JP3002104B2 - DNA encoding the ligand binding domain protein BC of granulocyte colony stimulating factor receptor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION The present invention relates to a protein of a ligand binding domain (i.e., a G-CSF binding domain 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 C-terminal domain, an expression vector containing the DNA, a transformant transformed with the vector,
And a method for producing a recombinant protein, comprising culturing the transformant in an appropriate medium and recovering the product from the culture.

[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 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, such as human It is also present in endothelial cells and the placenta, and elucidating the mechanism of the complex formation reaction between these ligands, G-CSF and G-CSF receptor, requires study, treatment or prevention of related diseases and abnormalities. It is effective for For example, it has been suggested that cancer cells such as myeloid leukemia cells proliferate by G-CSF,
It is possible to block it with substances that antagonize the G-CSF receptor. Already, mouse and human G-CS
The DNA encoding the F receptor has been cloned and sequenced [Fukunaga et al., Cell, 61 341-.
350 (1990); Proc. Natl. Acad, Sci. US
A. 87 8702-8706 ;; WO 91/1377
6 (released October 3, 1991) Fukunaga et al.
-Reported successful expression of the CSF receptor (Cell, supra; Proc. Natl. Acad, Sci. USA. Supra .; EMBO J. 10 2855-2865 (19).
91)) In each case, the entire sequence of the complementary DNA was expressed using animal cells, and the expression level was extremely low, which was not optimal for the production of a G-CSF receptor satisfying the demand. However, the research on the reaction mechanism could not be sufficiently performed.

[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.

[0004] 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
(Bazan Proc. Natl. Acad. Sci (1990) 6934-
6938]. In fact, this CRH region is also essential for the G-CSF receptor, and it is presently considered that this CRH region is one unit required for ligand binding and signaling of this family. This CRH region is further composed of two parts, an amino-terminal domain (hereinafter abbreviated as a BN domain) and a C-terminal domain (hereinafter abbreviated as a BC domain). When the BN domain is deleted, the 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 BC domain is also thought to be required. The above results indicate that, as the ligand binding domain protein in the present invention, the BC domain protein may also be involved in the ligand binding activity. In order to solve these problems, a large amount of the BC domain protein must be used. Need to be manufactured.

[0005] If Escherichia coli can be used to produce a protein having such an activity (eg, a ligand-binding region protein), the production of the protein, the ease of handling, and the advantage of producing a mutant would be difficult. Very convenient and significant. Conventionally, expression of the ligand binding site (CRH) of a cytokine receptor in Escherichia coli was considered to be extremely difficult. The reason for this is that this binding site contains many cysteine residues and proline residues and does not easily fold. Exceptionally, the human growth hormone receptor has been successfully expressed, but one of the reasons for the success is that the human growth hormone receptor has a higher cysteine residue than other similar receptors. It is believed that the content is low. For example, erythropoietin [Harr et al.
is), J. Biol. Chem. (1992) 15205-1520.
9] and interferon gamma [Fontrakis et al.
ulakis), J. Biol. Chem. (1990) 13268-13.
275], most of the products expressed in E. coli are insolubilized in the cells. Another reason that expression of these receptors is difficult is that these receptors
It may be formed by the combination of many complex domains. As described above, the G-CSF receptor also has a complex structure composed of a CRH region, an immunoglobulin-like region, and a fibronectin-like region. In the case of the human growth hormone receptor described above, the extracytoplasmic region consists only of the CRH region, which is also considered as another cause of the successful expression of the human growth hormone receptor. However,
The CRH region of this growth hormone receptor also consists of a BN domain and a BC domain, and cannot be declared to have a simple structure. Actually, the above-mentioned interferon γ and erythropoietin have almost no extracytoplasmic domain consisting only of the CRH region, but when expressed, most of them are insolubilized.

[0006] Signal transduction requires the entire CRH region including the BC domain (Fukunaga et al., EM, supra).
BO.J. 10 2855-2865 (1991)), and the BC domain may also retain the binding activity.
In other words, if only the BC domain of about 100 amino acid residues can be folded and expressed while maintaining almost the same structure as in nature, G-CS, which has been difficult to express conventionally,
It is possible to remove large amounts of functional domains of various cytokine receptors, including the F receptor. Then
By examining the ligand binding activity of the expression product, 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 a small-molecular-weight ligand-binding protein is extremely advantageous in higher-order structure analysis by X-ray or NMR. For the above reasons, the production of a ligand-binding region of a G-CSF receptor, in particular, a BC domain protein by a DNA recombination method, disclosed in the present invention,
It also opens the way to the production of proteins for the ligand binding region of other receptors, which has been difficult in the past.

[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 comprising about 100 amino acids as a BC 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. In addition, research on the binding between G-CSF and the G-CSF receptor (such as analysis of its three-dimensional structure) has been drastically developed.
It enables the synthesis of F 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 the CRH region of the G-CSF receptor, especially its BC domain. For the 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). BC, m
BC (mouse-derived BC) is defined as GC as defined above.
It refers to the ligand binding region of the SF receptor, but also refers to proteins in that region, and derivatives thereof, unless otherwise specified herein. The DNA encoding the G-CSF receptor ligand binding region protein refers to a DNA encoding an amino acid sequence constituting a region involved in binding to G-CSF in the G-CSF receptor. The DNA is G-
It contains DNA encoding the amino acid sequence constituting the CRH region of the CSF receptor, especially the BC domain. 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. DNA is complementary DN
A or any of synthetic DNAs.

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 , 3].
41-350 (1990)] and human [SEQ ID NO: 2 in the Sequence Listing; Fukunaga et al., Proc. Natl. Acad, Sci. USA. 87 , 8].
702-8706 (1990)]. In the present specification, the expression of the ligand binding region protein is shown using the complementary DNA of the region corresponding to the BC portion of the mouse G-CSF receptor, but it may be a synthetic DNA. As will be readily appreciated by those skilled in the art, even DNAs with different codons, as long as they encode the amino acid sequence of the protein of the present invention,
It is included in the scope 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 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. G-
As described above, the BC domain of the CSF receptor is G-
When the CRH region of the CSF receptor is divided into two, the region is the C-terminal region, and the genomic DNA of the G-CSF receptor (ge
nomic DNA) exons 7 and 8 [Seta et al.
Immunol. 148 , 259-266 (1992)]. Specifically, in the sequence disclosed by Fukunaga et al., Supra, in the mouse G-CSF receptor, 20
It refers to the part from the third leucine to the 308th lysine (in SEQ ID NO: 1, they correspond to the 107th leucine and 212th lysine, respectively). Similarly, in the human G-CSF receptor, 3-4
Arginine at position 09 (107 each in SEQ ID NO: 2)
(Corresponds to the leucine at position 212 and arginine at position 212)
Area. However, the start and end sites of the BC domain are not necessarily strict for the present invention, and do not significantly affect the three-dimensional structure of the entire BC domain. 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. Usually, such a slight difference in the primary structure is caused by the B
It is thought that the function is maintained without significantly affecting the three-dimensional structure of the entire C domain. Actually, in the examples described later, a DNA fragment encoding the BC domain of the mouse G-CSF receptor was used. This DNA fragment encodes an amino acid sequence starting from glycine-serine, passing through leucine at position 203 in the original sequence, and reaching lysine at position 308 at the C-terminus (SEQ ID NO: 3).

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. 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. In the Examples of the present specification, first, as a ligand binding domain protein, BC of mouse G-CSF receptor was used.
DNA encoding a domain (hereinafter, referred to as mBC) protein was used. DNA encoding the amino acid sequence of the mBC domain can be obtained from the known plasmid pBLJ17 by PCR. It can also be obtained by a DNA synthesis method.

In order to express the DNA encoding mBC in an E. coli host, this DNA must be linked to an E. coli gene expression system in advance. 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.
aniatis), (1982), Molecular Cloning: A Labor
Requirement Manual, Cold Spring Harbor Laboratory]. Also, basic methods such as protein purification are described in many books such as Biochemistry Laboratory Course (Nippon Biochemistry, Tokyo Chemical Dojin). The method of the present invention can be carried out by using the methods known in the literature, and will be briefly described below.

[0015] The mBC domain can be extracted by the polymerase chain reaction method (hereinafter abbreviated as PCR method). Next, the above BC domain gene is inserted into an E. coli-derived expression plasmid (FIG. 1). 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. Examples of such a gene include a tryptophan gene, a lactose gene, an alkaline phosphatase gene, and a tac gene which is a hybrid of a tryptophan gene and a lactose gene [Nakahara and Matsubara (1986), edited by The Japanese Biochemical Society, “Genetic Engineering”. Research Method II ", 1-172, Tokyo Kagaku Doujin]. 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.
There is a suitable expression system for each.

When the target product is a protein that is originally secreted extracellularly, the gene of the signal sequence of the secretory protein derived from Escherichia coli is linked to the N-terminus of the gene of the target product, and the expression product is converted to 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.

In an embodiment described later, the mBC domain is defined as MB
P and expressed as a fusion protein. In this embodiment, a tac promoter which is induced by the addition of IPTG is used as an expression promoter. MBP
Since the signal sequence is added to the N-terminal of
Expression products accumulate in the periplasm. Extraction of the fusion protein accumulated in the periplasm of the cell, hydrophobic chromatography,
S-Sepharose, Q-Sepharose, Factor Xa
Of a target peptide by a series of operations consisting of excision of a target peptide by S-sepharose and the use of S-Sepharose
Domains can be purified to a single substance. 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 one embodiment of the present invention, a BC domain gene was inserted downstream of DNA encoding MBP so as to be expressed as a fusion protein with MBP that is frequently expressed in E. coli. 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. By the above-mentioned method, an mBC protein having a natural amino acid sequence could be obtained. However, a mutant of the mBC having one or more amino acid mutations (deletion, insertion or substitution) was similarly transformed into E. coli. Can be expressed. Such mutations are intended to improve ligand binding activity, in vivo stability, and the like.

That is, the present invention preferably provides 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 transformant transformed with the expression plasmid. Provided transformants. 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 contains an mBC domain protein. When the physiological activity of the mBC domain produced by the method of the present invention using Escherichia coli as a host was measured using the binding ability to the ligand as an index, the binding ability of the mBC domain to the ligand was confirmed. This result indicates that the recombinant ligand binding domain (mBC domain) protein obtained by the method of the present invention retains the ligand binding activity of the G-CSF receptor, is physiologically active, and G-CSF with usefulness and three-dimensional structure analysis
It shows that the research of the receptor can lead to dramatic development.

[0020]

The present invention will be described in more detail with reference to the following examples. Example 1 Preparation of DNA Encoding BC Domain Protein of Mouse G-CSF Receptor and Escherichia coli Expression Vector
Construction of pMALp-mBC Based on the amino acid sequence of a known mouse G-CSF receptor [Fukunaga et al., Cell 61 , 341-350 (1990)], a DNA encoding the BC-domain (mBC domain) protein is represented by SEQ ID NO. 3 was designed. This DN
In preparing the fragment A, the mouse G-CSF receptor encodes lysine 203 to lysine 203 to leucine 308, and expresses that portion as a fusion protein with MBP.
The desired fusion protein was designed to be inserted into a vector encoding MBP so that a desired mBC domain protein could be isolated and purified from the expressed fusion protein. That is, with MBP
Ile-Glu which is a recognition site of factor Xa between mBC
It was designed such that the sequence encoding -Gly-Arg was further inserted at the N-terminal side. Thus, mBC expressed in a form fused with MBP is cleaved from MBP by cleavage with the restriction 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 tyrosine 203 to make the action of factor Xa more efficient. Further, a termination codon TAA was further added to the C-terminal. In order to ligate the DNA fragment to the BamHI and XbaI sites of pMAL ^™ p, the N-terminal and C-terminal sides of the DNA were further extended to form BamHI and XbaI recognition sites at the extended portions.

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: 4 and SEQ ID NO: 5, 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 dATP, dTTP, dGT
Add P, dCTP solution and 0.5 μl of Taq DNA polymerase to bring the final volume to 100 μl. Here, 10 × reaction buffer, dATP, dTTP, dGTP, dCTP, TaqD
The NA polymerase used was from a Takara Shuzo GeneAmp kit. This reaction solution was set in a DNA thermal cycler (model PJ2000, Takara Shuzo) and heated at 94 ° C for 1 hour.
The desired DNA fragment is synthesized by repeating the reaction 25 times with a cycle program of annealing at 37 ° C. for 2 minutes; heat treatment at 37 ° C. for 2 minutes. Further, the above P
The CR reaction solution is purified by phenol treatment.
About 1 μg of the purified DNA was mixed with 0.1 M salt and 10
50 μl of 50 mM Tris-HCl buffer (pH 7.5) containing mM magnesium chloride and restriction enzyme BamHI (Takara Shuzo)
And 5 units each of restriction enzyme XbaI (Takara Shuzo)
Incubate at 7 ° C. for 1 hour to digest DNA. After phenol treatment and ethanol precipitation of this digested juice,
Electrophoresis on a 1% agarose gel, cut out a band of about 0.3 kbp, put it on Sprek-01 (Takara Shuzo),
After freezing at -80 ° 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 converted into pMAL ^™ p
M (available from New England Biolabs)
Restriction protease factor X frozen downstream of BP
The recognition sequence a is inserted between the BamHI site and the XbaI site located on the N-terminal and C-terminal sides of the Ile-Glu-Gly-Arg (FIG. 1).

Specifically, about 1 μg of pMAL ^™ pDN
A containing 0.1 M salt and 10 mM magnesium chloride
Dissolve in 50 μl of 0 mM Tris-HCl buffer (pH 7.5)
By further adding 5 units of restriction enzymes BamHI and XbaI, respectively, and incubating at 37 ° C. for 1 hour,
Digest the added DNA. Next, the band corresponding to the larger DNA fragment was cut out by electrophoresis on a 1% agarose gel, and placed in Sprek-01 (Takara Shuzo).
After freezing at 80 ° 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, this is mixed with the above-prepared DNA fragment, and 10 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA is added 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 incubated at 0 ° C. for 30 minutes and further at 42 ° C. for 2 minutes, and transferred to Escherichia coli to construct expression plasmid pMALp-mBC. Escherichia coli K12 strain / pMALp-mBC 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: October 25, 1994; Deposit number: FE)
RM P-14602).

Example 2 Plasmid pMALp-mB
Production of mBC by E. coli transformed with C. Bactotripton (Difco) 10 g, yeast extract (Difco) 5 g, salt 5
g, 1 liter of medium containing 100 μg / ml ampicillin and 0.2% glucose,
Grow at 23 ° C. to the midlog phase (A600 = about 0.9). Then, IPTG (Sigma) is added so that the final concentration becomes 0.2 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.
Suspend in pH 7.5 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 (200 ml) is 20 mM
After adding a sodium phosphate buffer (pH 6.0), solid ammonium sulfate (22.8 g) is added to make 20% saturation. This is subjected to hydrophobic chromatography (30 ml; T
(SK gel Butyl-Toyopearl 650M; TOSOH) at a flow rate of 3 ml / min. Next, solution A [20 mM sodium phosphate buffer (pH 6.0) containing 20% saturated ammonium sulfate] was prepared as a mixer, and solution B (1 mM sodium phosphate buffer) was prepared as a reservoir, and the flow rate was 1.5.
The target product is eluted with a concentration gradient of ammonium sulfate from solution A to solution B for 20 minutes at ml / min. Continue developing with Solution B for another 120 minutes. When the target protein in the form of a fusion of MBP and BC is traced by absorbance at 280 nm, most of the protein elutes as a peak as the concentration of ammonium sulfate decreases after passing through the column. This can be confirmed by showing a molecular weight of about 54 kilodaltons in 15% polyacrylamide electrophoresis containing SDS.

After collection, the target substance is dialyzed overnight against a 20 mM sodium phosphate citrate buffer (pH 5.5). Next, the dialysate was added to 20 ml of S-Sepharose (S
-Sepharose FF; Pharmacia) at a rate of 1.5 ml / min. Elution is carried out with a concentration gradient of 0 M to 0.4 M salt (total 120 ml; flow rate 1 ml / min). The target substance was confirmed by polyacrylamide gel electrophoresis, collected,
Dialyze against mM sodium phosphate (pH 6.0). This was added to 10 ml of Q-Sepharose FF (Pharmacia) equilibrated with the same buffer, and
Elution is carried out with a concentration gradient of M to 0.4 M sodium chloride (total 240 ml; flow rate 1 ml / min). The target product can be confirmed by showing a molecular weight of about 12 kilodaltons by 15% polyacrylamide gel electrophoresis. The target fraction was collected and the salt concentration was finally adjusted to 0.1 M, and then protease factor Xa (New England Biolab) was added to a final concentration of 5 µg / ml, and the mixture was added at 16 ° C for 4 days. Let react. This releases the mBC from the MBP.

Next, the obtained fraction was reduced to a 5-fold amount of 20 mM.
S-Sepharose (5 ml; diluted with sodium phosphate citrate buffer (pH 5.5) and equilibrated with the same buffer)
-Sepharose Bio-Rad pre-packed column) at 1 ml / m
Add at in flow rate. Elution is performed with a concentration gradient of 0 M to 0.4 M salt (total 30 ml; flow rate 0.5 ml / min). As a result, the target substance is purified into a single standard. Finally 1l
Approximately 0.7 mg of purified protein is extracted from the culture.

Example 3 Binding Activity of Recombinant mBC with Ligand The biological activity of mBC 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 mBC and ¹²⁵ I
-CSF is mixed to form a complex of mBC 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 for the binding of labeled G-CSF with that of unlabeled, the dissociation constant of the complex of mBC and G-CSF can be measured. 0.5 μM mBC domain prepared in Example 2, 1.2 mM ¹²⁵ I-human G-CSF (100,000 cpm, Amsham) and various concentrations of unlabeled human G-CSF (10 ⁻⁵ -10 ⁻¹⁰⁾
M; provided by Kirin Co., Ltd.)
In a 0.1 M acetate buffer (pH 7.0) containing Tween 20, a final volume of 50 μl is maintained at room temperature for 90 minutes. Subsequently, 0.1 dissolved in 0.5 μl of dimethyl sulfoxide.
M dithiobissuccinimidyl propione (Pierce Chemical Co., Ltd.) is added, and the mixture is kept at 0 ° C. for 30 minutes. 5 μl of 2
The reaction is stopped by adding M Tris-HCl buffer. Subsequently, the reaction mixture is subjected to 15% polyacrylamide electrophoresis with SDS. 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 is
Appeared as a 30 kilodalton band on the gel. The dissociation constant of this complex was determined to be about 40 nM by comparison with the value in an experiment performed by adding unlabeled G-CSF (FIG. 2). This result indicates that the BC domain has a ligand binding activity similarly to the BN domain.

[0030]

[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 name: 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

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

SEQ ID NO: 3 Sequence length: 324 Sequence type: nucleic acid Number of strands: double-stranded Topology: linear Sequence type: cDNA to mRNA GGT TCT TTG GAG CCT CCC ATG CTG CAG GCC CTG GAC ATT GGC CCT GAT 48 Gly Ser Leu Glu Pro Pro Met Leu Gln Ala Leu Asp Ile Gly Pro Asp 1 5 10 15 GTA GTC TCT CAC CAG CCT GGC TGC CTG TGG CTG AGC TGG AAG CCA TGG 96 Val Val Ser His Gln Pro Gly Cys Leu Trp Leu Ser Trp Lys Pro Trp 20 25 30 AAG CCC AGT GAG TAC ATG GAA CAG GAG TGT GAA CTT CGC TAC CAG CCA 144 Lys Pro Ser Glu Tyr Met Glu Gln Glu Cys Glu Leu Arg Tyr Gln Pro 35 40 45 CAG CTC AAA GGA GCC AAC TGG ACT CTG GTG TTC CAC CTG CCT TCC AGC 192 Gln Leu Lys Gly Ala Asn Trp Thr Leu Val Phe His Leu Pro Ser Ser 50 55 60 AAG GAC CAG TTT GAG CTC TGC GGG CTC CAT CAG GCC CCA GTC TAC ACC 240 Lys Asp Gln Phe Glu Leu Cys Gly Leu His Gln Ala Pro Val Tyr Thr 65 70 75 80 CTA CAG ATG CGA TGC ATT CGC TCA TCT CTG CCT GGA TTC TGG AGC CCC 288 Leu Gln Met Arg Cys Ile Arg Ser Ser Leu P ro Gly Phe Trp Ser Pro 85 90 95 TGG AGC CCC GGC CTG CAG CTG AGG CCT ACC ATG AAG 324 Trp Ser Pro Gly Leu Gln Leu Arg Pro Thr Met Lys 100 105

SEQ ID NO: 4 Sequence length: 45 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA GGG GAT CCA TCG AGG GTA GGG GTT CTT TGG AGC CTC CCA TGC TGC 45

SEQ ID NO: 5 Sequence length: 36 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Synthetic DNA GGG TCT AGA TTA CTT CAT GGT AGG CCT CAG CTG CAG 36

[Brief description of the drawings]

FIG. 1. Expression vector pMALp-m for expression in Escherichia coli of a ligand binding domain protein of G-CSF receptor
It is a construction schematic diagram of BC.

FIG. 2 is a graph for measuring a dissociation constant between mBC and G-CSF produced by a recombinant method.

────────────────────────────────────────────────── (5) Continuation of the front page (51) Int.Cl. ⁷ identification code FI C12R 1:19) (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) EMBO J. J .; Vol. 10, No. 10, P.E. 2855-2865 (1991) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 15/00 C12N 1/21 C12P 21/02 C07K 14/715 BIOSIS (DIALOG)

Claims (12)

(57) [Claims] 1. Amino acid numbers 107-21 of SEQ ID NO: 1.
DNA encoding a protein consisting of the amino acid sequence represented by 2, or a mutant protein obtained by deletion, substitution or addition of one or several amino acids in the amino acid sequence, having a binding activity to a granulocyte colony stimulating factor DNA encoding a protein. 2. A DNA encoding a protein consisting of the amino acid sequence represented by SEQ ID NO: 3, or a mutant protein resulting from deletion, substitution or addition of one or several amino acids in said amino acid sequence, which stimulates granulocyte colony stimulation. DNA encoding a protein having a factor-binding activity. 3. Amino acid numbers 107-21 of SEQ ID NO: 2.
DNA encoding a protein consisting of the amino acid sequence represented by 2, or a mutant protein obtained by deletion, substitution or addition of one or several amino acids in the amino acid sequence, having a binding activity to a granulocyte colony stimulating factor DNA encoding a protein. 4. The DNA according to any one of claims 1 to 3,
An expression vector containing 5. The DNA according to any one of claims 1 to 3,
The expression vector according to claim 4, which is a recombinant plasmid containing E. coli under the control of an Escherichia coli gene expression system. 6. The DNA according to any one of claims 1 to 3.
6. The expression vector according to claim 5, wherein the expression vector is located downstream of the DNA encoding the Escherichia coli maltose binding protein, and the Escherichia coli can express a fusion protein of the ligand binding region protein of the granulocyte colony stimulating factor receptor and the Escherichia coli maltose binding protein. 7. The plasmid pMALp- shown in FIG.
The expression vector according to claim 6, which is mBC. A transformant transformed with the expression vector according to any one of claims 4 to 7. 9. The transformant according to claim 8, which is Escherichia coli. 10. The C-terminal domain of the ligand-binding region of the granulocyte colony-stimulating factor receptor, which comprises culturing the transformant according to claim 8 and recovering a recombinant product from the culture. A method for producing a protein. 11. Produced by the method of claim 10,
A substantially pure, ligand binding domain protein of a recombinant granulocyte colony stimulating factor receptor. 12. An amino acid number 107-2 of SEQ ID NO: 1.
A protein having the amino acid sequence of amino acid No. 107-212 of SEQ ID NO: 12 or a mutant protein obtained by deletion, substitution or addition of one or several amino acids in the amino acid sequence, which is a granulocyte colony stimulating factor A protein having an activity of binding to