the mouse G-CSF receptor BN domain (hereinafter, mBN) was used as the ligand-binding domain protein. The DNA encoding the protein was used. DNA encoding the amino acid sequence of the mBN domain can be obtained from the known plasmid pBLJ17 by PCR. It can also be obtained by a DNA synthesis method. In the examples described below, the mBN domain was expressed as a fusion protein with MBP that is frequently expressed in Escherichia coli.
In this embodiment, the tac promoter, which is induced by the addition of IPTG, was used as the expression promoter. M
Since the signal sequence is added to the N-terminus of BP, the expression product accumulates in the periplasm. For the purpose of expression by a method other than the fusion protein, the gene of the BN domain was linked to various promoters such as an alkaline phosphatase promoter, but no recombinant was obtained. This is considered to be because the recombinant protein had a lethal effect on the E. coli host even when the expression level was very small.

The fusion protein accumulated in the periplasm of cells is extracted, the target peptide is excised by Q-Sepharose and Factor Xa, and the target BN domain is purified by a series of operations consisting of S-Sepharose and gel filtration HPLC. be able to. These series of methods are merely examples, and the target substance can be obtained using other purification systems known to those skilled in the art. In particular,
The BN domain gene was inserted downstream of the DNA encoding MBP. The upstream of the MBP gene contains a portion corresponding to a signal sequence therefor, and the produced fusion protein is secreted and expressed from the cytoplasm into the periplasmic fraction. Therefore, when the E. coli host was transformed with the above expression plasmid and the resulting transformant was cultured, the fusion protein containing the target protein was accumulated in the periplasm of the host cell. Next, the fusion protein was extracted and purified by osmotic shock, and cut off from the maltose binding protein to obtain the target protein. In the above method,
Although an mBN protein having a natural amino acid sequence is obtained, a mutant of the mBN having one or more amino acid mutations (deletion, insertion or substitution) can be similarly expressed in E. coli. Such mutations are intended to improve ligand binding activity, in vivo stability, and the like.

For example, when trying to express an mBN domain having a native sequence, the products in the periplasm include:
In addition to the desired monomeric fusion protein, there are also macromolecular products with which they are associated. When these are cleaved with Factor Xa, the associated mBN
And no binding activity is observed. Many of these molecules show the same molecular weight as the monomer state when developed on a polyacrylamide gel containing SDS without a reducing agent. Therefore, cysteine residues present in the BN domain are cross-linked between molecules during the folding process. It is considered that this is not an aggregate formed but an aggregate formed by the association of a hydrophobic group. Therefore, 7 in the mBN domain
Mutating each of the 7th leucine and the 78th tyrosine to alanine makes the mutation less likely to cause association.
An attempt was made to produce mBN. For mBN-L77A, a DNA fragment encoding the same was designed as shown in SEQ ID NO: 9. This is obtained by changing the codon CTG encoding the 77th amino acid leucine of SEQ ID NO: 3 to the codon GCG codoning alanine. Also mBN-Y78A
As for the DNA fragment encoding this, SEQ ID NO: 1
It was designed as indicated by 0. This is the result of changing the codon TAC encoding the 78th amino acid tyrosine of sequence 3 to GCC.

The actual preparation of the above DNA fragment was performed by PCR. Specifically, a DNA primer having the nucleotide sequence of SEQ ID NO: 6 is synthesized as a N-terminal primer using a DNA synthesizer. This is a primer encoding a portion near the C-terminus in the MBP protein in the pMALp-mBN plasmid. As the C-terminal primer, a synthetic DNA having the nucleotide sequences of SEQ ID NO: 7 and SEQ ID NO: 8 was used. The primer of SEQ ID NO: 7 has CGC at positions 18-20 in the primer so that leucine at position 77 in the original amino acid sequence of SEQ ID NO: 3 is changed to alanine. Similarly, the primer of SEQ ID NO: 8
To change the tyrosine to alanine, 15-
The 17th has GGC. Further, it is constructed so as to have a recognition site for the restriction enzyme PfIMI immediately outside these mutated portions. Therefore, if a PCR reaction is performed using these primers and pMALp-mBN as a template, the restriction protein located immediately downstream from the region including the C-terminal of MBP of the fusion protein, including the mutated portion from the N-terminal of mBN, A region encoding a portion leading to the recognition site of the enzyme PfIMI is synthesized. Next, the product of the PCR reaction was combined with a restriction enzyme site BamHI upstream of the mBN portion,
Cleavage with PfIMI, BamHI-Nde of pMALp-mBN
When the I-cleavage and the NdeI-PfIMI-cleavage are ligated and ligated (see FIG. 5), the expression plasmids of the desired mutants pMALp-mBN-L77A and pMAL
p-mBN-Y78A can be constructed (FIG. 5).

On the other hand, in order to express DNA encoding the ligand binding site of the human G-CSF receptor in a host, the DNA was designed as shown in SEQ ID NO: 11. However, the natural BN domain cannot be removed in large quantities. This is because in the case of the mBN domain there are six cysteine residues, each of which is kept stable by forming three sets of disulfide bonds,
Another extra cysteine in the hBN domain (cysteine at position 67 in SEQ ID NO: 2, 6 in SEQ ID NO: 11)
(Corresponding to the ninth cysteine). That is, the extra cysteine residue is hB
In the process of expression and purification of the N domain, a difference occurs in the formation of a disulfide bond. As a result, the original disulfide bond is broken to form another disulfide bond, and the three-dimensional structure of the hBN domain is destroyed. Conceivable. In the case of mouse, the corresponding residue is serine (corresponding to serine 67 in SEQ ID NO: 1). Therefore, expression was attempted by modifying an extra cysteine in the hBN domain to serine. hBN-C6
9S is obtained by changing TGC encoding the 67th amino acid cysteine (corresponding to the 69th cysteine in SEQ ID NO: 11) of the DNA fragment encoding the hBN domain shown in SEQ ID NO: 2 to TCC encoding serine. (SEQ ID NO: 12). As described above, N-terminal and C-terminal
The region corresponding to exons 5 and 6 as hBN
As in the case of hBN, glycine-serine and lysine are added, respectively.

The natural hBN domain can be extracted from the known plasmid pHQ3 by PCR. Specifically, as an N-terminal primer, SEQ ID NO: 13
Is synthesized using a DNA synthesizer.
This is a primer encoding the N-terminal part in the hBN protein. SEQ ID NO: 14 as the C-terminal primer
A synthetic DNA having the following base sequence was used. By performing PCR using these as primers and pHQ3 as a template, hBN DNA can be obtained.

In the examples described later, hBN-C69
S was obtained by further mutating S by inserting a mutation from a natural hBN inserted into an expression plasmid derived from Escherichia coli (pMALp-hBN, FIG. 7) by PCR, but was obtained directly from pHQ3. Mutation plasmids can also be obtained by performing PCR while introducing mutations or by a DNA synthesis method. In this case, N
A DNA primer having the base sequence of SEQ ID NO: 6 is used as a terminal primer. This is pMALp-h
It encodes a portion near the C-terminus of the MBP protein in the BN plasmid. As the C-terminal primer, a synthetic DNA having the base sequence of SEQ ID NO: 15 was used. This changes the cysteine at position 69 to serine, so that 11
-Has a 13th GGA. PCR is performed using these as primers and pMALp-hBN as a template. As a result, DNA from the N-terminal half of hBN-C69S, that is, the DNA from the N-terminal to the mutated portion can be obtained. For the C-terminal half of hBN, the nucleotide sequences of SEQ ID NO: 16 and SEQ ID NO: 17
PCR is performed as a terminal primer. In this case, the former is used to change the cysteine at position 69 to serine.
The fifth base is TCC. The latter is pMAL
DNA encoding hBN in p-hBN plasmid
Encodes the DNA sequence in the expression vector immediately downstream of. As a result, DNA from the C-terminal half of hBN-C69S, that is, the DNA from the mutated portion to the C-terminal can be obtained. Finally, the PCR products were combined, and the DNA primer of SEQ ID NO: 6 and DN of SEQ ID NO: 17 were combined.
By adding the A primer and performing PCR, all hB
N-C69S DNA can be obtained.

Next, this DNA fragment was used for pMAL ^™ p
B (available from New England Biolabs)
The expression plasmid pMALp-hBN-C69S (FIG. 9) was inserted between the amHI and XbaI sites.
I got Escherichia coli cells transformed with plasmids pMALp-mBN-L77A and pMALp-mBN-L78A produced approximately 2-3 times more purified mN than hBN from E. coli transformed with the original pMALp-mBN.
BN-L77A and mBN-Y78A can be produced. Further, from the E. coli cells transformed with the original pMALp-hBN, only a very small amount of hBN can be extracted.
A large amount of hBN-C69S can be purified from E. coli cells transformed with MALp-hBN-C69S.

The expressed mutant protein, mBN-L77A
And mBN-Y78A, or hBN-C69S,
In the binding test with the ligand, the dissociation constant was the same as that of mBN or hBN. This indicates that these mutants retain the same ligand binding activity as the original mBN or hBN. The above results suggest that mBN-L77A and mBN-Y78A were less susceptible to degradation in Escherichia coli due to mutations in the hydrophobic residues, resulting in a less likely form of association. Further, as for hBN-C69S, as a result of replacement of an extra cysteine residue with serine,
This suggests that stability was increased as a result of eliminating disulfide bond differences that could adversely affect the conformational form. In this way, the increased stability of the product as a result of the mutation not only improves the yield, but also maintains the stability upon binding to other carriers, including in vivo administration. Therefore, it is significant that the handling of the BN domain protein is facilitated and its application range is widened.

That is, the present invention relates to an expression plasmid incorporating a DNA encoding a ligand binding site protein of a G-CSF receptor under the control of an Escherichia coli gene expression system, and a transformation transformed with the expression plasmid. Provide body. Further, the present invention provides a method for producing a ligand binding site protein using the transformant, and a ligand binding site protein obtained by the production method. The ligand binding site protein is preferably an mBN domain protein or an hBN domain protein. MBN domain or h produced by using Escherichia coli as a host according to the method of the present invention.
When the physiological activity of the BN domain was measured using the binding ability to the ligand as an index, the binding ability of both these domains to the ligand was recognized. This result indicates that the recombinant ligand binding region (mBN domain or h
(BN domain) protein retains the ligand-binding activity of G-CSF receptor, and has clinical utility. At the same time, the rapid development of G-CSF receptor research such as three-dimensional structure analysis is expected. It shows what can be brought.

[0029]

The present invention will be described in more detail with reference to the following examples. Example 1 Preparation of DNA encoding BN domain protein of mouse G-CSF receptor and Escherichia coli expression vector
Construction of pMALp-mBN Based on the amino acid sequence of a known mouse G-CSF receptor [Fukunaga et al., Cell, 61 , 341-350 (1990)]
DNA encoding the BN-domain (mBN domain) protein was designed as shown in SEQ ID NO: 3. This D
The NA fragment encodes mouse lysine from position 97 to position lysine 202 of the G-CSF receptor, expresses that portion as a fusion protein with MBP, and isolates a desired mBN domain protein from the expressed fusion protein. , So that it can be inserted into a vector encoding MBP so that it can be purified. That is, between MBP and mBN, Ile-Glu-Gl, which is a recognition site of factor Xa,
It is designed to insert a sequence encoding y-Arg. Therefore, mBN expressed in a form fused with MBP
Is cleaved from MBP by cleavage by the regulatory protease factor Xa. In the DNA fragment shown in SEQ ID NO: 3, GGTTCT which is a sequence encoding glycine-serine is added to the N-terminal side of the 97th tyrosine in order to make the effect of factor Xa more efficient. A termination codon TAA is added to the C-terminal. Furthermore, in order to ligate the prepared DNA fragment to the XbaI site of pMAL ^™ p, the C-terminal side of the DNA is further extended, and an XbaI recognition site is formed in the extension.

The actual preparation of the above DNA fragment used PCR. Specifically, first, an N-terminal primer and a C-terminal primer having the nucleotide sequences of SEQ ID NO: 4 and SEQ ID NO: 5, respectively, are prepared using a 380B DNA synthesizer manufactured by Applied Biosystems. Then
2 μM N-terminal primer (5 terminal phosphorylated), 2 μM C-terminal primer pJ17 DNA
(1 ng) 10 μl of 10 × reaction buffer, 0.25 mM d each
ATP, dTTP, dGTP, dCTP solution, Taq DNA
Add 0.5 μl of polymerase to bring the final volume to 100 μl. Where 10 × reaction buffer, dATP, dTTP, d
GTP, dCTP, and Taq DNA polymerase used were those of GeneAmp kit from Takara Shuzo. This reaction solution is
Set in a NA thermal cycler (model PJ2000, Takara Shuzo), heat treated at 94 ° C for 1 minute; annealing at 37 ° C for 2 minutes;
The desired DNA fragment is synthesized by repeating the reaction five times. Further, the PCR reaction solution is purified by phenol treatment. About 1μg of the purified DNA
With 0.1 M salt and 10 mM magnesium chloride
The DNA is digested by incubating with 50 μl of 0 mM Tris-HCl buffer (pH 7.5) and 5 units of restriction enzyme XbaI (Takara Shuzo) at 37 ° C. for 1 hour. This digested solution was subjected to phenol treatment and ethanol precipitation, and then subjected to 1% agarose gel electrophoresis. A band of about 0.3 kbp was cut out, put into Splect 01 (Takara Shuzo), frozen at −80 ° C. for 15 minutes, and then incubated at 37 ° C. Incubate for 5 minutes to thaw. This is centrifuged at 5000 rpm for 10 minutes, and the filtrate is collected. The target DNA fragment is removed from the filtrate by phenol treatment and ethanol precipitation.

Subsequently, the above DNA fragment was converted into pMAL ^™ p
MB (available from New England Biolabs)
Recognition sequence of restriction protease factor Xa existing downstream of P StuI existing at the end of Ile-Glu-Gly-Arg
Site and the XbaI site (FIG. 1). Specifically, about 1 μg of pMAL ^™ pDNA was added to 50 mM salt and 1 mM.
10 mM Tris-HCl buffer containing 0 mM magnesium chloride
(pH 7.5) Dissolve in 50 μl, add 5 units of restriction enzymes StuI and XbaI, respectively, and incubate at 37 ° C. for 1 hour to digest the added DNA.
Next, the band corresponding to the larger DNA fragment was cut out by electrophoresis on a 1% agarose gel, put into Splect 01 (Takara Shuzo), and frozen at -80 ° C for 15 minutes.
Incubate at 37 ° C for 5 minutes to thaw. This is 5
Centrifuge at 000 rpm for 10 minutes to collect the filtrate.
The target DNA fragment is removed from the filtrate by phenol treatment and ethanol precipitation. Subsequently, this was mixed with the DNA fragment prepared above and mixed with 10 mM containing 1 mM EDTA.
Add M Tris-HCl buffer (pH 7.4) to make 4 μl.
This is mixed with 16 μl of solution A (DNA ligation kit; Takara Shuzo) and 4 μl of solution B (DNA ligation kit; Takara Shuzo) and reacted at 16 ° C. for 30 minutes. Further, this was mixed with 100 μl of competent cells of Escherichia coli K12 strain, and the mixture was incubated at 0 ° C. for 30 minutes and further at 42 ° C. for 2 minutes, and transferred to E. coli to construct an expression plasmid pMALp-mBN. The Escherichia coli K12 strain / pMALp-mBN into which this plasmid has been introduced has been deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (Acceptance date: December 7, 1993; Accession number: FERM-P)
14002).

Example 2 Plasmid pMALp-mB
Production of mBN by Escherichia coli transformed with N 10 g of bactotripton (Difco), 5 g of yeast extract (Difco), and 5 g of sodium chloride supplemented with a very small amount of an antifoaming agent
g, 100 μg / ml ampicillin and 1 liter of medium containing 0.2% glucose,
Grow at 3 ° C. to midlog phase (A600 = about 0.9). In addition, IPTG (Sigma) was used at a final concentration of 0.
Add to 2 mM. Subsequently, after culturing for about 2 hours, the cells are collected. The collected cells were 100 ml of 20% sucrose 2 μg / ml aprotinin, 1 μg / ml pepstatin A,
30 mM Tris buffer containing 5 μg / ml leupeptin (p
H7.5) and incubate at room temperature for 10 minutes. The cells were then collected again, this time with 50 ml of 2 μg / ml aprotinin,
The suspension was suspended in 5 mM magnesium sulfate containing 1 μg / ml pepstatin A and 5 μg / ml leupeptin, and incubated at 0 ° C. for 10 minutes. After removing this by centrifugation at 8,000 rpm, the supernatant is collected and used as a periplasmic fraction.

The periplasmic fraction was added to a sodium phosphate buffer (pH 6.0) so that the concentration became 20 mM, and then 1 mM.
20 mM sodium phosphate buffer containing EDTA (pH 6.
0) (hereinafter referred to as A buffer) equilibrated with Q-Sepharose (2.2 cm diameter × 10 cm; Pharmacia) and eluted with a total of 500 ml of A buffer at a concentration gradient of 0-0.4 M salt. (FIG. 2). The target protein in a form in which MBP and mBN are fused is observed at an absorbance of 280 nm to be 0.3.
It is eluted as a peak near the 2M salt concentration and can be confirmed by showing a molecular weight of about 54 kilodaltons by 15% polyacrylamide electrophoresis containing SDS. The target fraction was concentrated to about 5 ml with PM-10 (Amicon), and the concentrated solution was diluted with 0.1 M Tris buffer (p.
H7.5). Further, 40 μl of protease factor Xa (New England Biolab) is added thereto and reacted at 16 ° C. for 20 hours. This releases the mBN from the MBP.

The reaction solution is diluted 10 times with the A buffer. This is added to S-sepharose (1.2 cm diameter × 5 cm; Pharmacia) equilibrated with A buffer, and eluted with a total of 200 ml of A buffer at a concentration gradient of 0-0.4 M sodium chloride. 28
When traced by the absorbance at 0 nm, the target substance, mBN, separated from the MBP is eluted near the 0.13 M salt concentration. This is subjected to 15% polyacrylamide gel electrophoresis containing SDS, and a band of about 13 kDa is detected, whereby it can be confirmed that the substance is the target substance. The molecular weight of MBP is about 42 kilodaltons and does not adsorb to S-Sepharose.
It is clearly distinguished from N. After concentration of the fraction containing the target substance, TSKgel G2000SW H, which is a gel filtration HPLC equilibrated with an A buffer containing 0.2 M salt, is used.
When added to PLC (7.6 mm diameter x 600 cm; Tosoh),
The mBN domain eluted (FIG. 3). Thus, 1
About 0.1 mg of mBN purified to a single standard was obtained from the cells cultured in 1 liter of culture solution. The N-terminal amino acid sequence of this protein is Gly-Ser-Tyr-Pro-Pro.
-Ala-Ser, which corresponds to the N-terminal sequence of the protein whose expression is expected. This is because the purified product is the desired mBN
It is shown that it is.

Example 3 Binding Activity of Recombinant mBN with Ligand The biological activity of mBN purified to single in Example 2 was measured by the following method, using the binding ability to G-CSF as a ligand as an index. . G radioactively labeled with mBN and ¹²⁵ I
-CSF is mixed to form a complex of mBN and G-CSF. After fixing the formed complex by adding a cross-linking agent, it is developed by SDS-containing polyacrylamide gel electrophoresis and separated as a band on the gel. The bands are observed by revealing the gel on an imaging plate. At this time, unlabeled G-CSF is added to the radiolabeled G-CSF.
By adding -CSF and competing the binding of labeled G-CSF with that of unlabeled, the dissociation constant of the complex of mBN and G-CSF can be measured.

The 0.5 μM mBN domain prepared in Example 2 and 1.2 mM ¹²⁵ I-human G-CSF (100,000 c
pm, Amsham) and various concentrations of unlabeled human G-
CSF (10 ^-5 -10 ^-10 M; provided by Kirin Co., Ltd.)
Is incubated for 90 minutes at room temperature in a final volume of 50 μl in 0.1 M acetate buffer (pH 7.0) containing 0.005% Tween 20. Next, 0.1 M dithiobissuccinimidyl propione (Pierce Chemical Co.) dissolved in 0.5 μl of dimethyl sulfoxide is added, and the mixture is kept at 0 ° C. for 30 minutes. 5 μl of 2M Tris-HCl buffer (pH 7.
The reaction is stopped by adding 5). Subsequently, the reaction mixture is transferred to SD
Perform 15% polyacrylamide electrophoresis with S. After the electrophoresis, the gel was exposed to a Fuji Imaging Plate (for analysis, type BA; Fuji Photo Film Co., Ltd.) and analyzed with an image analyzer (Bio-Image Analyzer BA100; Fuji Photo Film). The complex of CSF was revealed as a 30 kilodalton band on the gel. The dissociation constant of this complex was determined to be 30-80 nM by comparison with the value in an experiment performed with unlabeled G-CSF (FIG. 4).
This result was obtained from Fukunaga et al. (EMBO J. 10 , 2855-28).
65 (1991)) correspond well to the results of experiments performed using mutants in which only the C-terminal domain of the complementary DNA was deleted, and the protein obtained by the method of the present invention has the desired mB
This indicates that it is an N protein.

Thus, Fukunaga et al. Reported a dissociation constant of about 10 nM for the above mutant. The C-terminal domain-deletion mutant contains an immunoglobulin-like domain,
It is known that when only the immunoglobulin-like domain is deleted, the binding activity is reduced to about 1/10 to 1/20 that of the natural one (the dissociation constant is increased). Therefore, when only the BN domain is used, it is expected that the dissociation constant of the C-terminal domain-deletion mutant will be further increased from about 10 nM. Therefore, the value obtained in this example (30 to 80)
nM) well corresponds to this prediction.
That is, it is strongly suggested that the single purified mBN obtained in Example 2 has the same folding as mBN (ligand binding domain protein) in the natural G-CSF receptor.

Example 4 Preparation of DNA Encoding Mutant mBN Domains L77A and Y78A and E. coli Expression Vectors pMALp-mBN-L77A and pMALp
-Construction of -mBN-Y78A (1) Construction of pMALp mBN-L77A In order to produce DNA encoding the mutant mBN domain L77A, an N-terminal primer having the nucleotide sequence of SEQ ID NO: 6 and a C having the nucleotide sequence of SEQ ID NO: 7 The terminal primer is 380B DNA from Applied Biosystems.
It was prepared using a synthesizer. Using these primers and pMAL ^™ p-mBN as a template, PCR reaction was carried out in substantially the same manner as in Example 1 to prepare a mutant DNA. That is, 2 μM N-terminal primer, 2 μM C-
Terminal primer, pMALp ^™ -mBN DNA (1 ng),
10 μl of 10 × reaction buffer, 0.25 mM dATP, d
TTP, dGTP, dCTP solution and 0.5 μl of Taq DNA polymerase are added to make a final volume of 100 μl. Here, 10 × reaction buffer, dATP, dTTP, dGTP, dC
The TP and Taq DNA polymerases used were those of GeneAmp kit from Takara Shuzo. This reaction solution was set in a DNA thermal cycler (model PJ2000, Takara Shuzo), and
Heat treatment at 4 ° C. for 1 minute; annealing at 37 ° C. for 2 minutes; 72
The target DNA fragment is synthesized by repeating the reaction 25 times by a reaction cycle program at 3 ° C. for 3 minutes. Further, the PCR reaction solution is purified by phenol treatment. About 1 μg of the purified DNA was used together with 50 μl of 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 M salt and 10 mM magnesium chloride, 5 units of BamHI (Takara Shuzo) and 5 units of PfIMI (New England Biolab) for 37 hours. Incubate for 1 hour at C to digest the DNA. This digested solution was subjected to phenol treatment and ethanol precipitation, and then subjected to electrophoresis on a 1% agarose gel to cut out a band of about 0.2 kbp.
1 (Takara Shuzo), frozen at -80 ° C for 15 minutes,
Incubate at 7 ° C for 5 minutes to thaw. This is 50
Centrifuge at 00 rpm for 10 minutes and collect the filtrate. The target DNA fragment is removed from the filtrate by phenol treatment and ethanol precipitation.

On the other hand, about 1 μg of pMAL ^™ p-mBN
The DNA was dissolved in 50 μl of 10 mM Tris-HCl buffer (pH 7.5) containing 10 mM magnesium chloride and 10 mM magnesium chloride, and 5 units of each of the restriction enzymes BamHI and PfIMI were added thereto. To digest. Next, the band corresponding to the larger DNA fragment was cut out by electrophoresis on a 1% agarose gel, and the cut out was placed in Splect 01 (Takara Shuzo).
After 15 minutes at 37 ° C, incubate at 37 ° C for 5 minutes to thaw. This is centrifuged at 5000 rpm for 10 minutes, and the filtrate is collected. The target DNA fragment is removed from the filtrate by phenol treatment and ethanol precipitation. Subsequently, this was mixed with the DNA fragment prepared above,
10 mM Tris-HCl buffer (pH 7.
Add 4) to make 4 μl. This was mixed with 16 μl of solution A (DN
A ligation kit; Takara Shuzo) and 4 μl of solution B (DN
A ligation kit; Takara Shuzo)
Incubate for 0 minutes. Further, this was mixed with 100 μl of competent cells of E. coli K12 strain, and the mixture was mixed at 0 ° C. for 30 minutes.
The cells were further transferred to E. coli by incubating at 42 ° C. for 2 minutes, and the expression plasmid pMALp-mBN-L was expressed.
77A was constructed. The nucleotide sequence of the DNA fragment corresponding to the mutant BN domain in the plasmid is shown in SEQ ID NO: 9.
Escherichia coli K12 strain introduced with this plasmid / pMALp
-MBN-L77A has been deposited with the Research Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (Accepted date: December 1993)
7 days; accession number: FERM-P 14003).

(2) Construction of pMALp-mBN-Y78A Expression containing the target DNA fragment (SEQ ID NO: 10) was carried out in the same manner as in the above (1) except that a C-terminal primer having the nucleotide sequence of SEQ ID NO: 8 was used. Plasmid pMALp m
BN-Y78A was constructed. The Escherichia coli K12 strain / pMALp-mBN-Y78A into which this plasmid has been introduced has been deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry of Japan (Deposit date: December 7, 1993; Accession number: FERM)
-P 14004).

Example 5 Plasmid pMALp-mB
N-L77A or pMALp-mBN-L77AY7
Production of mutant mBN by Escherichia coli transformed with 8A and its activity The transformant was cultured in the same manner as in Example 2, and the desired mutant mBN was obtained from the periplasmic fraction. Thus, from the culture solution of each transformant cultured in 1 liter of the culture solution, mBN-L77A and mBN-Y78A, each purified to a single standard, were obtained in an amount of about 0.25 m.
g and about 0.25 mg were obtained. Thus, pMALp-m
From the culture of E. coli transformed with BN-L77A and pMALp-mBN-Y78A, the original pMALp-m
Approximately 2-3 times the amount of purified mBN-L77A and mN-L77A from the same amount of culture broth compared to E. coli transformed with BN.
BN-Y78A protein can be obtained. Each of these proteins is a single preparation. Next, in the same manner as in Example 3, the physiological activity of the mutant mBN purified to single was examined. As a result, mBN-L77A and mBN-
Y78A showed a dissociation constant that was not different from that of mBN (FIG. 6). This result indicates that the singly purified mutant mBN obtained in this example is the mBN at the native G-CSF receptor.
(Folder similar to (ligand binding region protein)) and strongly suggests that it has ligand binding ability.

Example 6 Preparation of DNA encoding BN domain protein of human G-CSF receptor and construction of E. coli expression vector pMALp-hBN Amino acid sequence of known human G-CSF receptor (Fukunaga et al.
Proc. Natl. Acad. Sci. USA, 87 , 8702.
-8706 (1990)), a DNA encoding the BN domain (hBN domain) protein was designed as shown in SEQ ID NO: 11. This DNA fragment encodes the human G-CSF receptor from tyrosine 98 to lysine 203 (Fukunaga et al., Supra), expresses that portion as a fusion protein with MBP, and expresses the desired fragment from the expressed fusion protein. The vector encoding MBP has been devised so that the hBN domain protein can be isolated and purified. That is, a sequence encoding Ile-Glu-Gly-Arg, which is a recognition site for Factor Xa, is inserted between MBP and hBN. Thus, hBN expressed in fusion with MBP is excised from MBP by the restriction protease factor Xa. In the DNA fragment represented by the hBN sequence, GGTTCT which is a sequence encoding glycine-serine is added to the N-terminal side of the 98th tyrosine in order to make the action of factor Xa more efficient. Further, a termination codon TAA is added to the C-terminal side. Further, the prepared DNA fragment was pMAL ^™
In order to ligate to the XbaI site of p, the C-terminal side of the DNA is further extended, and an XbaI recognition site is formed in the extension.

PCR was used to produce the above DNA. Specifically, DNA fragments represented by SEQ ID NO: 13 and SEQ ID NO: 14 were used as N-terminal and C-terminal primers, respectively, as 380B type D of Applied Biosystems.
It was prepared using an NA synthesizer. That is, 2 μM N-terminal primer, 2 μM C-terminal primer, pHQ3 DNA
(1 ng), 10 μl of 10 × reaction buffer, 0.25 m each
M dATP, dTTP, dGTP, dCTP solution, T
Add 0.5 μl of aq DNA polymerase to bring the final volume to 1
Make it 00 μl. Where 10X reaction buffer, dATP,
The dTTP, dGTP, dCTP solution, and Taq DNA polymerase used were those of Takara Shuzo's GeneAmp kit. This reaction solution was subjected to DNA thermal cycler (model PJ
2000, Takara Shuzo), and heat-treated at 94 ° C. for 1 minute; annealing at 37 ° C. for 2 minutes; Further, the PCR product is purified by phenol treatment. About 10n of this purified DNA
g with 5 units of restriction enzymes BamHI (Takara Shuzo) and XbaI (Takara Shuzo), respectively, in 0.1 M salt and 1 part.
The DNA is digested by incubating in 50 μl of 50 mM Tris buffer (pH 7.5) containing 0 mM magnesium chloride at 37 ° C. for 1 hour. The digested solution was subjected to phenol treatment and ethanol precipitation, and then subjected to electrophoresis on a 1% agarose gel. A band of about 0.3 kbp was cut out, put into Splect 01 (Takara Shuzo), frozen at -80 ° C for 15 minutes, and then incubated at 37 ° C. Incubate for 5 minutes to thaw. This is centrifuged at 5000 rpm for 10 minutes, and the filtrate is collected. The target DNA is extracted by phenol treatment and ethanol precipitation.

Subsequently, the above DNA fragment was ligated with pMAL ^™ p
It is inserted between the BamHI and XbaI sites at the end of the recognition sequence Ile-Glu-Gly-Arg of the restriction protease factor Xa present downstream of (available from New England Biolabs) (FIG. 7). Specifically, about 1 μg of pMAL ^™ pDNA was added to 50 mM saline and 10 mM.
The DNA is digested by dissolving in 50 μl of 10 mM Tris-HCl buffer (pH 7.5) containing mM magnesium chloride, adding 5 units of each of the restriction enzymes BamHI and XbaI, and incubating at 37 ° C. for 1 hour. Next, 1%
The mixture was subjected to agarose gel electrophoresis, and the larger band was cut out and placed in Sprek 01 (Takara Shuzo).
Freeze at 15 ° C for 15 minutes, then thaw by incubating at 37 ° C for 5 minutes. This is 5,000 revolutions 10
Centrifuge for minutes and collect the filtrate. The target DNA is extracted by phenol treatment and ethanol precipitation. Subsequently, this is mixed with the DNA fragment prepared above, and a 10 mM Tris buffer (pH 7.4) containing 1 mM EDTA is added to make 4 μl. This was mixed with 16 μl of solution A (DNA ligation kit; Takara Shuzo) and 4 μl of solution B (DN
A ligation kit; Takara Shuzo) and react at 16 ° C. for 30 minutes. Further, this was mixed with 100 μl of competent cells of Escherichia coli K12 strain, and incubated at 0 ° C. for 30 minutes and further at 42 ° C. for 2 minutes to transfer the cells into E. coli, and the expression plasmid pMALp-hB
N was constructed. Escherichia coli K1 with this plasmid
Two strains / pMALp-hBN have been deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry. (Contract date: 1994
May 23; Deposit No .: FERM-P 14321).

Example 7 Plasmid pMALp-hB
Production of hBN Using E. coli Transformed with N 10 g of bactotripton (Difco), 5 g of yeast extract (Difco), 5 g of sodium chloride, 1 g of 100 μg / ml ampicillin and 0.2% glucose Inoculate and grow at 23 ° C. to the midlog phase (A600 = about 0.9). And IP
TG (Sigma) is added to a final concentration of 0.2 mM. Subsequently, after culturing for about 2 hours, the cells are collected. The collected cells are suspended in 200 ml of 20% sucrose, 2 μg / ml aprotinin, 1 μg / ml pepstatin A, and 30 mM Tris buffer (pH 7.5) containing 5 μg / ml leupeptin, and incubated at room temperature for 10 minutes. Collect the bacteria again,
Suspend in 50 ml of 2 μg / ml aprotinin, 1 μg / ml pepstatin A, 5 mM magnesium sulfate containing 5 μg / ml leupeptin and incubate at 0 ° C. for 10 minutes. After removing this by centrifugation at 8000 rpm, the supernatant is collected and used as a periplasmic fraction. Periplasmic fraction 20 mM
After adding a sodium phosphate buffer (pH 6.0) so as to obtain a 20 mM sodium phosphate buffer (pH 6.0) containing 1 mM EDTA and 1 mM 2-mercaptoethanol.
6.0) (hereinafter abbreviated as A buffer) Q-Sepharose (2.2 cm diameter X 10 cm; Pharmacia)
To a total of 500-0.4M in A-buffer.
Elute with a salt gradient. The fusion protein of the desired MBP and hBN was eluted around 0.2M salt concentration and SD
This can be confirmed by showing a molecular weight of about 54 kilodaltons in 15% polyacrylamide gel electrophoresis containing S.

The target fraction was concentrated to about 5 ml with PM-10 (Amicon), and the concentrate was replaced with 0.1 M Tris buffer. Protease factor Xa
(New England Biolab) at 16 ° C
And react for 20 hours. This disconnects the hBN from the MBP. Then, the reaction solution was diluted with 1 mM EDTA, 1 m
S-Sepharose (1.2 cm diameter X5c) equilibrated with 20 mM sodium phosphate buffer (pH 5.5) containing M2-mercaptoethanol (hereinafter referred to as B buffer)
m; Pharmacia) and elute with a total of 200 ml of B buffer with a concentration gradient of 0-0.4M saline. 280
When traced by the absorbance of nm, the concentration around 0.2 M salt
The target substance hBN cleaved from the MBP was eluted. This can be confirmed as a band having a molecular weight of about 12 kilodaltons in SDS-containing -15% polyacrylamide gel electrophoresis. After the above target substance is concentrated,
Gel filtration HP equilibrated with A buffer containing 0.2 M salt
LC, TSK gel G2000SW HPLC
(7.6 mm diameter X 600 cm; Tosoh) to elute the hBN domain.

Example 8 Binding Activity of Recombinant hBN Domain to Ligand The physiological activity of hBN is the same as that of Example 3;
-The ability to bind to CSF was measured as an index. hBN and
A complex of ¹²⁵ I radiolabeled G-CSF is formed. After adding and fixing a crosslinking agent to the formed complex,
Develop by SDS-containing polyacrylamide gel electrophoresis and separate as a band on the gel. Observation of the band
This is done by revealing the gel on an imaging plate. At this time, unlabeled GC is added to the radiolabeled G-CSF.
The dissociation constant of the complex of hBN and G-CSF can be measured by adding SF and competing the binding of labeled G-CSF with that of unlabeled G-CSF. 0.5 mM hBN domain prepared in Example 6, 1.2 nM ¹²⁵ I-human G
-CSF (100,000 cpm, Amsham) and unlabeled human G-CSF (10 ^-5 -1) diluted to various concentrations
0 ^-10 M; Kirin Brewery donating than Ltd.) mixture of 0.1M acetate buffer containing 0.005% Tween 20 (p
Incubate at room temperature for 90 minutes in a final volume of 50 ml in H7.0). Then, 0.5 μl of 0.1 M dithiobissuccinimidyl propione (Pier S Chemical Co.)
And incubate at 0 ° C. for 30 minutes. The reaction is stopped by adding 5 μl of 2M Tris-HCl buffer (pH 7.5). Subsequently, the reaction mixture is electrophoresed on a 15% polyacrylamide gel containing SDS. After the electrophoresis, the gel is transferred to an imaging plate (for analysis, type BA;
When analyzed by Fuji Photo Film Co., Ltd., the generated hB
The complex of N and G-CSF appeared as a 30 kilodalton band on the gel. The dissociation constant of this complex was about 100 nM compared to the experiment in which unlabeled G-CSF was added.
(FIG. 8). The result is Fukunaga et al. (EMBO
J., 10 , 2855-2865 (1991)) well corresponds to the results obtained using mutants in which only the C-terminal domain of the CRH region of the complementary DNA was deleted.

Thus, Fukunaga et al. Reported a dissociation constant of about 10 nM for the above mutant of the complementary DNA. This variant contains an immunoglobulin-like domain, but if only this immunoglobulin-like domain is deleted,
It is known that the binding activity is reduced to about 1 / 10-1 / 20 of that of the natural one (the dissociation constant is increased). Therefore BN
In the case of only the domain, it is expected that the dissociation constant of the mutant of the complementary DNA will further increase from 10 nM. Therefore, 100 nM obtained in the present example corresponds well to this prediction. That is, it is strongly suggested that the hBN domain obtained in the present invention has a folding similar to that of the natural.

Example 9 Preparation of DNA Encoding Mutant hBN Domain C69S and Expression Vector pM
Construction of ALp-hBN-C69S PCR was used to produce DNA encoding the mutant hBN domain C69S. Specifically, the DNA fragments of SEQ ID NO: 6 and SEQ ID NO: 15 were
Applied Biosystems 3
It was prepared using an 80B type DNA synthesizer. Using these as primers, pMALp-hBND prepared in Example 6 was used.
Using NA as a template, a DNA encoding the N-terminal portion was produced by a PCR method. That is, 2 μM N
Terminal primer, 2 μM C-terminal primer, pMAL
p-hBN DNA (1 ng), 10X reaction buffer 10
μl each of 0.25 mM dATP, dTTP, dGT
P, dCTP solution, Taq DNA polymerase 0.5 μl
To a final volume of 100 μl. Here, 10X reaction buffer, dATP, dTTP, dGTP, dCTP solution, and Taq DNA polymerase
The kit was used. This was set in a DNA thermal cycler (model PJ2000, Takara Shuzo) and 94
Heat treatment for 30 seconds at 50 ° C; annealing for 30 seconds at 50 ° C;
The synthesis is carried out by repeating the reaction 20 times at 72 ° C. for 1.5 minutes. In addition, in order to produce DNA encoding the C-terminal portion, the DNA fragments represented by SEQ ID NO: 16 and SEQ ID NO: 17 were similarly synthesized as primers. The synthesized PCR products are 1
% Agarose gel electrophoresis, cut out a band of about 0.15 kbp, and put it in Splect 01 (Takara Shuzo).
After freezing at 80 ° C for 15 minutes, thaw by incubating at 37 ° C for 5 minutes. This is centrifuged at 5000 rpm for 10 minutes, and the filtrate is collected. Purify and recover by phenol treatment and ethanol precipitation. Subsequently, the recovered DNA fragments (1 ng each) were mixed to give a template, and the synthetic DNAs of SEQ ID NO: 6 and SEQ ID
Terminal C-terminal primer (2 μM each)
10 μl of 10 × reaction buffer, 0.25 mM dAT each
P, dTTP, dGTP, dCTP solution, Taq DNA
A PCR reaction was carried out by adding 0.5 μl of the polymerase to a final volume of 100 μl to prepare the entire DNA encoding hBN-C69S. The reaction is heat-treated at 94 ° C. for 30 seconds; annealing at 37 ° C. for 30 seconds; and repeated 20 times at 72 ° C. for 1 minute.

Further, the above PCR product is purified by phenol treatment. About 10n of this purified DNA
g together with 5 units of restriction enzymes BamHI (Takara Shuzo) and XbaI (Takara Shuzo), respectively, in 0.1 M salt and 1
The DNA is digested by incubating in 50 ml of 50 mM Tris buffer (pH 7.5) containing 0 mM magnesium chloride for 1 hour at 37 ° C. The digested solution was subjected to phenol treatment and ethanol precipitation, and then subjected to 1% agarose gel electrophoresis. A band of about 0.3 kbp was cut out, put into Splect 01 (Takara Shuzo), frozen at -80 ° C for 15 minutes, and then incubated at 37 ° C. Thaw by incubating for 5 minutes. This is centrifuged at 5000 rpm for 10 minutes, and the filtrate is collected. The DNA encoding the desired hBN-C69S is removed by phenol treatment and ethanol precipitation. Subsequently, the DNA fragment was ligated to the restriction protease factor Xa recognition sequence Ile-Glu-Gly downstream of pMAL ^™ p (available from New England Biolabs).
-Insert as described in Example 6 between the BamHI and XbaI sites present at the Arg end (FIG. 9).

Specifically, about 1 μg of pMAL ^™ p D
NA was dissolved in 50 μl of 10 mM Tris-HCl buffer (pH 7.5) containing 50 mM salt and 10 mM magnesium chloride, and 5 units of each of the restriction enzymes BamHI and X were added.
Add baI, incubate at 37 ° C for 1 hour and DN
Digest A. Next, this was subjected to electrophoresis on a 1% agarose gel, and the larger band was cut out.
1 (Takara Shuzo), frozen at -80 ° C for 15 minutes,
Incubate at 37 ° C for 5 minutes and thaw. This is 5
Centrifuge at 000 rpm for 10 minutes and collect the filtrate. DNA of interest in phenol treatment and ethanol precipitation
Take out. Subsequently, this is mixed with the DNA fragment prepared above, and 10 μl of Tris buffer (pH 7.4) containing 1 mM of EDTA is added to make 4 μl. This was mixed with 16 μl of solution A (DNA ligation kit; Takara Shuzo) and 4
Mix with μl of solution B (DNA ligation kit; Takara Shuzo) and react at 16 ° C. for 30 minutes. Further, this was mixed with 100 μl of competent cells of Escherichia coli K12 strain and incubated at 0 ° C. for 30 minutes and further at 42 ° C. for 2 minutes to transfer the cells into Escherichia coli to construct expression plasmid pMALp-hBN-C69S. Escherichia coli K12 / pMALp-hBN into which this plasmid was introduced
-C69S has been deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry. (Deposit date: May 23, 1994; Deposit number: FERM-P 14320).

Example 10 Plasmid pMALp-h
PMAL by E. coli transformed with BN-C69S
Production of p-hBN-C69S and its activity The transformant was cultured in the same manner as in Example 7, and the desired hBN-C69S was obtained from the periplasmic fraction. This is higher in expression level than native hBN, and is considered to be about 0.8 mg or more in one liter of culture. Next, when the activity was measured in the same manner as in Example 8, the activity was not different from that of the above-mentioned natural hBN (FIG. 10). The result is hBN-C69S
HBN at the natural human G-CSF receptor
It strongly suggests that it has the same folding and has ligand binding ability.

[0053]

According to the present invention, the ligand binding region protein of the present invention, that is, the mBN domain or the hBN domain is expressed by a gene recombination method using Escherichia coli as a host,
A method for extracting and purifying an mBN domain or an hBN domain from the host has been established. M thus obtained
The BN or hBN domain is the ligand G
-Retains the ability to bind to CSF, and is useful for research and treatment of diseases related to the interaction between G-CSF receptor and ligand. Clinically, it is useful for the treatment or prevention of G-CSF-dependent diseases and abnormalities, for example, leukemia caused by abnormal granulocyte proliferation, through antagonistic action on G-CSF receptors in vivo. The BN domain of the present invention
Since it is a small molecular weight protein consisting of about 100 amino acids, it is unlikely to produce antibodies when administered to the body, and has a wide range of applications such as binding to an appropriate carrier protein depending on the purpose of treatment. It is also advantageous for analysis of higher order structures by NMR or NMR. Further, the G-CSF receptor binding domain protein of the present invention can be used for screening a peptide G-CSF agonist or antagonist using a peptide library or the like, and for screening a non-peptide G-CSF agonist or antagonist. Enable, G-CSF and G-CS
It is useful for research and manufacture of drugs such as G-CSF agonists and antagonists by dramatically developing research on F receptor binding (such as analysis of its three-dimensional structure). Currently G
-CSF is useful as a therapeutic agent for restoring leukopenia in cancer patients who have been treated with radiation therapy or anticancer drugs, based on its induction of differentiation of myeloid leukemia cells and the effect of enhancing the function of mature granulocytes. As expected, the above G-CSF
It is anticipated that agonists or antagonists will be able to exert the same or better effects than G-CSF in treating these leukopenic patients.

[0054]

[Sequence list]

SEQ ID NO: 1 Sequence length: 639 Sequence type: number of nucleic acid chains: double-stranded Topology: linear Sequence type: cDNA to mRNA origin Organism: Mouse Cell type: NFS-60 sequence TAT CCC CCT GCC AGC CCC TCA AAC CTA TCC TGC CTC ATG CAC CTC ACC 48 Tyr Pro Pro Ala Ser Pro Ser Asn Leu Ser Cys Leu Met His Leu Thr 1 5 10 15 ACC AAC AGC CTG GTC TGC CAG TGG GAG CCA GGT CCT GAG ACC CAC CTG 96 Thr Asn Ser Leu Val Cys Gln Trp Glu Pro Gly Pro Glu Thr His Leu 20 25 30 CCC ACC AGC TTC ATC CTA AAG AGC TTC AGG AGC CGC GCC GAC TGT CAG 144 Pro Thr Ser Phe Ile Leu Lys Ser Phe Arg Ser Arg Ala Asp Cys Gln 35 40 45 TAC CAA GGG GAC ACC ATC CCG GAT TGT GTG GCA AAG AAG AGG CAG AAC 192 Tyr Gln Gly Asp Thr Ile Pro Asp Cys Val Ala Lys Lys Arg Gln Asn 50 55 60 AAC TGC TCC TCC ATC CCC CGA AAA AAC TTG CTC CTG TAC CAG TAT ATG GCC 240 Asn Cys Ser Ile Pro Arg Lys Asn Leu Leu Leu Tyr Gln Tyr Met Ala 65 70 75 80 ATC TGG GTG CAA GCA GAG AAT ATG CTA GGG TCC AGC GAG TCC CCA AAG 288 Ile Trp Val Gln Ala Glu Asn Met Leu Gly Ser Ser Glu Ser Pro Lys 85 90 95 CTG TGC CTC GAC CCC ATG GAT GTT GTG AAA TTG GAG CCT CCC ATG CTG 336 Leu Cys Leu Asp Pro Met Asp Val Val Lys Leu Glu Pro Pro Met Leu 100 105 110 CAG GCC CTG GAC ATT GGC CCT GAT GTA GTC TCT CAC CAG CCT GGC TGC 384 Gln Ala Leu Asp Ile Gly Pro Asp Val Val Ser His Gln Pro Gly Cys 115 120 125 CTG TGG CTG AGC TGG AAG CCA TGG AAG CCC AGT GAG TAC ATG GAA CAG 432 Leu Trp Leu Ser Trp Lys Pro Trp Lys Pro Ser Glu Tyr Met Glu Gln 130 135 140 GAG TGT GAA CTT CGC TAC CAG CCA CAG CTC AAA GGA GCC AAC TGG ACT 480 Glu Cys Glu Leu Arg Tyr Gln Pro Gln Leu Lys Gly Ala Asn Trp Thr 145 150 155 160 CTG GTG TTC CAC CTG CCT TCC AGC AAG GAC CAG TTT GAG CTC TGC GGG 528 Leu Val Phe His Leu Pro Ser Ser Lys Asp Gln Phe Glu Leu Cys Gly 165 170 175 CTC CAT CAG GCC CCA GTC TAC ACC CTA CAG ATG CGA TGC ATT CGC TCA 576 Leu His Gln Ala Pro Val Tyr Thr Leu Gln Met Arg Cys Ile Arg Ser 180 185 190 TCT CTG CCT GGA TTC TGG AGC CCC TGG AGC CCC GGC CTG CAG CTG AGG 624 Ser Leu Pro Gly Phe Trp Ser Pro Trp Ser Pro Gly Leu Gln Leu Arg 195 200 205 CCT ACC ATG AAG GCC 639 Pro Thr Met Lys Ala 210 213

SEQ ID NO: 2 Sequence length: 639 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism name: Homo sapiens (human) Tissue type: Placenta or U937 sequence TAC CCT CCA GCC ATA CCC CAC AAC CTC TCC TGC CTC ATG AAC CTC ACA 48 Tyr Pro Pro Ala Ile Pro His Asn Leu Ser Cys Leu Met Asn Leu Thr 1 5 10 15 ACC AGC AGC CTC ATC TGC CAG TGG GAG CCA GGA CCT GAG ACC CAC CTA 96 Thr Ser Ser Leu Ile Cys Gln Trp Glu Pro Gly Pro Glu Thr His Leu 20 25 30 CCC ACC AGC TTC ACT CTG AAG AGT TTC AAG AGC CGG GGC AAC TGT CAG 144 Pro Thr Ser Phe Thr Leu Lys Ser Phe Lys Ser Arg Gly Asn Cys Gln 35 40 45 ACC CAA GGG GAC TCC ATC CTG GAC TGC GTG CCC AAG GAC GGG CAG AGC 192 Thr Gln Gly Asp Ser Ile Leu Asp Cys Val Pro Lys Asp Gly Gln Ser 50 55 60 CAC TGC TGC ATC CCA CGC AAA CAC CTG CTG TTG TAC CAG AAT ATG GGC 240 His Cys Cys Ile Pro Arg Lys His Leu Leu Leu Tyr Gln Asn Met Gly 65 70 75 80 ATC TGG GTG CAG GCA GAG AAT GCG CTG GGG ACC AGC ATG TCC CCA CAA 288 Ile Trp Val Gln Ala Glu Asn Ala Leu Gly Thr Ser Met Ser Pro Gln 85 90 95 CTG TGT CTT GAT CCC ATG GAT GTT GTG AAA CTG GAG CCC CCC ATG CTG 336 Leu Cys Leu Asp Pro Met Asp Val Val Lys Leu Glu Pro Pro Met Leu 100 105 110 CGG ACC ATG GAC CCC AGC CCT GAA GCG GCC CCT CCC CAG GCA GGC TGC 384 Arg Thr Met Asp Pro Ser Pro Glu Ala Ala Pro Pro Gln Ala Gly Cys 115 120 125 CTA CAG CTG TGC TGG GAG CCA TGG CAG CCA GGC CTG CAC ATA AAT CAG 432 Leu Gln Leu Cys Trp Glu Pro Trp Gln Pro Gly Leu His Ile Asn Gln 130 135 140 AAG TGT GAG CTG CGC CAC AAG CCG CAG CGT GGA GAA GCC AGC TGG GCA 480 Lys Cys Glu Leu Arg His Lys Pro Gln Arg Gly Glu Ala Ser Trp Ala 145 150 155 160 CTG GTG GGC CCC CTC CCC TTG GAG GCC CTT CAG TAT GAG CTC TGC GGG 528 Leu Val Gly Pro Leu Pro Leu Glu Ala Leu Gln Tyr Glu Leu Cys Gly 165 170 175 CTC CTC CCA GCC ACG GCC TAC ACC CTG CAG ATA CGC TGC ATC CGC TGG 576 Leu Leu Pro Ala Thr Ala Tyr Thr Leu Gln Ile Arg Cys Ile Arg Trp 180 185 190 CCC CT G CCT GGC CAC TGG AGC GAC TGG AGC CCC AGC CTG GAG CTG AGA 624 Pro Leu Pro Gly His Trp Ser Asp Trp Ser Pro Ser Leu Glu Leu Arg 195 200 205 ACT ACC GAA CGG GCC 639 Thr Thr Glu Arg Ala 210 213

SEQ ID NO: 3 Sequence length: 327 Sequence type: number of nucleic acid chains: double-stranded Topology: linear Sequence type: cDNA to mRNA GGT TCT TAT CCC CCT GCC AGC CCC TCA AAC CTA TCC TGC CTC ATG CAC 48 Gly Ser Tyr Pro Pro Ala Ser Pro Ser Asn Leu Ser Cys Leu Met His 1 5 10 15 CTC ACC ACC AAC AGC CTG GTC TGC CAG TGG GAG CCA GGT CCT GAG ACC 96 Leu Thr Thr Asn Ser Leu Val Cys Gln Trp Glu Pro Gly Pro Glu Thr 20 25 30 CAC CTG CCC ACC AGC TTC ATC CTA AAG AGC TTC AGG AGC CGC GCC GAC 144 His Leu Pro Thr Ser Phe Ile Leu Lys Ser Phe Arg Ser Arg Ala Asp 35 40 45 TGT CAG TAC CAA GGG GAC ACC ATC CCG GAT TGT GTG GCA AAG AAG AGG 192 Cys Gln Tyr Gln Gly Asp Thr Ile Pro Asp Cys Val Ala Lys Lys Arg 50 55 60 CAG AAC AAC TGC TCC ATC CCC CGA AAA AAC TTG CTC CTG TAC CAG TAT 240 Gln Asn Asn Cys Ser Ile Pro Arg Lys Asn Leu Leu Leu Tyr Gln Tyr 65 70 75 80 ATG GCC ATC TGG GTG CAA GCA GAG AAT ATG CTA GGG TCC AGC GAG TCC 288 Met Ala Ile Trp Val Gln Ala Glu Asn Met L eu Gly Ser Ser Glu Ser 85 90 95 CCA AAG CTG TGC CTC GAC CCC ATG GAT GTT GTG AAA TAA 327 Pro Lys Leu Cys Leu Asp Pro Met Asp Val Val Lys 100 105 108

SEQ ID NO: 4 Sequence length: 27 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GGTTCTTATC CCCCTGCCAG CCCCTCCA
27

SEQ ID NO: 5 Sequence length: 35 Sequence type: number of nucleic acid chains: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCTCTAGATT ATTTCACAAC ATCATCATGGGG TCG
AG 35

SEQ ID NO: 6 Sequence length: 26 Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence CGCCGCCAGC GGTCGTCAGA CTGTCG 26

SEQ ID NO: 7 Sequence length: Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GTGGCCATAT ACTGGTACGC GAGCAAGTTT TTTCGGGGGA T41

SEQ ID NO: 8 Sequence length: Sequence type: Number of nucleic acid chains: Single strand Topology: Linear Sequence type: Synthetic DNA sequence GTGGCCATAT ACTGGGCCAG GAGCAAGTTT TTTCGGGG 38

SEQ ID NO: 9 Sequence length: 327 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence characteristics: 229-231: mutation sequence GGT TCT TAT CCC CCT GCC AGC CCC TCA AAC CTA TCC TGC CTC ATG CAC 48 Gly Ser Tyr Pro Pro Ala Ser Pro Ser Asn Leu Ser Cys Leu Met His 1 5 10 15 CTC ACC ACC AAC AGC CTG GTC TGC CAG TGG GAG CCA GGT CCT GAG ACC 96 Leu Thr Thr Asn Ser Leu Val Cys Gln Trp Glu Pro Gly Pro Glu Thr 20 25 30 CAC CTG CCC ACC AGC TTC ATC CTA AAG AGC TTC AGG AGC CGC GCC GAC 144 His Leu Pro Thr Ser Phe Ile Leu Lys Ser Phe Arg Ser Arg Ala Asp 35 40 45 TGT CAG TAC CAA GGG GAC ACC ATC CCG GAT TGT GTG GCA AAG AAG AGG 192 Cys Gln Tyr Gln Gly Asp Thr Ile Pro Asp Cys Val Ala Lys Lys Arg 50 55 60 CAG AAC AAC TGC TCC ATC CCC CGA AAA AAC TTG CTC GCG TAC CAG TAT 240 Gln Asn Asn Cys Ser Ile Pro Arg Lys Asn Leu Leu Ala Tyr Gln Tyr 65 70 75 80 ATG GCC ATC TGG GTG CAA GCA GAG AAT ATG CTA GGG TC C AGC GAG TCC 288 Met Ala Ile Trp Val Gln Ala Glu Asn Met Leu Gly Ser Ser Glu Ser 85 90 95 CCA AAG CTG TGC CTC GAC CCC ATG GAT GTT GTG AAA TAA 327 Pro Lys Leu Cys Leu Asp Pro Met Asp Val Val Lys 100 105 108

SEQ ID NO: 10 Sequence length: 327 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence characteristics: 232-234: mutation sequence GGT TCT TAT CCC CCT GCC AGC CCC TCA AAC CTA TCC TGC CTC ATG CAC 48 Gly Ser Tyr Pro Pro Ala Ser Pro Ser Asn Leu Ser Cys Leu Met His 1 5 10 15 CTC ACC ACC AAC AGC CTG GTC TGC CAG TGG GAG CCA GGT CCT GAG ACC 96 Leu Thr Thr Asn Ser Leu Val Cys Gln Trp Glu Pro Gly Pro Glu Thr 20 25 30 CAC CTG CCC ACC AGC TTC ATC CTA AAG AGC TTC AGG AGC CGC GCC GAC 144 His Leu Pro Thr Ser Phe Ile Leu Lys Ser Phe Arg Ser Arg Ala Asp 35 40 45 TGT CAG TAC CAA GGG GAC ACC ATC CCG GAT TGT GTG GCA AAG AAG AGG 192 Cys Gln Tyr Gln Gly Asp Thr Ile Pro Asp Cys Val Ala Lys Lys Arg 50 55 60 CAG AAC AAC TGC TCC ATC CCC CGA AAA AAC TTG CTC CTG GCC CAG TAT 240 Gln Asn Asn Cys Ser Ile Pro Arg Lys Asn Leu Leu Leu Ala Gln Tyr 65 70 75 80 ATG GCC ATC TGG GTG CAA GCA GAG AAT ATG CTA GGG TCC AGC GAG TCC 288 Met Ala Ile Trp Val Gln Ala Glu Asn Met Leu Gly Ser Ser Glu Ser 85 90 95 CCA AAG CTG TGC CTC GAC CCC ATG GAT GTT GTG AAA TAA 327 Pro Lys Leu Cys Leu Asp Pro Met Asp Val Val Lys 100 105 108

SEQ ID NO: 11 Sequence length: 327 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA sequence GGT TCT TAC CCT CCA GCC ATA CCC CAC AAC CTC TCC TGC CTC ATG AAC 48 Gly Ser Tyr Pro Pro Ala Ile Pro His Asn Leu Ser Cys Leu Met Asn 1 5 10 15 CTC ACA ACC AGC AGC CTC ATC TGC CAG TGG GAG CCA GGA CCT GAG ACC 96 Leu Thr Thr Ser Ser Leu Ile Cys Gln Trp Glu Pro Gly Pro Glu Thr 20 25 30 CAC CTA CCC ACC AGC TTC ACT CTG AAG AGT TTC AAG AGC CGG GGC AAC 144 His Leu Pro Thr Ser Phe Thr Leu Lys Ser Phe Lys Ser Arg Gly Asn 35 40 45 TGT CAG ACC CAA GGG GAC TCC ATC CTG GAC TGC GTG CCC AAG GAC GGG 192 Cys Gln Thr Gln Gly Asp Ser Ile Leu Asp Cys Val Pro Lys Asp Gly 50 55 60 CAG AGC CAC TGC TGC ATC CCA CGC AAA CAC CTG CTG TTG TAC CAG AAT 240 Gln Ser His Cys Cys Ile Pro Arg Lys His Leu Leu Leu Tyr Gln Asn 65 70 75 80 ATG GGC ATC TGG GTG CAG GCA GAG AAT GCG CTG GGG ACC AGC ATG TCC 288 Met Gly Ile Trp Val Gln Ala Glu Asn Ala Leu Gly Thr Ser Met Ser 85 90 95 CCA CAA CTG TGT CTT GAT CCC ATG GAT GTT GTG AAA TAA 327 Pro Gln Leu Cys Leu Asp Pro Met Asp Val Val Lys 100 105 108

SEQ ID NO: 12 Sequence length: 327 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Sequence characteristics: 205-207: mutation sequence GGT TCT TAC CCT CCA GCC ATA CCC CAC AAC CTC TCC TGC CTC ATG AAC 48 Gly Ser Tyr Pro Pro Ala Ile Pro His Asn Leu Ser Cys Leu Met Asn 1 5 10 15 CTC ACA ACC AGC AGC CTC ATC TGC CAG TGG GAG CCA GGA CCT GAG ACC 96 Leu Thr Thr Ser Ser Leu Ile Cys Gln Trp Glu Pro Gly Pro Glu Thr 20 25 30 CAC CTA CCC ACC AGC TTC ACT CTG AAG AGT TTC AAG AGC CGG GGC AAC 144 His Leu Pro Thr Ser Phe Thr Leu Lys Ser Phe Lys Ser Arg Gly Asn 35 40 45 TGT CAG ACC CAA GGG GAC TCC ATC CTG GAC TGC GTG CCC AAG GAC GGG 192 Cys Gln Thr Gln Gly Asp Ser Ile Leu Asp Cys Val Pro Lys Asp Gly 50 55 60 CAG AGC CAC TGC TCC ATC CCA CGC AAA CAC CTG CTG TTG TAC CAG AAT 240 Gln Ser His Cys Ser Ile Pro Arg Lys His Leu Leu Leu Tyr Gln Asn 65 70 75 80 ATG GGC ATC TGG GTG CAG GCA GAG AAT GCG CTG GGG ACC AGC ATG TCC 288 Met Gly Ile Trp Val Gln Ala Glu Asn Ala Leu Gly Thr Ser Met Ser 85 90 95 CCA CAA CTG TGT CTT GAT CCC ATG GAT GTT GTT AAA TAA 327 Pro Gln Leu Cys Leu Asp Pro Met Asp Val Val Lys 100 105 108

SEQ ID NO: 13 Sequence length: 48 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: synthetic DNA sequence GGGGATCCAT CGAGGGTAGG GGTTCTTACC CTCCAGCCAT ACCCCACA 48

SEQ ID NO: 14 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GGGTCTAGAT TATTTCACAACATCCATGGGG ATC
AAG 36

SEQ ID NO: 15 Sequence length: 30 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: synthetic DNA sequence TGCGTGGGAT GGAGCAGTGG CTCTGCCCCGT
30

SEQ ID NO: 16 Sequence length: 22 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCTCCATCCC ACGCAAACAC CT 22

SEQ ID NO: 17 Sequence length: 24 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence CGCCGAGGTT TTCCCAGTCA CGAC
24

[Brief description of the drawings]

FIG. 1. Expression vector p for expression in E. coli of a mouse-derived G-CSF receptor ligand binding domain protein.
It is a construction schematic diagram of MALp-mBN.

FIG. 2: Cultured transformant, E. coli K12 strain / pM
MBN-M expressed in the periplasm of ALp-mBN
It is a mimetic diagram of the elution pattern of BP fusion protein by Q-Sepharose.

FIG. 3 is a mimic diagram of an elution pattern of mBN domain protein expressed by a transformant by TSKgel G2000SW HPLC.

FIG. 4 is a graph for measuring the dissociation constant between mBN and G-CSF produced by a recombinant method.

FIG. 5: Expression vectors pMALp-mBN-L77A and pMALp for expression in Escherichia coli of a derivative of a ligand-binding domain protein of a G-CSF receptor derived from a mouse.
FIG. 3 is a schematic diagram showing the construction of -mBN-Y78A.

FIG. 6 is a graph for measurement of the dissociation constant between G-CSF and mBN-L77A and mBN-Y78A produced by a recombinant method.

FIG. 7: Expression vector pM for expression in Escherichia coli of a human-derived G-CSF receptor ligand binding domain protein
It is a construction schematic diagram of ALp-hBN.

FIG. 8 is a graph for measuring the dissociation constant between hBN and G-CSF produced by a recombinant method.

FIG. 9 is a schematic diagram showing the construction of an expression vector pMALp-hBN-C69S for expressing a human-derived derivative of a ligand-binding domain protein of a G-CSF receptor in Escherichia coli.

FIG. 10 shows hBN-C69S produced by recombinant method and G-
It is a graph for measurement of a dissociation constant with CSF.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification symbol FI C12R 1:19) (C12N 15/09 ZNA C12R 1:91) (C12P 21/02 C12R 1:19) (58) (Int. Cl. ⁶ , DB name) C12N 15/00-15/90 BIOSIS (DIALOG) WPI (DIALOG) GenBank / EMBL / DDBJ EPAT (QUESTEL)

Claims (7)

(57) [Claims] 1. A DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3 or 11, or a mutant protein obtained by deletion, substitution or addition of an amino acid in the amino acid sequence represented by SEQ ID NO: 3 or 11. DNA encoding a protein having a binding activity to granulocyte colony stimulating factor. 2. The DNA according to claim 1, wherein the mutant protein has an amino acid sequence represented by SEQ ID NO: 9, 10 or 12. 3. A recombinant plasmid containing the DNA according to claim 1 or 2 under the control of an Escherichia coli gene expression system. 4. The fusion of the ligand-binding domain protein of the granulocyte colony-stimulating factor receptor and the Escherichia coli maltose-binding protein to Escherichia coli, comprising the DNA according to claim 1 or 2 downstream of the DNA encoding the Escherichia coli maltose-binding protein. The recombinant plasmid according to claim 3, which can express a protein. 5. An Escherichia coli transformed with the plasmid according to claim 3 or 4. 6. A method for producing a ligand-binding domain protein of a granulocyte colony-stimulating factor receptor, which comprises culturing the Escherichia coli according to claim 5 and collecting a recombinant product accumulated in the Escherichia coli. 7. A ligand-binding domain protein of a granulocyte colony stimulating factor receptor produced by the method according to claim 6.