JP3081761B2 - DNA encoding a protein in the ligand-binding region including the CRH region of the granulocyte colony-stimulating factor receptor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a ligand binding protein (i.e., a G-CSF binding protein) in the extracytoplasmic domain of a granulocyte colony stimulating factor receptor (hereinafter referred to as "G-CSF receptor"). More specifically, a DNA encoding a protein of the cytokine receptor complementary region or a DNA encoding a protein of the immunoglobulin-like region and the protein of the cytokine receptor complementary region
A, an expression vector containing the DNA, a transformant transformed with the vector, and a recombinant protein comprising culturing the transformant in an appropriate medium and recovering the product from the culture. It relates to a manufacturing method.

[0002] Granulocyte colony stimulating factor (G-CSF) is a member of the colony stimulating factor involved in blood cell proliferation and differentiation and plays an important role in the proliferation and differentiation of neutrophilic granulocytes. This factor has been shown to be deeply involved in controlling neutrophil concentration in blood, as well as in activating mature neutrophils. For example, G-CSF acts on a neutrophil precursor cell or the like via a receptor (G-CSF receptor) or the like to stimulate its proliferation or differentiation to give mainly neutrophil 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 non-blood cells. For example, it is also present in human endothelial cells and placenta. Recent studies suggest that the receptor forms a multimeric structure, such as a dimer, when expressing the activity. Elucidation of the complex formation reaction between these ligands (that is, G-CSF) and the G-CSF receptor and the mechanism of multimerization 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.

[0004] Accordingly, the present invention is useful for the study and treatment of diseases related to the interaction between G-CSF receptor and ligand.
Through antagonism of the receptor, G-CSF-dependent diseases and abnormalities, such as leukemia caused by abnormal granulocyte proliferation, can be treated or prevented.

Further, 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.

[0006] At present, G-CSF is used for restoring leukopenia in cancer patients who have received radiotherapy or anticancer drug treatment, based on their induction of differentiation of myeloid leukemia cells and the effect of enhancing the function of mature granulocytes. Although expected to be useful as a therapeutic agent, the above-mentioned G-CSF agonists or antagonists are used in the treatment of these leukopenic patients in G-CSF.
It is expected that an effect equivalent to or higher than F can be exerted.

[0007]

2. Description of the Related Art Mouse and human G-CSF
The DNA encoding the receptor has been cloned and sequenced [Fukunaga et al., Cell, 61 341-3.
50 (1990); Proc. Natl. Acad. Sci. US
A. 87 8702-8706 (1990); WO91 /
14776 (released October 3, 1991)].

Have reported the successful expression of the G-CSF receptor (Cell, supra, Proc. Nat).
l. Acad, Sci. USA. Supra, EMBO J .; 10
2855-2865 (1991)) in which all of the complementary DNA sequences are expressed using animal cells, the expression level of which is extremely low, and is optimal for the production of G-CSF receptor that satisfies demand. In addition, the structural analysis and the study of the reaction mechanism could not be performed sufficiently.

Have succeeded in expressing the ligand binding site of the G-CSF receptor using an E. coli maltose fusion protein (J. Biol. Chem. 269, 22412).
22419 (1994); Japanese Patent Application No. 6-116252).
However, in this case, since the minimum unit of the ligand binding site was removed, a decrease in binding activity was inevitably observed as compared with the natural G-CSF receptor. In addition, multimerization of the receptor, which is considered to occur when the activity of the receptor is expressed, was not observed. Further, several steps of processing such as Q-Sepharose and Factor Xa processing were required for purification.

[0010]

SUMMARY OF THE INVENTION In order to achieve the above-mentioned various objects, the present inventors have studied the binding and multimerization of all G-CSF receptor molecules, in particular, G-CSF. Focusing on the usefulness of proteins in the necessary regions, research has been conducted to establish an effective method for producing such proteins.

[0011] 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. That is, Fukunaga et al. Cloned a cDNA (complementary DNA) encoding the G-CSF receptor, and this receptor comprises an immunoglobulin-like region (hereinafter abbreviated as Ig region) and a cytokine receptor complementary region (hereinafter abbreviated as CRH region). (Cell, supra; Proc. Natl. Acad, Sci.
USA. Supra).

The CRH region is interleukin 2-
7, erythropoietin, growth hormone, GM-CSF
Furthermore, a complementary region consisting of about 200 amino acids found in the extracytoplasmic region of cytokine receptors such as interferon α, β, γ, etc., is considered to be a ligand binding site of these receptors. For this reason, these receptors have been termed the cytokine receptor family [Bazan (B
azan Proc. Natl. Acad. Sci 6934-693
8 (1990)]. In fact, even in the G-CSF receptor, this CR
The H region is essential, and the CRH region is currently considered to be one unit required for ligand binding and signaling in this family.

The CRH region further comprises two parts, an amino-terminal domain (hereinafter abbreviated as a BN domain) and a C-terminal domain. When the BN domain is deleted,
Activity is completely lost. This suggests that the BN domain plays the most important role in ligand binding, but it is not yet clear whether the activity is retained only by the BN domain, for example, growth hormone and interleukin. In the case of 6 receptors, a C-terminal domain is also considered necessary. Hiraoka et al. Expressed this BN domain as a ligand binding region (Hiraoka et al., Supra).

In addition to the G-CSF receptor, the Ig region has a cytokine receptor family, in particular, interleukin 3,5,6, and GM-CSF, which are found in about 10 receptors.
It is thought that the region consisting of 0 amino acids forms an immunoimmunoglobulin-like amino acid sequence. The role of this region in the function of each receptor is unknown. However, as an extracytoplasmic region, this Ig region and CR
The G-CSF receptor consisting only of the H region (these are abbreviated as Ig-CRH) retains the signal sequence and has the same ligand binding ability as the natural receptor. Further, the CRH region alone has a lower ligand binding ability than the natural G-CSF receptor, but has a higher ligand binding ability than the BN domain described above. (See Examples 5 and 10 described later.)

The above results indicate that the ligand-binding region protein of the present invention contains a protein containing a CRH region or an Ig region in addition to a CRH region (Ig-CRH).
Region) suggests that the protein is useful,
There is no example of expression only in the H region or Ig-CRH region, and only in the CRH region or Ig-CRH region,
It was unclear what level of binding activity it had and its ability to multimerize. In order to solve these problems, it is necessary to produce a large amount of a CRH domain protein or an Ig-CRH domain protein as a G-CSF receptor binding domain.

The Ig-CRH region of the G-CSF receptor is considered to be very difficult to express. This is because this binding site contains many cysteine residues (1 in mouse origin).
(4 residues, 17 residues from human)). In addition, the CRH region contains 10 cysteine residues from mouse and 12 cysteine residues from human, and it is considered that expression is difficult because it does not easily fold. However, if the Ig-CRH region can be expressed for the above-mentioned reason, whether or not the expressed region retains almost the same activity as the natural receptor, and further, retains the multimerization ability Can be clarified.

For the above reasons, G-
Production of the ligand binding domain protein of the CSF receptor by DNA recombination has been difficult to produce a highly functional receptor containing a protein having almost the same function as the natural G-CSF receptor, which has been difficult in the past. It opens the way.

[0018]

The present inventor has proposed a G-CSF.
Ligand binding region of receptor, especially CRH region and Ig
In expressing the gene encoding the CRH region,
Insect-derived virus, baculovirus, was used as a vector, and insect-derived cells were used as hosts. As a result, the target product was successfully secreted and produced efficiently, and the present invention was completed.

The Ig-CRH region ligand-binding domain protein of the G-CSF receptor produced by the method of the present invention comprises:
As will be described later, it exhibited ligand binding activity of almost the same strength as the natural G-CSF receptor, and also exhibited ligand-dependent multimerization ability. In addition, the CRH area is
It showed higher binding activity than the N domain alone.

[0020] The present invention provides a GC
SF receptor ligand binding domain protein, specifically, CR
A plasmid for producing an H region protein or an Ig-CRH region protein, in which a DNA fragment encoding the region is incorporated into a baculovirus-derived gene control system. And a method for producing a hormone-binding domain protein of a growth hormone receptor using an insect cell as a host, using the recombinant baculovirus vector incorporated under the above-mentioned method.

For purposes of the present invention, the terms used herein are defined below.

In the context of the present invention, the term G-CSF receptor is used to refer to GCs of any natural mammalian origin, including humans.
It is intended to mean the SF receptor and also includes variants of these natural G-CSF receptors made by genetic engineering.

The ligand-binding domain protein of the G-CSF receptor refers to a protein constituting a domain involved in binding to G-CSF in the G-CSF receptor. The protein is G
The amino acid sequence constituting the CRH region in the ligand binding region of the CSF receptor;
It has an amino acid sequence constituting an Ig-CRH region including an Ig region in addition to the H region. For the purpose of the invention,
Useful are 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. Therefore, in the present 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).

The DNA encoding the ligand binding region protein of the G-CSF receptor is defined as G-CSF receptor
-Refers to DNA encoding an amino acid sequence that constitutes a region involved in binding to CSF. The DNA is G-CSF
It encodes a ligand binding region including the CRH region of the receptor, and DNAs encoding a ligand binding region including the CRH region and the Ig region are also included in this definition. Similar to the definition of the protein described above, the DNA not only encodes 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.
Is also included. The DNA may be either a complementary DNA or a synthetic DNA.

The Ig-CRH region of the G-CSF receptor refers to an immunoglobulin-like region and a region complementary to a cytokine receptor, and unless otherwise specified herein, also refers to a protein in the region or a derivative thereof. The amino acid sequence corresponding to this region and its signal sequence is mouse [SEQ ID NO: 1 in the Sequence Listing; Fukunaga et al., Cell 61 , 341-350 (1
990)] and human [SEQ ID NO: 2 in the Sequence Listing; Fukunaga et al., Pr.
oc. Natl. Acad, Sci. USA. 87 , 8702-8
706 (1990)]. As is well known to those skilled in the art, a signal sequence is an amino acid sequence required when an extracellular protein is secreted from inside a cell to outside a cell. It is linked to the N-terminus of the secreted protein, and is cleaved and removed as the protein is secreted out of the cell. This sequence is considered to correspond to serine 25 from position 1 of methionine in SEQ ID NO: 1 and leucine 23 to position 1 from methionine in SEQ ID NO: 2. In the present specification, the expression of the ligand binding region protein was shown using the complementary DNA of the regions corresponding to the CRH region and the Ig-CRH region of the mouse G-CSF receptor.
A may be used. As will be readily understood by those skilled in the art, DNAs having different codons are included in the scope of the present invention as long as they encode the amino acid sequence of the protein of the present invention. Moreover, it may be of human origin and is not limited to the description in the examples.

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)]. This plasmid pBLJ17 is FERM
It is available from Escherichia coli strain K12, which is deposited under the BP-3312 (Deposit date: March 9, 1990) with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry.

The Ig-CRH region of the G-CSF receptor is specifically defined as the sequence disclosed by Fukunaga et al.
In the mouse G-CSF receptor, it refers to the portion from cysteine 1 to alanine 309 (corresponding to cysteine 26 and alanine 334, respectively, in SEQ ID NO: 1). Similarly, in the human G-CSF receptor, it is a region from glutamic acid at position 1 to alanine at position 310 (corresponding to glutamic acid at position 24 and alanine at position 333 in SEQ ID NO: 2, respectively).

The CRH region is expressed by mouse GC
In the SF receptor, it refers to the portion from tyrosine at position 97 to alanine at position 309 (in SEQ ID NO: 1, they correspond to tyrosine at position 122 and alanine at position 334, respectively). Similarly, in the human G-CSF receptor, it is a region from tyrosine 98 to alanine 310 (corresponding to tyrosine 121 to alanine 333 in SEQ ID NO: 2).

However, the CRH region and Ig-C
The start and end sites of the RH region are not necessarily critical for the invention, and the CRH region and the Ig-CRH
It does not significantly affect the three-dimensional structure of the entire region.
The N-terminus and / or C-terminus may be shifted in either direction, or several amino acids may be added to the N-terminus and / or C-terminus, provided that the function is maintained. Are also included. In fact, Ig-C
The first amino acid at the N-terminus of the RH region is cysteine in mouse, but the residue corresponding to this cysteine is third cysteine in human due to amino acid homology.
In other words, exactly where the first amino acid is is not completely determined. Regarding the C-terminus, such a slight difference in the primary structure usually does not greatly affect the three-dimensional structure of the entire Ig-CRH region, and it is considered that the function is maintained.

In the examples described later, the CRH region is also expressed using a DNA sequence (shown in SEQ ID NO: 5) in which a considerable portion of the Ig region has been deleted from the entire Ig-CRH region. In this case, when expressed in insect cells, the amino acid sequence derived from the Ig region is expressed at the N-terminus in addition to the CRH region. However, the remaining Ig region is easily cleaved by a protease in insect cells, and the final purified product is a protein in which several amino acids are added to the CRH region starting from tyrosine. This is because the CRH region alone is
The amino acid sequence of the Ig region remaining at the N-terminus alone does not probably maintain the structure, as a result of maintaining the proper three-dimensional structure, and as a result, it is easily cut by proteases in insect cells. It is thought to be due to the existence.

Furthermore, 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.

For expression, D which encodes the target part
If the NA fragment also contains a signal peptide portion for secretory production outside the cell, the produced ligand binding region protein is secreted outside the cell. This signal sequence is also contained in the natural G-CSF receptor gene. In the mouse receptor, the -25th methionine to the 1st serine (corresponding to the 1st methionine to the 25th serine in SEQ ID NO: 1) In the human receptor, it is -1st leucine from -23th methionine (corresponding to 23rd leucine from 1st methionine in SEQ ID NO: 2). In the expression of the mouse receptor described below, a protein corresponding to the natural one is used, but signal sequences of other proteins derived from insect cells or animal cells can also be used.

In order to express the DNA fragment encoding the ligand-binding region of the G-CSF receptor, particularly the Ig-CRH region and the protein of the CRH region, in an insect cell as a host, the DNA fragment must be expressed in advance by gene expression. It must be connected to the system. As a gene expression system for insect cells, nuclear polyhedrosis virus (Nuclear Polyhedrosis), a kind of baculovirus using insects as a host, is known.
virus: are the expression system of the NPV) is known, Autographa
California NPV (AcNPV) and Bombyx mori N
There are two types of PV (BmNPV). As a host, a cell derived from a moth is used. In the case of AcNPV, a cell Spodoptera
frugiperda (SF cells) or Trichoplusia ni
The (TN cell), also in the case of BmNPV, cell Bomb yx
mori N (BmN cells) is used. Both baculoviruses both contain a polyhedron gene, which is under the control of a strong polyhedron promoter. Therefore, when expression is performed using these viral vectors, a recombinant baculovirus in which a desired gene is inserted downstream of the polyhedron promoter is prepared, and the baculovirus is infected with a host cell and cultured. Express the protein to be expressed. In preparing this expression virus vector, the target gene cannot be directly inserted into the virus because the virus is as large as about 130 kilobase pairs. Therefore, as an actual procedure, first, only a gene expression control region, that is, a polyhedron gene portion containing a polyhedron promoter is cut out, and a translocation vector inserted into a vector using Escherichia coli as a host, for example, pUC8 is used.

As will be described later, Ig is used as a transposition vector.
In the expression of -CRH region, pAcYM1 is used, and in the expression of CRH region, pVL1393 is used. However, there are many similar vectors, and the present invention is not limited to these vectors.

Next, the gene of interest is inserted downstream of the polyhedron promoter in the transposition vector. Subsequently, the translocated vector DNA incorporating the gene of interest is simultaneously transferred to insect cells together with the wild-type baculovirus genomic DNA and cultured, and intracellular DNA recombination is performed in the insect cells to obtain a recombinant baculovirus. Get. In this case, the transfer of the DNA to the insect cells also requires Ig-C
The calcium phosphate method is used for expression of the RH region, and the lipofectin method is used for expression of the CRH region, but there is essentially no difference between the two. In addition, regarding the baculovirus genomic DNA to be transferred as a transposition vector, wild-type baculovirus genomic DNA, C
For expression of the RH region, baculogold DNA, which is a mutant genomic DNA, is used, but there is no essential difference therebetween.

Further, there is a method of infecting larvae with the obtained recombinant baculovirus and extracting a target substance from the larvae. In this specification, the AcNPV vector system is used,
f. A method for obtaining a recombinant baculovirus from cells, infecting the cells with TN cells, and extracting the target substance from the culture solution and cells after culture is described, but the present invention is not limited thereto. . Genes encoding the signal sequence and the ligand binding region protein can be extracted by the polymerase chain reaction (hereinafter abbreviated as PCR). Regarding the genetic manipulation method using Escherichia coli used in the examples described later, a book (Mani
atis et al. (1982) Molecular Cloning: A Labor.
atory Manual, Cold Spring Harbor Laborato
ry). For experiments using baculovirus and its host,
Book [Summers and Smith (1987), A manual of m
ethods for baculovirus vectors and insect ce
ll culture procedures., Texas Agricultural
Experiment Station Bulletin No. 1555, T
exas A & M University] or [Maeda (198
9), Experimental Medicine, Vol. 7, pp. 146-151], describes the procedure in detail.

The host cells infected with the recombinant baculovirus were prepared from a known Sf-900IISFM medium (GIBC
O). After removing the cells from the obtained culture solution, the resultant is subjected to affinity column chromatography and gel filtration HPLC using G-CSF as a ligand to obtain a pure G-CSF receptor ligand-binding region. The affinity column is Fukunaga et al.
(J. Biol. Chem. 265 , 14008-14015).
(1990)). The resulting expression product is larger than the molecular weight predicted from the amino acid sequence encoded by the inserted DNA. This indicates the addition of a sugar chain to the product. In fact, in the presence of tunicamycin, which is a sugar biosynthesis inhibitor, an expression product having a molecular weight almost predicted from the amino acid sequence is obtained.

Preparation of affinity column or G-
For non-radioactively labeled G-CSF used for measuring the ligand binding activity of the CSF receptor, those provided by Kirin Brewery Co., Ltd. were used, but natural G-CSF or Fukunaga et al. (J. Biol. Chem. 265, 14008-140
15 (1990)] can be used, and the present invention is not limited to those described herein.

[0039]

EXAMPLES Example 1 Construction of the Translocation Vector pAcIg-CRH Plasmid The translocation vector pAcYM1 containing a portion encoding the polyhedral protein of AcNPV (Matsuura et al., J. Gen. Virol.
(1987), 68 , 1233-1250), the ligand binding region of the G-CSF receptor downstream of the polyhedron promoter.
(Hereinafter, abbreviated as Ig-CRH region) is inserted.

The amino acid sequence of a known mouse G-CSF receptor [Fukunaga et al., Cell, 61 , 341-350 (199)
0)], the DNA encoding the Ig-CRH region protein of the present invention was replaced with the G-CSF receptor 26 of SEQ ID NO: 1.
The protein was designed to be a protein encoding cysteine number 334 to alanine number 334. In the expression of this Ig-CRH region, the Ig-CRH region is secreted so that the expression product is secreted into the culture medium.
In preparing the DNA encoding the H region, the following additions have been made. In other words, a sequence encoding No. 1 methionine to No. 25 serine corresponding to the signal sequence is linked to the N-terminus, and a termination codon TAA for protein synthesis is added to the C-terminus. In order to insert the DNA fragment into the vector pAcYM1, the N-terminal and C-terminal thereof were further extended, and the extended portion was restricted with the restriction enzyme BamH.
It is devised so that an I recognition site is formed.

PCR was used for the actual preparation of the DNA fragment. Specifically, first, an N-terminal primer and a C-terminal primer having the nucleotide sequences of SEQ ID NO: 3 and SEQ ID NO: 4, respectively, are prepared using a 380B DNA synthesizer manufactured by Applied Biosystems. Then, 2 μM N-terminal primer, 2 μM C-terminal primer pBLJ17 DNA (1 ng), 10 μl of 10 × reaction buffer, 0.25 mM each of dATP, dTTP, dGTP,
Add the dCTP solution and 0.5 μl of Taq DNA polymerase to a final volume of 100 μl. Here, 10 × reaction buffer, dATP, dTTP, dGTP, dCTP, Taq DNA
The polymerase used was one from Takara Shuzo's GeneAmp kit. This reaction solution was set in a DNA thermal cycler (model PJ2000, Takara Shuzo), heat-treated at 94 ° C. for 1 minute; annealing at 37 ° C. for 2 minutes; Synthesize the fragments. Further, the PCR reaction solution is purified by phenol treatment. Its purification D
About 1 μg of NA is made up with 50 mM Tris-HCl buffer (pH 7.5) containing 0.1 M salt and 10 mM magnesium chloride.
3 with 50 μl and 5 units of restriction enzyme BamHI (Takara Shuzo)
Incubate at 7 ° C. for 1 hour to digest DNA. After phenol treatment and ethanol precipitation of this digested juice,
The resultant was subjected to 1% agarose gel electrophoresis, and a band of about 1 kbp was cut out and placed in Sprek-01 (Takara Shuzo).
After freezing at 0 ° C for 15 minutes, 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, the above DNA fragment was transferred to the translocation vector pAcY.
Insert into the BamH site of M-1.

Specifically, about 1 μg of pAcYM1 DN
A containing 50 mM salt and 10 mM magnesium chloride
The DNA thus obtained is dissolved in 50 μl of 0 mM Tris-HCl buffer (pH 7.5), added with 5 units of restriction enzyme BamHI (Takara Shuzo), and incubated at 37 ° C. for 1 hour to digest the added DNA. Next, the target DNA fragment is extracted by phenol treatment and ethanol precipitation.
This was dissolved in 50 μl of 0.1 M Tris-HCl buffer (pH 8.0) containing 5 mM magnesium chloride, and 3 units of alkaline phosphatase (alkaline phosphatase, E. coli C75; Takara Shuzo) was added, followed by incubation at 37 ° C. for 1 hour. By doing so, the DNA is attached to the 5 ′ of the DNA and the target DNA is taken out. The band corresponding to the larger DNA fragment was cut out by electrophoresis on a 1% agarose gel, put into Sprek-01 (Takara Shuzo), and stored at -80 ° C.
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. Next, the target DNA fragment is extracted from the filtrate by phenol treatment and ethanol precipitation.

Subsequently, this was mixed with the DNA fragment prepared above and mixed with a 10 mM Tris-HCl buffer containing 1 mM EDTA.
(pH 7.4) to make 4 μl. Add 16 μl of this solution
(DNA ligation kit; Takara Shuzo) and 4 μl of solution B
(DNA Ligation Kit; Takara Shuzo) and mix
Incubate at 30 ° C for 30 minutes. Further, this was mixed with 100 μl of competent cells of E. coli K12 strain, and
E. coli was transferred by incubating at 0 ° C for 2 minutes at 42 ° C to construct a translocation vector pAcIg-CRH plasmid. The Escherichia coli K12 strain / pAcIg-CRH into which this plasmid has been introduced has been deposited with the Institute of Biotechnology and Industrial Technology of the Ministry of International Trade and Industry of Japan.
November 8, 2012; Accession number: FERM P-14618)

Example 2 Recombinant virus AcIg-CR
Preparation of H Plasmid pAcIg-CRHD obtained in Example 1
A recombinant baculovirus is prepared from NA. That is, this plasmid DNA is transferred together with wild-type AcNPV DNA into insect cells by the calcium chloride-phosphate precipitation method, and a recombinant baculovirus is prepared by recombination in the cells. The outline is shown in FIG. First, 1 μg of wild-type baculovirus AcNPV DNA [AcNPV was obtained from M. D. Summers (Depatrment of Entomology, Te
xas Agricultural Experimentation Station and Tex
as A & M University College Station, Texas
s, 77483-2475) and its D
The extraction of NA is based on Maeda's method (Experimental Medicine Vo17, No13,
146-151, 1989)] and pAcIg-CRH.
25 μg of DNA (purified twice by the cesium chloride density gradient method) are precipitated in a Hepes-phosphate buffer (pH 7.05) containing calcium chloride. On the other hand, insect cells Spodoptera frugiperda 9 (American Type Culture)
ture Collection, 12301, Parklawn Drive,
Rockvlle, available from MD20852 / 1776,
The following S. f. 9. ) In a Grace's medium (Gibco) containing 10% fetal calf serum (about 1 to 1.5 × 10
⁽⁶ cells / 2 ml) was placed in a 35 mm cell culture dish (Falcon 3001) and allowed to stand for 1 hour to culture the cells. Next, the cell supernatant is taken out, the above-mentioned DNA precipitate is added dropwise, and left for 1 hour. Further, the cell supernatant was removed, and a fresh Grace's medium containing 10% fetal bovine serum was added.
Add 2 ml and incubate at 26.5 ° C for 4 days. The obtained culture solution contains the desired recombinant baculovirus AcIg-CRH in addition to the wild-type AcNPV.

A plaque assay is performed to remove AcIg-CRH from the culture. First, S. cerevisiae grown in the same manner as above. f. The medium of 9 cells (10 ⁶ cells / 35 mm petri dish) was withdrawn, and the culture solution was diluted to about 4 to 10,000 times with a culture solution of 0.1%.
Add 1 ml. After standing for about 1 hour, remove the culture solution,
1 ml of a 3% agarose solution [3% agarose (low melting point sea plaque agarose, FMC) and a Grace's medium containing 10% fetal bovine serum at a ratio of 1: 2 and kept at 37 ° C.] are overlaid. After solidification of agarose, 1 ml of 1
Grace's medium containing 0% fetal calf serum was further overlaid,
Incubate at 6.5 ° C. for 4 days to generate viral plaques. In the resulting plaque, rinse the one with a transparent center with agarose with the tip of a pipette etc., 1 ml of 10%
Isolate by suspending in Grace's medium containing fetal calf serum (wild-type virus has a white turbidity in the center of the plaque and the recombinant is clear). When transparent plaque is difficult to find, adjust to an appropriate concentration so that plaque can be easily identified.
Dilute the original culture and repeat the plaque assay. The plaque suspension of the transparent plaque that has been spread is diluted about 100- to 1000-fold, respectively.
0.2 ml of the solution was used in the same manner as described above. f. The plaque assay is repeated by overlaying 9 cells to ensure that no plaques such as other wild-type viruses are mixed. The thus isolated product is a recombinant baculovirus AcIg-CR
H.

Example 3 Ig- using AcIg-CRH
Secretion expression of CRH region Recombinant virus AcIg-CRH was transferred to TN cells (Invitrog).
en; HIGH FIVE ^™ CELLS) to Sf-900I
225-cm ² flask with ISFM medium (GIBCO)
After growing to confluence using (Coastar)
After diluting 10-fold with the same medium, culture at 27 ° C. for 1 day. To this was added the recombinant virus AcIg-CRH (MOI
1-10) Culture was performed at 18 ° C. for 9 days, and the culture solution was collected.

The secretory production of Ig-CRH in the culture solution is as follows.
It can also be detected by Western blotting. The Western blot method can be performed by a method described in a compendium (Seizoku Kagaku Jikken Koza Vol. 1, Japan Biochemistry, Tokyo Kagaku Dojin) or the like. Here, Bio-Rad kit (Immun-Bl
ot, Goat Anti-Rabbit IgG (H + L) Alkaline
(Phosphatase Conjugate) was used, and the procedure was also performed according to the instructions attached thereto. Specifically, after a culture solution (about 10 μl) was electrophoresed by SDS-PAGE, it was then transferred to a cellulose acetate membrane (Schlei).
cher &Schchuell; BA85). The membrane is an anti-G-CSF receptor CRH antibody (anti-M1 serum;
After incubation in a Tris buffer pH 7.5 containing Osaka Bioscience Research Institute (provided by Mr. Nagata), the mixture was further allowed to act on an anti-rabbit IgG (H + L) -alkaline phosphatase conjugate, and Ig-CRH was added as a band on the same membrane. To detect.

As a result, an Ig-CRH band was detected at a position corresponding to about 45 kilodaltons, which was larger than the value predicted from the amino acid sequence encoded by the transferred DNA. This suggests that a sugar chain is added to the produced Ig-CRH. In fact, when tunicamycin, which is an inhibitor of sugar chain biosynthesis, is added,
This band of about 45 kilodaltons disappears and about 40-4
A two kilodalton band appears. This approximately corresponds to the size of the band predicted from the amino acid sequence. This confirms that a sugar chain has been added to the 45 kilodalton band.

Example 4 Ig-CR of G-CSF receptor
Purification of H region The G-CSF receptor in the culture solution can be purified by G-CSF-affinity column chromatography using G-CSF as a ligand. A method for producing G-CSF-affinity column chromatography is described by Fukunaga et al. [J. Biol. C
hem., 265 , 14008-14015]. Specifically, 10 g of human G-CSF (provided by Kirin Brewery) was added to 0.4 g of AF-Tresyl- in 0.1 M sodium bicarbonate buffer (pH 8.0) containing 0.5 M salt.
Mix with Toyopearl gel resin (Tosoh) to a final volume of about 2 ml and stir at room temperature for 4 hours. After further stirring overnight at 4 ° C., the gel resin contained 0.1M NaCl 0.1%.
Stir for an additional 3 hours at room temperature with M Tris buffer (pH 8.0). Finally, it is made by washing with PBS phosphate buffered saline.

That is, the AcIg-CRH prepared in Example 3
Is added to a G-CSF affinity column (1 × 5 cm). After addition, wash with PBS phosphate buffered saline and add 0.2M saline containing
Elute with 1 M glycine hydrochloride buffer. The eluted protein is 2
Immediately neutralize with M trizma base. Then change this to 0.
Gel filtration HPLC (7.6 mm id × 60 cm; TSK) equilibrated with 20 mM sodium phosphate buffer containing 2 M salt
(GEL G3000 SWHPLC column, Tosoh)
And elute with the same buffer. Most of the eluted protein is eluted at 85 kDa. The peak at 85 kDa becomes a single band at 45 kDa in SDS-PAGE, and is stained by Western blot.
It is considered a dimer of the g-CRH region. According to the N-terminal amino acid sequencing method, the N-terminal of this sample is Cys
-Gly-His-Ile-Ser-Pro-Pro-, which was consistent with the expected N-terminal sequence of the Ig-CRH region shown by Fukunaga et al. [Cell, supra]. Also 8
In addition to the 5 kDa peak, two small peaks are 45 kDa and 2
Elution occurs at the position of 00-250 kDa.
S-PAGE shows a single 45 kDa band and staining by Western blot, suggesting that it is a monomer (45 kDa) of Ig-CRH domain protein and a tetramer (200-250 kDa). Expression levels ranged from 0.2 mg to 0.5 mg / l.

Example 5 Activity measurement of Ig-CRH region The activity of the purified Ig-CRH region protein (85 kDa) was measured. The activity was measured by Fukunaga et al. [J. Biol. Chem., 26
5 , 140008-14015 (1990)] with some modifications. That is, Ig-CRH domain protein, 12.5, 25, 50, 100, 250, 500, 100 in 50 μl of PBS.
0 pM, ¹²⁵ IG-CSF of each concentration (Amsham), 1
0% fetal calf serum, 0.1% chaps (CHAPS, 3-
[(3-cholamidopropyl) dimethylammonio] -1-propan
esulfonic acid (Dojin Co.)
A reaction solution in the presence and absence of M unlabeled G-CSF is prepared, and reacted at room temperature for 90 minutes. To this is added 150 μl of a solution containing 100 μg of γ-globulin in PBS, and further 200 μl of a solution containing 30% (w / v) polyethylene glycol 6000 in PBS. This is 0
After incubation at 30 ° C. for 30 minutes, the mixture is centrifuged at 12,000 rpm for 30 minutes to precipitate the Ig-CRH.G-CSF complex. And ¹²⁵ I-G contained in the precipitate.
-The radioactivity of CSF is counted with a γ-counter (Cobra manufactured by Packard). The result is shown in FIG. As a result, the ligand binding activity of the Ig-CRH region was bilayer, with the high binding activity having a dissociation constant of about 100 pM and the low binding activity having a dissociation constant of about 2.5 nM. This high binding activity is almost the same as the activity exhibited by the G-CSF receptor on natural cells.

Example 6 Multimerization of Ig-CRH region In order to measure the multimerization ability of an Ig-CRH region protein, Ig-CRH.G-CSF by gel filtration HPLC was used.
The size of the complex was analyzed. First, the Ig-CRH region and G-CSF were mixed at 1:10, 1: 1, 1: 0.5, 1: 0.1, in 20 mM sodium phosphate buffer containing 0.2 M salt.
The mixture was mixed at a molar ratio of 1: 0 (the concentration of the Ig-CRH region was 1.6.
(fixed to μM) and eluted as a mixture of 80 μl on gel filtration TSK gel G3000SW HPLC. The results are shown in FIG. From the figure, when the ratio is 1: 1, 200-2
It can be seen that the complex was eluted at the position of 50 kDa. The position does not change when the ratio is 1:10. Also, 1: 0.
At 5.1: 0.1, the complex was also eluted at a position of 95 kDa. The above results show that the Ig-CRH region as a dimer is tetramerized by the addition of G-CSF as a ligand.

Example 7 Construction of the Translocation Vector pAcCRH Plasmid The CRH ligand-binding region of the G-CSF receptor (hereinafter referred to as the G-CSF receptor downstream of the polyhedron promoter of the translocation vector pVL1393 (Invitrogen) containing the portion encoding the polyhedral protein of AcNPV. , Abbreviated as CRH region)
Insert The DNA that expresses the CRH region in insect cells must be designed so that the CRH region is linked to the signal peptide region. Such a DNA fragment is called plasmid pBOS @ Ig (Fukunaga et al., EMBO.J. supra).
It can be more easily obtained by the PCR method. In other words, the DNA fragment corresponding to the G-CSF receptor in this plasmid is the Ig-
In the CRH region, serine 109 is deleted from glutamic acid 30 to replace glycine codon GG.
C is inserted. In other words, as a result of the deletion of most of the Ig region, the sequence is linked to the CRH region only by cutting out the 16 amino acid sequence following the signal sequence.
Therefore, the sequence shown in SEQ ID NO: 5 was designed as a DNA fragment expressing the CRH region shown in this example.

That is, in the sequence of SEQ ID NO: 5, the amino acid sequence of SEQ ID NO: 1 is the same as that of methionine at position 1 to isoleucine at position 29 except for serine at position 26 (cysteine at position 26 in SEQ ID NO: 1). The sequence from glycine at position 30 to alanine at position 255 following glycine at position 30 is the same as that of alanine at position 334 from position 110 at valine in SEQ ID NO: 1. SEQ ID NO: 1
The cysteine at position 26 has been converted to serine in SEQ ID NO: 5. In plasmid pBOS @ Ig, three out of four cysteine residues present in the Ig region
Up to the cysteine residue present in the CRH region as free cysteine upon expression, which causes a difference in the SS bond between the cysteine residue and the cysteine residue. The above transformation was performed because it is thought to inhibit expression. Further, a termination codon TAA for protein synthesis is added to the C-terminal. In order to insert the DNA fragment into the vector pVL1393, its N-terminal side and C-terminal side are further extended, and a restriction enzyme BamHI at the N-terminal side and an XbaI recognition site at the C-terminal side are formed in the extended part. It is devised as follows.

PCR was used for the actual preparation of the above DNA fragment. Specifically, first, an N-terminal primer and a C-terminal primer having the nucleotide sequences of SEQ ID NO: 6 and SEQ ID NO: 7, respectively, are prepared using a 380B DNA synthesizer manufactured by Applied Biosystems. In SEQ ID NO: 7, the 17th to 19th bases have an AGA sequence in order to change the cysteine at position 26 to serine. Then 2 μM N-terminal primer, 2 μM
M C-terminal primer, pBOS @ IgDNA (1 n
g) 10 μl of 10 × reaction buffer, 0.25 mM dAT each
Add P, dTTP, dGTP, dCTP solution and 0.5 μl of Taq DNA polymerase to bring the final volume to 100 μl. Where 10 × reaction buffer, dATP, dTTP, dG
TP, dCTP and Taq DNA polymerase are from Takara Shuzo's Ge.
The neAmp kit was used. This reaction solution is
It is set in a thermal cycler (model PJ2000, Takara Shuzo) and heat-treated at 94 ° C. for 1 minute; annealing at 37 ° C. for 2 minutes; and repeating the reaction 25 times with a reaction program at 72 ° C. for 3 minutes to synthesize a target DNA fragment.
Further, the PCR reaction solution is purified by phenol treatment. Thereby, the N-terminal side of the target DNA fragment is synthesized.

Next, an N-terminal primer and a C-terminal primer having the nucleotide sequences of SEQ ID NO: 8 and SEQ ID NO: 9 were synthesized, and a PCR reaction was carried out in the same manner using pBOS @ Ig as a template. Is synthesized at the C-terminal side. In SEQ ID NO: 8, the tenth to eleventh sequences are TC
It is T. Next, N-terminal DNA and C-terminal DNA
And DN as SEQ ID NO: 6 as an N-terminal primer.
The DNA of SEQ ID NO: 9 is added as A and C-terminal primers, and a PCR reaction is performed again to purify.

About 1 μg of the purified DNA was added to 0.1 M
The DNA is digested by incubating with 50 µl of 50 mM Tris-HCl buffer (pH 7.5) containing salt and 10 mM magnesium chloride and 5 units of restriction enzymes BamHI (Takara Shuzo) and 5 units of XbaI (Takara Shuzo) 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 0.8 kbp was cut out and placed in Sprek-01 (Takara Shuzo) at -80 ° C.
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, the above DNA fragment was transferred to the transposition vector pVL13.
Insert at 93 BamHI and XbaI sites.

Specifically, about 1 μg of pVL1393 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,
Further, 5 units of restriction enzymes BamHI (Takara Shuzo) and XbaI (Takara Shuzo) are added, and the mixture is incubated at 37 ° C. for 1 hour to digest the added DNA. Next, this was subjected to electrophoresis on a 1% agarose gel to cut out a band corresponding to the larger DNA fragment, put into Splect-01 (Takara Shuzo), frozen at -80 ° C for 15 minutes, and then incubated at 37 ° C for 5 minutes. And thaw. This is centrifuged at 5000 rpm for 10 minutes, and the filtrate is collected. Next, the target DNA fragment is extracted from the filtrate by phenol treatment and ethanol precipitation.

Subsequently, this was mixed with the DNA fragment prepared above, and 10 mM Tris-HCl buffer containing 1 mM EDTA was added.
(pH 7.4) to make 4 μl. Add 16 μl of this solution
(DNA ligation kit; Takara Shuzo) and 4 μl of solution B
(DNA Ligation Kit; Takara Shuzo) and mix
Incubate at 30 ° C for 30 minutes. Further, this was mixed with 100 μl of competent cells of E. coli K12 strain, and
E. coli was transferred by incubating for 0 minutes and further at 42 ° C. for 2 minutes to construct a translocation vector pAcCRH plasmid. E. coli K with this plasmid introduced
12 strains / pAcCRH have been deposited with the Institute of Biotechnology and Industrial Technology, Ministry of International Trade and Industry (Accepted date: November 8, 1994)
Date; accession number: FERM P-14617)

Example 8 Construction of Recombinant Virus AcCRH and Expression of CRH Region Using It Plasmid pAcIg-CRHD obtained in Example 1
A recombinant baculovirus is prepared from NA. That is, this plasmid DNA was used as a mutant AcNPV genomic DNA.
Was transferred to insect cells by the lipofectin method together with Baculogold (Invitrogen). Specifically, 1 μg of plasmid pAcCRH DNA and 100 ng of baculogold DNA (Invitrogen) were adjusted to 10 μl with distilled water, and then 4 μl of distilled water and 4 μl of lipofectin (BRL).
Is added little by little to 8 μl of the mixture, and left at room temperature for 15 minutes.

On the other hand, insect cells Spodoptera frugiperda
9 (American Type Culture Collection, 12
301, Parklawn Drive, Rockville, MD2
0852/1776; f. 9. 3) (approximately 1-1.5 × 10 ⁶ cells / ml) in a Grace's medium (Gibco) containing 10% fetal bovine serum.
The cells are placed in a 5 mm cell culture dish (Falcon 3001) and left to stand for 1 hour to culture the cells. Next, the cell supernatant is taken out, the above-mentioned DNA / lipofectin mixed solution is dropped, and left for 1 hour. Further, the cell supernatant was removed, and a fresh Grace's medium containing 10% fetal bovine serum was added.
Add 2 ml and incubate at 26.5 ° C for 4 days. The obtained culture solution contains the desired recombinant baculovirus AcIg-CRH in addition to the wild-type AcNPV.

A plaque assay is performed to remove AcIg-CRH from the culture. First, S. cerevisiae grown in the same manner as above. f. The medium of 9 cells (10 ⁶ cells / 35 mm petri dish) was withdrawn, and the culture solution was diluted to about 4 to 10,000 times with a culture solution of 0.1%.
Add 1 ml. After standing for about 1 hour, remove the culture solution,
1 ml of a 3% agarose solution [3% agarose (low melting point sea plaque agarose, FMC) and Grace's medium containing 10% fetal bovine serum at a ratio of 1: 2 and kept at 37 ° C.] are overlaid. After solidification of agarose, 1 ml of 1
Grace's medium containing 0% fetal calf serum was further overlaid,
Incubate at 6.5 ° C. for 4 days to generate viral plaques. The plaque suspension of the plaque that has been picked up is diluted about 100- to 1000-fold, respectively.
2 ml of S.I. f. The plaque assay is repeated by overlaying 9 cells. The thus isolated product is the recombinant baculovirus AcCRH.

Example 9 Secretion and Expression of CRH Region Using AcCRH and Purification of CRH Protein CRH was produced using TN cells in the same manner as in Example 3 using this recombinant baculovirus AcCRH. I can do it. As a result, the target CRH band at a position corresponding to about 29 kilodaltons can be detected by Western blot in the culture solution. This CRH region of about 29 kilodaltons can be purified by a G-CSF affinity column as in Example 4. 75% of its N-terminal amino acid sequence is Asp-Gln-Ala-Glu-Leu, 25
% Is Ala-Gly-Tyr-Pro-Pro, which corresponds to the sequence beginning with aspartic acid at position 35 and alanine at position 41 of SEQ ID NO: 5. In SEQ ID NO: 5, serine at position 25 from methionine No. 1 corresponds to the signal sequence. Therefore, if only the signal sequence is cleaved in the same manner as in the expression using AcIg-CRH, the Ig region corresponding to the 26th serine to the 42nd glycine in addition to the 43rd target CRH region. Should remain. However, the actual results indicate that the desired product was obtained in a form that contained the Ig region portion of 8 residues and 2 residues in addition to the CRH region starting from the 43rd position in SEQ ID NO: 5. This, as mentioned above,
The sequence from position 26 to position 42 derived from the Ig region is a sequence largely deleted from the complete Ig region, and therefore cannot retain a significant higher-order structure by itself. This is presumably because the enzyme was cleaved with a protease and most of the protein was removed. The expression level here was about 10 mg / l.

Example 10 Activity Measurement of CRH Region The ligand binding ability of this CRH region can be measured in the same manner as in Example 5. The dissociation constant of the binding of the CRH domain protein to the ligand G-CSF was about 2.5 nM, similar to the low binding activity of the Ig-CRH domain (FIG. 4). This indicates that the binding activity is 10 times stronger than the binding activity of the BN domain. In addition, this value is the value of the plasmid pBOS △
The Ig showed almost the same ligand binding activity (Fukunaga et al., EMBO.J. supra) of the substance obtained by expression performed using animal cells.

Further, by gel filtration HPLC, CR
The size of the HG-CSF complex was measured in the same manner as in Example 6. The result is shown in FIG. From FIG. 5, the CRH region is 2
9 kDa and G-CSF is 19 kDa. The complex elutes at 40 kilodaltons. From the above results, the CRH region showed binding with the ligand at a 1: 1 ratio, and the CRH region alone did not cause multimerization such as tetramerization in the case of the Ig-CRH region.

[0066]

As described above, the present invention provides a G-CSF
CRH region and Ig among the ligand binding regions of the receptor
Constructing an expression vector containing a DNA encoding a CRH region protein, transforming insect cells with the vector, culturing the resulting transformant, and recovering the expression product secreted into the culture solution; Thus, a method for producing a recombinant CRH region protein and an Ig-CRH region protein has been newly established. The recombinant protein obtained according to the present invention is compared with the conventional BN,
Retains high binding activity to CSF, especially Ig-CR
The H region has the same ligand-binding ability as the natural G-CSF receptor and also has a multimerization ability. Moreover, according to the present invention, it is possible to obtain a large amount of a high-purity standard without containing impurities normally associated with the same region protein obtained by an extraction method from a natural product, which is caused by granulocyte proliferation. It can greatly contribute to the treatment of leukemia and the development and / or study of drugs such as agonists and antagonists that can be replaced with G-CSF.

[0067]

[Sequence list]

SEQ ID NO: 1 Sequence length: 1002 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: cDNA to mRNA origin Organism name: Mouse Cell type: NFS-60 sequence ATG GTA GGG CTG GGA GCC TGC ACC CTG ACT GGA GTT ACC CTG ATC TTC 48 Met Val Gly Leu Gly Ala Cys Thr Leu Thr Gly Val Thr Leu Ile Phe 1 5 10 15 TTG CTA CTC CCC CGA AGA AGT CTG GAG AGC TGT GGA CAC ATC GAG ATT TCA 96 Leu Leu Leu Pro Arg Ser Leu Glu Ser Cys Gly His Ile Glu Ile Ser 20 25 30 CCC CCT GTT GTC CGC CTG GGG GAC CCT GTC CTG GCC TCT TGC ACC ATC 144 Pro Pro Val Val Arg Leu Gly Asp Pro Val Leu Ala Ser Cys Thr Ile 35 40 45 AGC CCA AAC TGC AGC AAA CTG GAC CAA CAG GCA AAG ATC TTA TGG AGA 192 Ser Pro Asn Cys Ser Lys Leu Asp Gln Gln Ala Lys Ile Leu Trp Arg 50 55 60 CTG CAA GAT GAG CCC ATC CAA CCT GGG GAC AGA CAG CAT CAT CTG CCT 240 Leu Gln Asp Glu Pro Ile Gln Pro Gly Asp Arg Gln His His Leu Pro 65 70 75 80 GAT GGG ACC CAA GAG TCC CTC ATC A CT CTG CCT CAC TTG AAC TAC ACC 288 Asp Gly Thr Gln Glu Ser Leu Ile Thr Leu Pro His Leu Asn Tyr Thr 85 90 95 CAG GCC TTC CTC TTC TGC TTA GTG CCA TGG GAA GAC AGC GTC CAA CTC 336 Gln Ala Phe Leu Phe Cys Leu Val Pro Trp Glu Asp Ser Val Gln Leu 100 105 110 CTG GAT CAA GCT GAG CTT CAC GCA GGC TAT CCC CCT GCC AGC CCC TCA 384 Leu Asp Gln Ala Glu Leu His Ala Gly Tyr Pro Pro Ala Ser Pro Ser 115 120 125 AAC CTA TCC TGC CTC ATG CAC CTC ACC ACC AAC AGC CTG GTC TGC CAG 432 Asn Leu Ser Cys Leu Met His Leu Thr Thr Asn Ser Leu Val Cys Gln 130 135 140 TGG GAG CCA GGT CCT GAG ACC CAC CTG CCC ACC AGC TTC ATC CTA AAG 480 Trp Glu Pro Gly Pro Glu Thr His Leu Pro Thr Ser Phe Ile Leu Lys 145 150 155 160 AGC TTC AGG AGC CGC GCC GAC TGT CAG TAC CAA GGG GAC ACC ATC CCG 528 Ser Phe Arg Ser Arg Ala Asp Cys Gln Tyr Gln Gly Asp Thr Ile Pro 165 170 175 GAT TGT GTG GCA AAG AAG AGG CAG AAC AAC TGC TCC ATC CCC CGA AAA 576 Asp Cys Val Ala Lys Lys Arg Gln Asn Asn Cys Ser Ile Pro Arg Lys 180 185 190 AAC TTG CTC CTG TAC TAC CAG TAT ATG GCC ATC TGG GTG CAA GCA GAG AAT 624 Asn Leu Leu Leu Tyr Gln Tyr Met Ala Ile Trp Val Gln Ala Glu Asn 195 200 205 ATG CTA GGG TCC AGC GAG TCC CCA AAG CTG TGC CTC GAC CCC ATG GAT 672 Met Leu Gly Ser Ser Glu Ser Pro Lys Leu Cys Leu Asp Pro Met Asp 210 215 220 GTT GTG AAA TTG GAG CCT CCC ATG CTG CAG GCC CTG GAC ATT GGC CCT 720 Val Val Lys Leu Glu Pro Pro Met Leu Gln Ala Leu Asp Ile Gly Pro 225 230 235 240 GAT GTA GTC TCT CAC CAG CCT GGC TGC CTG TGG CTG AGC TGG AAG CCA 768 Asp Val Val Ser His Gln Pro Gly Cys Leu Trp Leu Ser Trp Lys Pro 245 250 255 TGG AAG CCC AGT GAG TAC ATG GAA CAG GAG TGT GAA CTT CGC TAC CAG 816 Trp Lys Pro Ser Glu Tyr Met Glu Gln Glu Cys Glu Leu Arg Tyr Gln 260 265 270 CCA CAG CTC AAA GGA GCC AAC TGG ACT CTG GTG TTC CAC CTG CCT TCC 864 Pro Gln Leu Lys Gly Ala Asn Trp Thr Leu Val Phe His Leu Pro Ser 275 280 285 AGC AAG GAC CAG TTT GAG CTC TGC GGG CTC CAT CAG GCC CCA GTC TAC 912 Ser Lys Asp Gln Phe Glu Leu Cys Gly Leu His Gln Ala Pro Val Tyr 290 295 300 ACC CTA CAGATG CGA TGC ATT CGC TCA TCT CTG CCT GGA TTC TGG AGC 960 Thr Leu Gln Met Arg Cys Ile Arg Ser Ser Leu Pro Gly Phe Trp Ser 305 310 315 320 CCC TGG AGC CCC GGC CTG CAG CTG AGG CCT ACC ATG AAG GCC 1002 Pro Trp Ser Pro Gly Leu Gln Leu Arg Pro Thr Met Lys Ala 325 330

SEQ ID NO: 2 Sequence length: 999 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 ATG GCA AGG CTG GGA AAC TGC AGC CTG ACT TGG GCT GCC CTG ATC ATC 48 Met Ala Arg Leu Gly Asn Cys Ser Leu Thr Trp Ala Ala Leu Ile Ile 1 5 10 15 CTG CTG CTC CCC GGA AGT CTG GAG GAG TGC GGG CAC ATC AGT GTC TCA 96 Leu Leu Leu Pro Gly Ser Leu Glu Glu Cys Gly His Ile Ser Val Ser 20 25 30 GCC CCC ATC GTC CAC CTG GGG GAT CCC ATC ACA GCC TCC TGC ATC ATC 144 Ala Pro Ile Val His Leu Gly Asp Pro Ile Thr Ala Ser Cys Ile Ile 35 40 45 AAG CAG AAC TGC AGC CAT CTG GAC CCG GAG CCA CAG ATT CTG TGG AGA 192 Lys Gln Asn Cys Ser His Leu Asp Pro Glu Pro Gln Ile Leu Trp Arg 50 55 60 CTG GGA GCA GAG CTT CAG CCC GGG GGC AGG CAG CAG CGT CTG TCT GAT 240 Leu Gly Ala Glu Leu Gln Pro Gly Gly Arg Gln Gln Arg Leu Ser Asp 65 70 75 80 GGG ACC CAG GAA TCT ATC ATC ACC CTG CCC CAC CTC AAC CAC ACT CAG 288 Gly Thr Gln Glu Ser Ile Ile Thr Leu Pro His Leu Asn His Thr Gln 85 90 95 GCC TTT CTC TCC TGC TGC CTG AAC TGG GGC AAC AGC CTG CAG ATC CTG 336 Ala Phe Leu Ser Cys Cys Leu Asn Trp Gly Asn Ser Leu Gln Ile Leu 100 105 110 GAC CAG GTT GAG CTG CGC GCA GGC TAC CCT CCA GCC ATA CCC CAC AAC 384 Asp Gln Val Glu Leu Arg Ala Gly Tyr Pro Pro Ala Ile Pro His Asn 115 120 125 CTC TCC TGC CTC ATG AAC CTC ACA ACC AGC AGC CTC ATC TGC CAG TGG 432 Leu Ser Cys Leu Met Asn Leu Thr Thr Ser Ser Leu Ile Cys Gln Trp 130 135 140 GAG CCA GGA CCT GAG ACC CAC CTA CCC ACC AGC TTC ACT CTG AAG AGT 480 Glu Pro Gly Pro Glu Thr His Leu Pro Thr Ser Phe Thr Leu Lys Ser 145 150 155 160 TTC AAG AGC CGG GGC AAC TGT CAG ACC CAA GGG GAC TCC ATC CTG GAC 528 Phe Lys Ser Arg Gly Asn Cys Gln Thr Gln Gly Asp Ser Ile Leu Asp 165 170 175 TGC GTG CCC AAG GAC GGG CAG AGC CAC TGC TGC ATC CCA CGC AAA CAC 576 Cys Val Pro Lys Asp Gly Gln Ser His Cys Cys Ile Pro Arg Lys His 180 185 190 CTG CT G TTG TAC CAG AAT ATG GGC ATC TGG GTG CAG GCA GAG AAT GCG 624 Leu Leu Leu Tyr Gln Asn Met Gly Ile Trp Val Gln Ala Glu Asn Ala 195 200 205 CTG GGG ACC AGC ATG TCC CCA CAA CTG TGT CTT GAT CCC ATG GAT GTT 672 Leu Gly Thr Ser Met Ser Pro Gln Leu Cys Leu Asp Pro Met Asp Val 210 215 220 GTG AAA CTG GAG CCC CCC ATG CTG CGG ACC ATG GAC CCC AGC CCT GAA 720 Val Lys Leu Glu Pro Pro Met Leu Arg Thr Met Asp Pro Ser Pro Glu 225 230 235 240 GCG GCC CCT CCC CAG GCA GGC TGC CTA CAG CTG TGC TGG GAG CCA TGG 768 Ala Ala Pro Pro Gln Ala Gly Cys Leu Gln Leu Cys Trp Glu Pro Trp 245 250 255 CAG CCA GGC CTG CAC ATA AAT CAG AAG TGT GAG CTG CGC CAC AAG CCG 816 Gln Pro Gly Leu His Ile Asn Gln Lys Cys Glu Leu Arg His Lys Pro 260 265 270 270 CAG CGT GGA GAA GCC AGC TGG GCA CTG GTG GGC CCC CTC CCC TTG GAG 864 Gln Arg Gly Glu Ala Ser Trp Ala Leu Val Gly Pro Leu Pro Leu Glu 275 280 285 GCC CTT CAG TAT GAG CTC TGC GGG CTC CTC CCA GCC ACG GCC TAC ACC 912 Ala Leu Gln Tyr Glu Leu Cys Gly Leu Leu Pro Ala Thr Ala Tyr Thr 290 29 5 300 CTG CAG ATA CGC TGC ATC CGC TGG CCC CTG CCT GGC CAC TGG AGC GAC 960 Leu Gln Ile Arg Cys Ile Arg Trp Pro Leu Pro Gly His Trp Ser Asp 305 310 315 320 TGG AGC CCC AGC CTG GAG CTG AGA ACT ACC GAA CGG GCC 999 Trp Ser Pro Ser Leu Glu Leu Arg Thr Thr Glu Arg Ala 325 330

SEQ ID NO: 3 Sequence length: 28 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence CGGGATCCAT GGTAGGGCTG GGAGCCTG 28

SEQ ID NO: 4 Sequence length: 31 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCGGATCCTT AGGCCTTCAT GGTAGGCCTC A 31

SEQ ID NO: 5 Sequence length: 765 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA Origin Organism: Mouse Cell type: NFS-60 sequence ATG GTA GGG CTG GGA GCC TGC ACC CTG ACT GGA GTT ACC CTG ATC TTC 48 Met Val Gly Leu Gly Ala Cys Thr Leu Thr Gly Val Thr Leu Ile Phe 1 5 10 15 TTG CTA CTC CCC CGA AGA AGT CTG GAG AGC TCT GGA CAC ATC GGC GTC CAA 96 Leu Leu Leu Pro Arg Ser Leu Glu Ser Ser Gly His Ile Gly Val Gln 20 25 30 CTC CTG GAT CAA GCT GAG CTT CAC GCA GGC TAT CCC CCT GCC AGC CCC 144 Leu Leu Asp Gln Ala Glu Leu His Ala Gly Tyr Pro Pro Ala Ser Pro 35 40 45 TCA AAC CTA TCC TGC CTC ATG CAC CTC ACC ACC AAC AGC CTG GTC TGC 192 Ser Asn Leu Ser Cys Leu Met His Leu Thr Thr Asn Ser Leu Val Cys 50 55 60 CAG TGG GAG CCA GGT CCT GAG ACC CAC CTG CCC ACC AGC TTC ATC CTA 240 Gln Trp Glu Pro Gly Pro Glu Thr His Leu Pro Thr Ser Phe Ile Leu 65 70 75 80 AAG AGC TTC AGG AGC CGC GCC GAC TGT CAG TAC CAA GGG GAC ACC ATC 288 Lys Ser Phe Arg Ser Arg Ala Asp Cys Gln Tyr Gln Gly Asp Thr Ile 85 90 95 CCG GAT TGT GTG GCA AAG AAG AGG CAG AAC AAC TGC TCC ATC CCC CGA 336 Pro Asp Cys Val Ala Lys Lys Arg Gln Asn Asn Cys Ser Ile Pro Arg 100 105 110 AAA AAC TTG CTC CTG TAC CAG TAT ATG GCC ATC TGG GTG CAA GCA GAG 384 Lys Asn Leu Leu Leu Tyr Gln Tyr Met Ala Ile Trp Val Gln Ala Glu 115 120 125 AAT ATG CTA GGG TCC AGC GAG TCC CCA AAG CTG TGC CTC GAC CCC ATG 432 Asn Met Leu Gly Ser Ser Glu Ser Pro Lys Leu Cys Leu Asp Pro Met 130 135 140 GAT GTT GTG AAA TTG GAG CCT CCC ATG CTG CAG GCC CTG GAC ATT GGC 480 Asp Val Val Lys Leu Glu Pro Pro Met Leu Gln Ala Leu Asp Ile Gly 145 150 155 160 CCT GAT GTA GTC TCT CAC CAG CCT GGC TGC CTG TGG CTG AGC TGG AAG 528 Pro Asp Val Val Ser His Gln Pro Gly Cys Leu Trp Leu Ser Trp Lys 165 170 175 CCA TGG AAG CCC AGT GAG TAC ATG GAA CAG GAG TGT GAA CTT CGC TAC 576 Pro Trp Lys Pro Ser Glu Tyr Met Glu Gln Glu Cys Glu Leu Arg Tyr 180 185 190 CAG CCA CAG CTC AAA GGA GCC AAC TGG ACT CTG GTG TTC CAC CTG CCT 624 Gln Pro Gln Leu Lys Gly Ala Asn Trp Thr Leu Val Phe His Leu Pro 195 200 205 TCC AGC AAG GAC CAG TTT GAG CTC TGC GGG CTC CAT CAG GCC CCA GTC 672 Ser Ser Lys Asp Gln Phe Glu Leu Cys Gly Leu His Gln Ala Pro Val 210 215 220 TAC ACC CTA CAG ATG CGA TGC ATT CGC TCA TCT CTG CCT GGA TTC TGG 720 Tyr Thr Leu Gln Met Arg Cys Ile Arg Ser Ser Leu Pro Gly Phe Trp 225 230 235 240 AGC CCC TGG AGC CCC GGC CTG CAG CTG AGG CCT ACC ATG AAG GCC 765 Ser Pro Trp Ser Pro Gly Leu Gln Leu Arg Pro Thr Met Lys Ala 245 250

SEQ ID NO: 6 Sequence length: 28 Sequence type: nucleic acid Number of strands: single strand Topology: linear Sequence type: synthetic DNA sequence CGGGATCCAT GGTAGGCCTG GGAGCCTG 28

SEQ ID NO: 7 Sequence length: 39 Sequence type: nucleic acid Number of strands: single stranded Topology: linear Sequence type: synthetic DNA sequence GGACGCCGAT GTGTCCAGAG CTCTCCAGAC TTCTGGGGA 39

SEQ ID NO: 8 Sequence length: 36 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence CTGGAGAGCT CTGGACACAT CGGCGTCCAA CTCCTG 36

SEQ ID NO: 9 Sequence length: 31 Sequence type: nucleic acid Number of strands: single-stranded Topology: linear Sequence type: synthetic DNA sequence GCTCTAGATT AGGCCTTCAT GGTAGGCCTC A 31

[Brief description of the drawings]

BRIEF DESCRIPTION OF THE FIGURES FIG. 1. C-ligand binding region of G-CSF receptor
Recombinant baculovirus Ac for expressing RH domain protein and Ig-CRH domain protein in insect cells
FIG. 3 is a schematic diagram showing the construction of CRH and AcIg-CRH.

FIG. 2. Ig-expressed by the baculovirus system.
FIG. 4 is a simulated view of the results of Scatchard analysis, showing the binding activity of CRH domain proteins to G-CSF.

FIG. 3 Ig-CRH • G in gel filtration HPLC
FIG. 3 is a schematic view of an elution pattern of a CSF complex.

FIG. 4. CRH expressed by baculovirus system
It is a mimetic diagram of the result of Scatchard analysis which shows the binding activity of the domain protein to G-CSF.

FIG. 5: CRH-G-CS in gel filtration HPLC
It is a mimic diagram of the elution pattern of F complex.

──────────────────────────────────────────────────続 き Continuation of the front page (56) References WO 91/14776 (WO, A1) Cell, Vol. 61, p. 341-350 (1990) Proc. Natl. Acad. Sc i. USA, Vol. 87, p. 8702-8706 (1990) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00 C07K 14/715 C12N 5/00 C12P 21/00 BIOSIS (DIALOG) WPI (DIALOG)

Claims (14)

(57) [Claims] 1. Amino acid numbers 26 to 334 of SEQ ID NO: 1.
D encoding a protein consisting of the amino acid sequence represented by
DNA encoding a mutant protein resulting from deletion, substitution or addition of one or more amino acids in NA or the amino acid sequence, the protein having a binding activity to granulocyte colony stimulating factor. 2. Amino acid numbers 122-33 of SEQ ID NO: 1.
DNA encoding a protein consisting of the amino acid sequence represented by No. 4, or a mutant protein obtained by deletion, substitution or addition of one or more amino acids in the amino acid sequence, having a binding activity to a granulocyte colony stimulating factor DNA encoding a protein. 3. Amino acid numbers 26-255 of SEQ ID NO: 5.
D encoding a protein consisting of the amino acid sequence represented by
DNA encoding a mutant protein resulting from deletion, substitution or addition of one or more amino acids in NA or the amino acid sequence, the protein having a binding activity to granulocyte colony stimulating factor. 4. Amino acid numbers 24-333 of SEQ ID NO: 2
D encoding a protein consisting of the amino acid sequence represented by
DNA encoding a mutant protein resulting from deletion, substitution or addition of one or more amino acids in NA or the amino acid sequence, the protein having a binding activity to granulocyte colony stimulating factor. 5. Amino acid numbers 121-33 of SEQ ID NO: 2
DNA encoding a protein consisting of the amino acid sequence represented by No. 3, or a mutant protein obtained by deletion, substitution or addition of one or more amino acids in the amino acid sequence, having a binding activity to a granulocyte colony stimulating factor DNA encoding a protein. 6. The DNA according to any one of claims 1 to 5,
An expression vector containing 7. The method according to claim 1, which is downstream of the polyhedron promoter.
The expression vector according to claim 6, which is a recombinant baculovirus into which the DNA according to any one of 5 is incorporated. 8. The pAcCRH or pAcI according to FIG.
The expression vector according to claim 7, which is g-CRH. 9. A transformant transformed with the expression vector according to any one of claims 6 to 8. 10. The transformant according to claim 9, which is a cell derived from an insect. 11. A cytokine receptor complementation of a ligand-binding region of a granulocyte colony-stimulating factor receptor, which comprises culturing the transformant according to claim 9 or 10 and recovering a recombinant product from the culture. A method for producing a protein of a region or an immunoglobulin-like region and a region complementary to a cytokine receptor. 12. Produced by the method of claim 11,
A substantially pure, ligand binding domain protein of a recombinant granulocyte colony stimulating factor receptor. 13. Amino acids 26-33 of SEQ ID NO: 1
4 or 122-334, amino acid number 2 of SEQ ID NO: 5
6-255, or amino acid numbers 24-3 of SEQ ID NO: 2
A protein having the amino acid sequence represented by SEQ ID NO: 33 or 121-333, or a mutant protein resulting from deletion, substitution or addition of one or more amino acids in the amino acid sequence, and having a binding activity to a granulocyte colony-stimulating factor 13. The protein according to claim 12, which is a protein. 14. Amino acid numbers 35-25 of SEQ ID NO: 5
5. A protein having an amino acid sequence represented by 5 and having a binding activity to granulocyte colony stimulating factor.