JP2001078777A - New protein and its production - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new protein which comprises a protein containing a specific amino acid sequence, consists of an analog of adipogenesis inhibitory factor that controls the differentiation or muturation of the fat cell and is useful as a pharmaceutical composition for prophylaxis or treatment for obesity or an antigen for immunologic diagnosis. SOLUTION: This protein is the new protein containing at least 25th-302nd amino acids in the amino acid sequence represented by formula, consists of an analog of adipogenesis inhibitory factor(ADIF) comprising a protein exhibiting differentiation and/or muturation inhibitory activity in the fat cell and is useful as the pharmaceutical composition for prophylaxis and/or treatment for obesity or the antigen for establishing immunologic diagnosis. This protein is obtained by carrying out PCR using a primer comprising the partial sequence of natural type ADIF, using the resultant cDNA fragment as a probe to screen out a cDNA library derived from the human fetal lung fibroblast and transducing the resultant gene into an expression system to express.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to an adipogenesis inhibitory factor (Adipogenesis Inhibitory Factor), which is a protein having an activity of suppressing adipocyte differentiation and / or maturation.
The present invention relates to a novel protein which is an analog of ADIF), a DNA encoding the protein, a method for producing the protein using the DNA, a C-terminal deletion mutant of the protein, and a DNA encoding the same. The protein of the present invention is useful as a pharmaceutical composition for preventing and / or treating obesity, or as an antigen for establishing an immunological diagnosis.

[0002]

2. Description of the Related Art Obesity is a risk factor for diabetes, hypertension, heart disease and the like, and is a threat to the health of citizens in developed countries. Obesity refers to a physical condition in which adipose tissue has accumulated more than normal. Adipose tissue is a special organ that stores excess energy in the body as fat, that is, triglyceride, and is composed of fat cells, fibroblasts including their precursor cells, macrophages, perivascular cells, blood cells, and the like. I have. Fat cells are said to occupy 1/3 to 2/3 of the cells present in adipose tissue,
It accumulates fat, triglyceride, in its cells. Adipocytes are mesothelial pluripotent stem cells, lipoblasts that have acquired the base of adipocytes, preadipocytes that have no adipocyte initial markers, and adipocytes that have no adipocytes. It is said that the cells will differentiate and mature into mature fat cells and mature fat cells in which a large amount of fat has accumulated. In a living body of a mildly obese adult, the amount of fat accumulated in individual fat cells, that is, triglyceride, is increased and the cells are enlarged. As the degree of obesity increases, so does the number of fat cells. Therefore, by suppressing the differentiation and maturation of adipocytes and reducing the number of adipocytes, and by suppressing the enlargement of mature adipocytes, it suppresses the increase in the amount of accumulated fat, stops the progression of obesity, and reduces obesity. It is expected to be treated. It has been revealed that the regulation of adipocyte differentiation in vivo is positively or negatively controlled by a number of factors derived from environmental factors such as food intake and exercise. Tumor necrosis factor-α as a cytokine that suppresses the differentiation of preadipocytes into adipocytes; (TNF-α: T
orti FM et al., Science, Vol.229, p867 (1985)),
Transforming growth factor-β (transforming gro
wth factor- β; Ignotz RA et al., Proc. Natl. A
cad. Sci. USA, Vol. 82, p8530 (1985)), preadipocyte factor-1 (Pref-1, Preadipocyte facto).
r-1: Smas CM et al., Cell, Vol. 73, p725 (1993)
) Have been reported. In addition, leptin, a translation protein of the ob gene cloned in recent years,
It has been reported that food intake and adipose tissue weight are reduced, possibly through the central nervous system (Levin N. et al. Pro
c. Natl. Acad. Sci. USA, Vol. 93. p1726, 1996).
Furthermore, a peptide in the brain that has a strong food intake promoting action
Neuropeptide Y and its receptor are also attracting attention as powerful tools for the development of drugs for suppressing obesity (Sain
sburg A. et al. Diabetologia, Vol. 39, p353, 199
6). These cytokines are expected to be therapeutic agents for obesity due to the inhibitory action of fat cells on fat accumulation, and some of the above-mentioned cytokines such as leptin are undergoing clinical trials as therapeutic agents or preventive agents for obesity. . At present, Redax (American Home Products) is already on the market in the United States as a therapeutic or preventive drug for obesity. Meridia (Knoll) and Xenical (Roche) are also expected to be approved in the United States as a therapeutic agent for obesity or a fat absorption inhibitor. However, the therapeutic methods using these drugs are not always satisfactory in their effects and therapeutic results, and there has been a demand for the development of new therapeutic agents that are more effective and have fewer side effects instead of these.

The present inventors have already reported that a novel substance having a new anti-obesity activity or an obesity-improving activity has been introduced into a culture medium of human fetal lung fibroblast IMR-90 (ATCC deposition-accession number CCL186). A protein having an activity of suppressing cell formation, that is, an activity of suppressing differentiation and / or maturation of adipocytes was found, and this protein was named as an adipogenesis inhibitory factor (ADIF). Further, a method for producing the protein, characterized in that it is purified from a culture solution obtained by culturing human fibroblasts by ion exchange column, affinity column, and reverse phase column chromatography, and as a carrier for adherent cells. The present inventors have found an efficient method for producing the protein by culturing cells using alumina ceramic pieces. Furthermore, they have found a DNA encoding the protein and a method for producing the protein using the DNA. The properties of natural ADIF are shown below. (a) activity: having an activity to suppress adipocyte differentiation and / or maturation (b) molecular weight (by SDS-PAGE): about 45 kDa (under non-reducing conditions) about 28 kDa and / or about 23 kDa (under reducing conditions) (c) Affinity: has affinity for heparin. (d) It contains a sugar chain. (e) N-terminal amino acid sequence: Xaa Ser Gln Gln Lys Gly Arg Leu Ser Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Cys Leu Val Asn Ala Gly Asp Val Gly Cys Gly Val Phe Glu 20 25 30 Cys Phe Glu Asn Asn Xaa Xaa Glu 35 40 (However, Xaa indicates an unidentified amino acid.)

[0004]

The present inventors have further searched for a substance having the same activity as the above-mentioned protein, and as a result of intensive search, have found that a recombinant AD having a different N-terminal site from ADIF.
We have found an IF analog protein. That is, the present invention relates to a novel protein having an activity of inhibiting adipocyte differentiation and / or maturation, which is an adipogenesis inhibitor (Adipogenesis
Novel protein which is an analog of Inhibitory Factor (ADIF), DNA encoding the protein, and its DNA
An object of the present invention is to provide a method for producing the protein by using the method.

[0005]

SUMMARY OF THE INVENTION The present invention provides a novel protein, Adipogenesis Inhibitor, which is a novel protein having an activity of suppressing adipocyte differentiation and / or maturation.
y Factor; ADIF). The present invention also relates to a DNA encoding the protein. Further, the present invention relates to a method for producing the protein using the DNA. More specifically, the present invention relates to a protein comprising the amino acid sequence described in SEQ ID NO: 13 of the Sequence Listing and a DNA encoding the same, particularly a DNA represented by the nucleotide sequence of SEQ ID NO: 12 in the Sequence Listing. It is also represented by a protein obtained by genetically expressing these DNAs, in particular, the amino acid sequence of SEQ ID NO: 13 or an amino acid sequence in which one or more amino acids have been deleted, substituted or added. The present invention relates to a protein and a protein having an activity of inhibiting adipocyte differentiation and / or maturation obtained by expressing a DNA encoding an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 13 in the Sequence Listing. . The DNA
And a method for producing a protein having the following properties by genetic engineering. (a) activity: having an activity of suppressing the differentiation and / or maturation of adipocytes. (b) Affinity: has affinity for heparin. (c) It contains a sugar chain. The protein of the present invention is useful as a pharmaceutical composition for preventing and / or treating obesity, or as an antigen for establishing an immunological diagnosis.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION The protein of the present invention can be produced by a genetic engineering technique. That is, the above-mentioned natural type A
Based on the information on the amino acid sequence of DIF, a cDNA encoding the protein of the present invention was cloned, and this cDN
The protein of the present invention can be obtained by expressing A by a genetic engineering technique. For details, see Natural AD
The N-terminal amino acid sequence of IF is analyzed to produce a mixture of oligonucleotides encoding this sequence. next,
Using the prepared oligonucleotide mixture as a primer,
Using PCR (preferably RT-PCR), A
Obtain a DIF cDNA fragment. By cloning this cDNA fragment as a probe using a cDNA library prepared from IMR-90 cells, AD
A full-length cDNA of an IF analog can be obtained (SEQ ID NO: 12 in the Sequence Listing). Furthermore, the obtained full-length cDNA is inserted into an appropriate expression vector to prepare an ADIF analog expression plasmid, which is introduced into various bacteria or animal cells to express the recombinant ADIF analog. Obtainable. The recombinant ADIF analog thus obtained has the same physicochemical properties as the above-mentioned natural ADIF, and its N-terminal amino acid sequence is shown as follows. N-terminal amino acid sequence: Thr Asp Ala Thr Asn Pro Pro Glu Gly Pro Gln Asp Arg Ser Ser 1 5 10 15

[0007] The biological activity of the protein of the present invention is determined by Kodama H. et al.
al. method (Journal of Cellular Physiology, Vol. 11
2, p83 (1982)), that is, the suppression of adipocyte formation in the presence of dexamethasone using mouse preadipocyte cell lines as target cells can be confirmed by evaluating the inhibition of triglyceride accumulation.

The protein of the present invention is useful as a pharmaceutical composition for preventing and / or treating obesity or as an antigen for establishing an immunological diagnosis. The protein of the present invention can be safely administered to humans and animals. The protein of the present invention can be formulated and administered orally or parenterally. Examples of the form of the pharmaceutical composition include an injection composition, an infusion composition, a suppository, a nasal agent, a sublingual agent, a transdermal absorbent, and the like. These preparations should use pharmacologically acceptable carriers, excipients, stabilizers, coloring agents, surfactants, and / or other additives according to known pharmaceutical manufacturing methods. Thus, the desired preparation can be obtained. In the case of an injectable composition, a pharmacologically effective amount of the adipocyte formation inhibitory factor of the present invention,
And a mixture with pharmaceutically acceptable excipients / activators, such as amino acids, saccharides, cellulose derivatives, and other organic / inorganic compounds. When an injection is prepared using the protein of the present invention and these excipients / activators, a pH adjuster, a buffer, a stabilizer, a solubilizing agent, etc. are added, if necessary, according to a conventional method. Various injections can be prepared.

[0009]

EXAMPLES The present invention will be described in more detail with reference to the following examples, which are merely illustrative, and do not limit the present invention in any way.

[0010]

Example 1 Preparation of Human Fibroblast IMR-90 Culture Solution Human embryonic lung fibroblast IMR-90 (ATCC deposition-accession number CL186) was prepared using a roller bottle (490c).
80 g in m ² , 110 × 171 mm, Corning)
Was adhered to an alumina ceramic piece (alumina 99.5%, Toshiba Ceramic Co., Ltd.) and cultured. Use 40 roller bottles for culture, 5% per roller bottle
Using 500 ml of DMEM (Gibco BRL) supplemented with newborn bovine serum and 10 mM HEPES buffer, 37
The culture was allowed to stand for 7 to 10 days in the presence of 5% CO ₂ at 7 ° C. After the culture, the culture was collected and fresh medium was added to obtain a 20 L IMR-90 culture per culture cycle. The obtained culture solution was used as Sample 1.

[0011]

Example 2 Method for Measuring Adipocyte Formation Inhibitory Activity ADIF and the activity of the protein of the present invention were measured by Kodama H. et al.
al. method (Journal of Cellular Physiology, Vol. 11
2, p83, 1982). That is, mouse preadipocyte cell line MC3T3-G2 / PA6 cells (RIKEN GENE BANK, RCB1127
) Was used as a target cell, and the formation of adipocytes in the presence of dexamethasone was tested using triglyceride accumulation as an index, and the inhibitory activity was measured.
That is, 50 μl of a sample diluted with α-MEM (Gibco BRL) containing 10% fetal bovine serum was placed in a 96-well microplate, and then the mouse preadipocyte cell line MC3T3-
G2 / PA6 cells were suspended in α-MEM medium containing 2 × 10 ⁻⁷ M dexamethasone and 10% fetal bovine serum, and seeded at a cell density of 3 × 10 ³ cells / 50 μl / well.
The cells were cultured for one week at 5% CO2, 37 ° C and 100% humidity. After 7 days of culture, the medium was removed by suction, air-dried, and the amount of triglyceride accumulated in the fat cells was measured using a triglyceride measurement kit (Triglyceride G-Test Wako, Wako Pure Chemical Industries, Ltd.). OD510nm decrease AD
IF activity.

[0012]

Example 3 Purification of ADIF i) Purification by Heparin Sepharose CL-6B About 60 liters of an IMR-90 culture solution (sample 1) was used.
0.22 μm filter (Hydrophilic Millidisk, 2,
000 cm ² , Millipore) and divided into three portions of 10 mM Tris-HC containing 0.3 M NaCl.
Heparin-Sepharose CL-6B column (5 x 4.1 cm, gel volume 8) equilibrated with 1 buffer, pH 7.5
0 ml). 10 mM at a flow rate of 500 ml / hr
Tris-HCl buffer, pH 7.5 (hereinafter referred to as Tri
s-HCl), elution was performed with 10 mM Tris-HCl containing 2 M NaCl, pH 7.5, and heparin-Sepharose CL-6B adsorbed fraction 1.5
One liter was obtained and designated as Sample 2.

Ii) 10 mM of the heparin-Sepharose-adsorbed fraction (sample 2) purified by HiLoad-SP / HP
After dialysis against Tris-HCl, pH 7.5,
CHAPS was added to a concentration of 0.1%, and the mixture was allowed to stand at 4 ° C. overnight. The cation exchange column (6 parts) was equilibrated with 50 mM Tris-HCl containing 0.1% CHAPS, pH 7.5, and equilibrated (6 times). 1. HiLoad-SP / HP;
(6 × 10 cm, Amersham Pharmacia). 50 mM Tris-H containing 0.1% CHAPS
Cl, pH 7.5, then NaCl for 120 minutes
Elution was performed at a linear gradient of 0.5 M at a flow rate of 6 ml / min, and fractionation was performed at 12 ml / fraction. Using 30 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and about 0.15 to 0.25 MNa
936 ml of an ADIF-active fraction eluted with Cl was obtained as Sample 3.

Iii) Purification by HiLoad-Q / HP The obtained sample 3 was washed with 50 mM containing 0.1% CHAPS.
After diluting with Tris-HCl, pH 7.5, 1900 ml, the mixture was divided into six portions and the 50 m containing 0.1% CHAPS was added.
Anion exchange column (HiLoad-Q / HP, 2.6 × 10 6) equilibrated with M Tris-HCl, pH 7.5
cm, Amersham Pharmacia Pharmacia). 50 mM Tri with 0.1% CHAPS
After washing with s-HCl, pH 7.5, N
Elution was performed at a linear gradient with an aCl concentration of 0.5 M, at a flow rate of 6 ml / min, and fractionation was performed at 12 ml / fraction. Using 30 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and about 0.1 to 0.18
936 ml of the ADIF active fraction eluted with MNaCl was obtained and used as sample 4.

Iv) Purification by HiLoad-SP / HP The obtained sample 4 was treated with 50 mM containing 0.1% CHAPS.
BisTris-HCl buffer, pH 6.0 (hereinafter, referred to as BisTris-HCl buffer)
BisTris-HCl), diluted with 1900 ml, and divided into three portions of 50 mM containing 0.1% CHAPS.
Cation exchange column (HiLoad-SP / HP, 2.6, equilibrated with BisTris-HCl, pH 6.0)
× 10 cm, Amersham Pharmacia). 50 mM BisTri with 0.1% CHAPS
After washing with s-HCl, pH 6.0, N
Elution was performed at a linear gradient from 0.1 M to 0.6 M in aCl concentration, at a flow rate of 6 ml / min, and fractionation was performed at 12 ml / fraction. ADIF activity was measured using 30 μl of each fraction according to the method described in
ADIF active fraction 3 eluted at ~ 0.45M NaCl
60 ml was obtained and used as Sample 5.

V) Purification by Resource S The obtained sample 5 was subjected to 50 mM containing 0.1% CHAPS.
BisTris-HCl, pH 6.0, 1080 ml
Cation exchange column (Resource S, equilibrated with 50 mM BisTris-HCl, pH 6.0 containing 0.1% CHAPS, divided into three portions)
0.5 × 5 cm, Amersham Pharmacia). 50 mM BisTr containing 0.1% CHAPS
After washing with is-HCl, pH 6.0, N
Elution was performed at a linear gradient with an aCl concentration of 0.6 M at a flow rate of 1 ml / min, and fractionation was performed at 1 ml / fraction.
Using 10 μl of each fraction, the ADIF activity was measured according to the method described in Example 2, and about 0.2 to 0.3 M N
30 ml of the ADIF active fraction eluted with aCl was obtained and used as sample 6.

Vi) Purification by Superose 12 The obtained sample 6 is transferred to a centrifugal concentrator (Centricon 10,
After concentrating to about 600 μl with Millipore Co., Ltd., the solution was divided into three portions and containing 50% containing 0.1% CHAPS and 0.5 M NaCl.
Gel filtration column (Superose 12, 1.0 × 60 c) equilibrated with mM Tris-HCl, pH 7.5
m, Amersham-Pharmacia), 0.1%
50 mM Tr containing CHAPS and 0.5 M NaCl
It was developed with is-HCl, pH 7.5 at a flow rate of 0.5 ml / min, and fractionated at 0.5 ml / fraction. Using 10 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and 9 ml of an ADIF active fraction eluted at an elution time of about 25 to 30 minutes was obtained as Sample 7.

Vii) Purification by Heparin 5PW The obtained sample 7 was subjected to 50 mM containing 0.1% CHAPS.
After dilution with 41 ml of BisTris-HCl, pH 6.0, 50 mM BisTr containing 0.1% CHAPS was used.
Heparin affinity column (Heparin 5PW, 0.5 × 5) equilibrated with is-HCl, pH 6.0
cm, Tosoh Corporation). After washing with 50 mM BisTris-HCl containing 0.1% CHAPS, pH 6.0, elution was performed at a linear gradient of NaCl concentration from 0.2 M to 0.8 M in 120 minutes, at a flow rate of 0.5 ml / min. Fractionation was performed at 1 ml / fraction (FIG. 1).
Using 10 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and about 0.2 to 0.3 M Na
16 ml of an ADIF active fraction eluted with Cl was obtained and used as sample 8.

Viii) Purification by Polycat A The obtained sample 8 is centrifuged (centricon 10,
After concentrating to about 300 μl with Millipore, 600 μl
50 mM BisTris containing 0.1% CHAPS
-HCl, pH 6.0, and diluted with 50 mM BisTris-HCl, pH 6.0 containing 0.1% CHAPS, weak cation exchange column (Polycat A, 0.
46 × 20 cm, Polyersie). 0.1
50 mM BisTris-HC with% CHAPS
After washing with 1, pH 6.0, elution was performed at a flow rate of 1.0 ml / min with a linear gradient of 0.4 M NaCl concentration in 50 minutes, and fractionation was performed at 1 ml / fraction (FIG. 2). . Using 10 μl of each fraction, ADIF activity was measured according to the method described in Example 2, and about 0.3 to 0.4
ADIF fraction No. eluted with MNaCl. Three
7-46 were obtained.

Ix) Purification by reverse phase column The obtained polycat A fraction No. 1 ml of 43
Was added to a reverse phase column (C4, 2.1 × 250 mm, Baydac) equilibrated with 30% acetonitrile containing 0.1% TFA after adding 10 μl of 20% TFA (trifluoroacetic acid). 5 minutes of acetonitrile in 50 minutes
Elution was performed at a linear gradient of 5% and a flow rate of 0.2 ml / min, and each protein peak was collected (FIG. 3). Using 35 μl of each peak fraction, ADIF activity was measured according to the method described in Example 2, and peak 5 concentration-dependent activity was detected. FIG. 4 shows the results.

[0021]

Example 4 Measurement of ADIF Molecular Weight SDS-polyacrylamide gel electrophoresis was performed under reducing and non-reducing conditions using 100 μl of the peak 5 in which ADIF activity was recognized. That is, 50 μl of each of the peak 5 fractions was separated into two tubes, concentrated under reduced pressure, and then concentrated to 1 mM EDTA, 2.5% SDS, and 0.0%.
10 mM Tris containing 1% bromophenol blue
-HCl, pH 8, 1.5 μl, and each was left under non-reducing conditions and reducing conditions (in the presence of 5% 2-mercaptoethanol) at 37 ° C. overnight.
1 was loaded on SDS-polyacrylamide gel electrophoresis. For electrophoresis, a 10-15% acrylamide gradient gel (Amersham Pharmacia) was used, and an electrophoresis apparatus Phast System (Amersham Pharmacia) was used.
This was performed using As molecular weight markers, phosphorylase b (94 kDa), bovine serum albumin (67 kD
a), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa), α-lactalbumin (14.4)
kDa) was used. After the electrophoresis, Phast Gel Silver
Silver staining was performed using Stain Kit (Amersham Pharmacia). In addition, the molecular weight of the protein of the peak 5 was calculated as follows. That is, for each molecular weight marker, the migration distance from the upper end of the separation gel was measured, and the measured migration distance was plotted against the molecular weight on a semilogarithmic graph to prepare a standard straight line. In addition, peak 5
The migration distance of the protein was measured, and the molecular weight was calculated using the prepared standard line. FIG. 5 shows the results. As a result, only the protein band of 45 kDa was observed in peak 5 under non-reducing conditions, and about 28 kDa and about 23
Only the kDa protein band was detected.

[0022]

Example 5 Removal and analysis of N-linked sugar chains from ADIF
A purified using reverse-phase column with molecular weight measured in Example 3-ix) methods
Samples containing about 0.2 μg of DIF were concentrated under reduced pressure.
Add 0.5 M sodium phosphate buffer, pH
8.6, 1 μl, 0.5 μl 2-mercaptoethanol 1 μl,
1 μl of water and 250 U / ml N-glycanase solution (Genzyme
After adding 1 μl and stirring well, the mixture was allowed to stand at 37 ° C. for 24 hours. Add 2mM EDTA, 5% SDS, 0.02% to this sample
After adding 2 μl of 20 mM Tris-HCl buffer solution containing 8.0% of bromophenol blue and 5% 2-mercaptoethanol, pH 8.0, the mixture was stirred well, and then left at 37 ° C. for 24 hours. 1 μl of this sample was subjected to SDS by the method described in Example 4.
-Silver staining after polyacrylamide electrophoresis. As a control, 0.2 μg of untreated purified ADIF prepared by the method described in Example 3-ix) was subjected to electrophoresis in the same manner, followed by silver staining. FIG. 6 shows the results. As a result, N- determined by SDS-PAGE under reducing conditions
The apparent molecular weight of the ADIF protein containing no linked sugar chain was about 18 kDa. SDS-P under reducing conditions
Since the apparent molecular weight of untreated ADIF determined by AGE is about 23 kDa and / or about 28 kDa, it is clear that ADIF is a glycoprotein containing an N-linked sugar chain in the molecule. became.

[0023]

Example 6 Determination of N-Terminal Amino Acid Sequence Polycat A fraction No. obtained in Example 3-viii) 37 to 46, a centrifugal concentrator (Centricon 1)
0, Millipore Co., Ltd.)
1% 25% TFA, 30% with 0.1% TFA
Reversed phase column (C4, 2.1) equilibrated with acetonitrile
× 250 mm, Vydac), a linear gradient of 55% acetonitrile in 50 minutes, flow rate 0.2 ml
The elution was carried out at a rate of 5 min / min. The obtained peak 5 was concentrated under reduced pressure with a centrifugal concentrator, and then 1 mM EDT
A, 10 mM Tris-HCl, pH 8, containing 2.5% SDS and 0.01% bromophenol blue,
After dissolving in 0.5 μl and leaving overnight at 37 ° C. under reducing conditions (in the presence of 5% 2-mercaptoethanol), 4 μl is SD
S-polyacrylamide gel electrophoresis was loaded. Electrophoresis was performed using a 10-15% acrylamide gradient gel (Amersham Pharmacia) and an electrophoresis apparatus Phas.
The measurement was performed using t System (Amersham Pharmacia). After the electrophoresis, a PVDF membrane (Pr) was used using a blotting device Phast Transfer (Amersham Pharmacia).
oBlot, PerkinElmer), 20V, 25m
The proteins were transcribed at A for 30 minutes. After transfer, the cells were stained with a 0.2% Coomassie blue / 40% methanol / 10% acetic acid solution, and excess dye was decolorized with a 60% methanol solution.
Bands of about 23 kDa and about 28 kDa were cut out and subjected to N-terminal amino acid sequence analysis using a protein sequencer (Procise, Model 492, Perkin Elmer). The results are shown in SEQ ID NO: 1 in the Sequence Listing.

The polycat A fraction No. obtained from the IMR-90 culture solution of about 80 liters by the same method as described above. 10 μl of 20% for each of 37-46
TFA was added, and a reverse phase column (C4, 10%) equilibrated with 30% acetonitrile containing 0.1% TFA was divided into 10 times.
2.1 x 250 mm, Baydac), a linear gradient of 55% acetonitrile in 50 minutes, flow rate 0.2
Elution was performed at ml / min, and peak 5 was collected. After the resulting peak 5 was subjected to reductive pyridylethylation, N-terminal amino acid sequence analysis was performed using a protein sequencer (Procise, Model 492, Perkin Elmer). As a result, the amino acid sequence of the ADIF up to the 40th amino acid from the N-terminal could be determined. The results are shown in SEQ ID NO: 2 in the Sequence Listing.

[0025]

Embodiment 7Cloning of cDNA fragment of the protein of the present invention i) Isolation of poly (A) ⁺ RNA from IMR-90 cells Fast Track mRNA Isolation Kit (Inv
1) according to the manual.
× 10⁸About 10 μg of poly (A) from one IMR-90 cell ⁺RNA
Isolated.

Ii) Preparation of Mixed Primers Based on the N-terminal amino acid sequence described in SEQ ID NO: 2 obtained in Example 6, the following two types of mixed primers were synthesized. That is, 19 from the 13th (Thr) from the N-terminal
A mixture of oligonucleotides (mix primers, TAE / F) having all nucleotide sequences capable of encoding the amino acid sequence up to the first (Cys) was synthesized. 29th from N-terminal
A mixture (mix primer, CFE / R) of complementary oligonucleotides to all nucleotide sequences capable of encoding the amino acid sequence from (Gly) to 35th (Glu) was synthesized.
Table 1 shows the base sequences of the used mix primers.

[0027]

[Table 1]

Iii) Amplification of ADIF cDNA fragment by PCR Using 1 μg of poly (A) ⁺ RNA obtained in Example 7-i) as a template and a Superscript II cDNA synthesis kit (Gibco BRL), the protocol of the company was used. A single-stranded cDNA is synthesized according to the procedure described above, PCR is performed using this cDNA and the primers described in Example 8-ii), and ADIFcD
An NA fragment was obtained. The composition of the reaction solution is shown below. 10X Ex Taq buffer (Takara Shuzo) 10 μl 2.5 mM dNTP 8 μl cDNA solution 2.5 μl Ex Taq (Takara Shuzo) 1 μl Distilled water 73.5 μl 20 μM Primer TAE / F 2.5 μl 20 μM Primer CFE / R 2.5 μl Trace amount of the above solution After mixing in a centrifuge tube, PCR was performed under the following conditions. After pretreatment at 95 ° C. for 3 minutes, a three-step reaction consisting of 95 ° C. for 30 seconds, 50 ° C. for 30 seconds, and 72 ° C. for 1 second was repeated 40 times, followed by incubation at 72 ° C. for 5 minutes. A part of the reaction solution was analyzed by agarose electrophoresis, and it was confirmed that a cDNA fragment of about 68 bp predicted from the N-terminal amino acid sequence was obtained.

[0029]

Example 8 Cloning of ADIF cDNA fragment amplified by PCR
The cDNA fragment obtained in Example 7-iii) was subjected to DNA ligation kit Ver. PT using 2 (Takara Shuzo)
7 Insert into Tvector (Novagen) and use E. coli XL2Bl
ue (Stratagene) was transformed. The obtained transformant is grown and about 68 bp of ADIFcDN.
The plasmid into which the fragment A was inserted was purified according to a conventional method.
This plasmid was named pBSADIF, and the nucleotide sequence of ADIF cDNA inserted into this plasmid was determined using a DNA sequencing kit (Perkin Elmer). The amino acid sequence consisting of 23 amino acids predicted from this nucleotide sequence could be found in the N-terminal amino acid sequence of ADIF used to design the mix primer (SEQ ID NO: 2 in the Sequence Listing). based on the above results,
The cloned 68 bp cDNA is ADIFcD
It was confirmed to be an NA fragment.

[0030]

Example 9 Preparation of DNA Probe Using the plasmid inserted with the 68 bp ADIF cDNA fragment prepared in Example 8 as a template and performing PCR under the conditions of Example 6-iii), this ADIF cDNA
The fragment was amplified. About 68 bp by agarose electrophoresis
After isolation of the ADIF cDNA fragment of QIAEX II DN
Purification was performed using an A isolation kit (Qiagen). This cDNA was purified using a Megaprime DNA Labeling Kit (Amersham Pharmacia) [α ³²
It was labeled with [P] dCTP and used as a probe to screen for full-length ADIF cDNA.

[0031]

[Example 10] Preparation of cDNA library Using poly (A) ⁺ RNA obtained in Example 7-i), 2.5 µg as a template, and using a Great Strength cDNA synthesis kit (Clontech) according to the company's protocol, oligo
cDNA synthesis using (dT) primer, EcoRI-SalI-Not-
After addition of I adapter and cDNA size fractionation, ethanol precipitation was performed, followed by dissolution in 10 μl of TE buffer. 0.1 μg of the obtained adapter-added cDNA
Pre-cut with EcoRI using T4 DNA ligase1
μg of λZAP express vector (Stratagene) was inserted. The cDNA recombinant phage DNA solution thus obtained was subjected to an in vitro packaging reaction using Gigapack Gold II (Stratagene) to prepare a λZAP express recombinant phage.

[0032]

Example 11 Screening of Recombinant Phage The recombinant phage obtained in Example 10 was infected with Escherichia coli XL1-Blue MRF '(Stratagene) at 37 ° C. for 15 minutes, and then heated to 50 ° C. at 0.7%. It was added to NZY medium containing agar and poured onto NZY agar plate. After overnight incubation at 37 ° C, place Hybond N on the plate with plaques.
(Amersham Pharmacia) was adhered for about 30 seconds.
The filter was alkali-denatured according to a conventional method, neutralized, immersed in 2XSSC solution, and immobilized on the filter by UV crosslink (Stratagene).
The obtained filter was immersed in a rapid hybridization buffer (Amersham Pharmacia) containing 100 μg / ml of salmon sperm DNA, pretreated at 65 ° C. for 1 hour, and then heat-denatured DNA probe (2 × 10 ⁵ cpm / ml) ) Was added to the above buffer and hybridization was carried out at 65 ° C. overnight. After the reaction, filter with 2XSSC
The plate was washed twice with a 0.1X SSC and 0.1% SDS solution twice at 65 ° C for 10 minutes. Some of the obtained positive clones were purified by one more screening. Those having an insert of about 2.0 kb were used below. The purified phage was infected with Escherichia coli XL1-Blue MRF 'according to the protocol of the λZAP Express Cloning Kit (Stratagene), followed by multiple infections with the helper phage ExAssist (Stratagene), and the culture supernatant was used to transform the culture supernatant into Escherichia coli XLOLR. (Stratagene)
By picking up kanamycin-resistant strains after infection
A transformant having the plasmid pBKADIF in which the above-mentioned 2.0 kb insert was inserted into pBKCMV (Stratagene) was obtained. This transformant was designated as XLOLR / pBKADIF by the Institute of Biotechnology, Ministry of International Trade and Industry on August 1, 1998.
Deposited one day under accession number FERM BP-6449. Propagating a transformant carrying this plasmid,
The plasmid was purified by a conventional method.

[0033]

Example 12 Encoding the entire amino acid sequence of the protein of the present invention
Determination of Nucleotide Sequence of cDNA to be Performed The nucleotide sequence of the cDNA of the protein of the present invention obtained in Example 11 was determined using a DNA sequencing kit (Perkin Elmer). The primers used were T3 and T7 primers (Stratagene), and synthetic primers designed based on the nucleotide sequence of ADIF cDNA,
The sequences are shown in SEQ ID NOs: 4 to 11 in the sequence listing. The nucleotide sequence of the protein of the present invention thus determined is represented by SEQ ID NO: 1 in the Sequence Listing.
2 shows the amino acid sequence deduced from the sequence, and SEQ ID NO: 13 in the sequence listing. This amino acid sequence was confirmed to start from 13 residues further upstream from the N-terminal Ser of native ADIF.

[0034]

Example 13 Recombinant type using 293 / EBNA cells
Production of ADIF Analog i) Preparation of ADIF cDNA Expression Plasmid The plasmid pBKADIF into which the approximately 2.0 kb ADIF cDNA obtained in Example 10 was inserted was digested with a restriction enzyme SalI,
The cDNA containing the ADIF analog of the present invention is cut out, separated by agarose electrophoresis, and then QIAEXII DNA
Purification was performed using an isolation kit (Qiagen). The ADIF analog cDNA was digested with a restriction enzyme XhoI in advance and treated with an alkaline phosphatase-treated expression plasmid pCEP4 (Invitrogen) using a ligation kit Ver. 2 (Takara Shuzo) to transform E. coli DH5α (Gibco BRL). The obtained transformant was grown and the expression plasmid pCEPADIF into which the ADIF analog cDNA was inserted was purified using a Qiagen column (Qiagen). After the ADIF analog expression plasmid pCEPADIF was precipitated with ethanol, it was dissolved in sterile distilled water and used for the following procedure.

Ii) Transfection of ADIF analog cDNA
Using the ADIF analog expression plasmid pCEPADIF obtained in Example 13-i), a recombinant ADIF analog was expressed by the method described below, and its activity was measured.
2 × 10 ⁵ 293 / EBNA cells (Invitrogen) were suspended and seeded in each well of a 24-well plate in IMDM medium (Gibco BRL) containing 10% fetal bovine serum (Gibco BRL). . On the next day, I was prepared in advance according to the protocol attached to the transfection reagent FuGENE ^™ 6 (Boehringer Mannheim).
FuGENE ^™ 6 diluted with MDM medium
After mixing pCEPADIF, this mixture was added to the cells in each well. The amounts of pCEPADIF and FuGENE ^™ 6 used were 0.5 μg and 1 μl, respectively. After 72 hours, the medium was recovered and used as a sample for measuring ADIF activity.
The measurement of ADIF activity was performed as follows. Using the mouse preadipocyte cell line MC3T3-G2 / PA6 cells as target cells, the formation of adipocytes in the presence of dexamethasone was tested using triglyceride accumulation as an index, and its inhibitory activity was measured. That is, 10% is added to a 96-well microplate.
50 μl of a sample diluted with an α-MEM medium (Gibco BRL) containing fetal bovine serum was added, and 3 × 10 ³ mouse preadipocyte MC3T3-G2 / PA6 cells were placed in 50 μl of 2 × 10 3
Α-M containing ^-7 M dexamethasone and 10% fetal calf serum
The cells were suspended in an EM medium, inoculated, and cultured at 5% CO ₂ , 37 ° C. and 100% humidity for one week. After 7 days of culture, the medium was removed by suction, air-dried, and the triglyceride accumulated in the fat cells was measured using a triglyceride measurement kit (Triglyceride G-Test Wako, code number 274-69802, Wako Pure Chemical Industries, Ltd.). The decrease in OD 510 nm was defined as ADIF activity. As a result, as shown in Table 2, the IMR-
The activity similar to that of the native ADIF obtained from the culture medium of No. 90 was measured for the 293 / EBNA into which the ADIF analog gene was introduced.
It was confirmed that the cell culture solution had.

[0036]

[Table 2]

[0037]

Embodiment 14ADIF activity using 3T3 / LI cells
Measurement Mouse preadipocyte cell line 3T3-LI cells (ATCC Deposit-Deposit
No. CL173) as a target cell, dexamethasone
And 1-methyl-3-isobutylxanthine in the presence of
Adipocyte formation based on triglyceride accumulation
ADIF by testing and measuring its inhibitory activity
Activity was evaluated. That is, 96-well micro play
Α-MEM medium containing 10% fetal bovine serum (Gibco B
(RL)) and put 50 µl of the diluted sample
3x3-LI cells 5x10 ^Three50 μl of 4 ×
10^-7M dexamethasone, 2 × 10^-FiveM 1-methyl-
Contains 3-isobutylxanthine and 10% fetal calf serum
The cells were suspended and seeded in an α-MEM medium, and 5% CO_Two, 37
The cells were cultured at 100 ° C. and 100% humidity for one week. Culture after 7 days of culture
The ground was removed by suction, air-dried, and the trig accumulated in fat cells.
Use the triglyceride measurement kit (triglyceride)
Ride G-Test Wako, code number 274-69802, Wako
The measurement was carried out by using Junyaku Industry Co. OD510nm decrease AD
IF activity. As a result, as shown in Table 3, 3T3-LI
When cells are used as target cells, ADIF analogs
The culture solution of 293 / EBNA cells into which the gene has been introduced is AD
It was confirmed to have IF activity.

[0038]

[Table 3]

[0039]

Example 15 Recombinant type using 293 / EBNA cells
Production of FL-ADIF analog i) Construction of FLAG-tagged ADIF analog expression plasmid Plasmid pBKADIF obtained in Example 11 was used as a template,
ExTaq (Takara Shuzo) using ADIFF2 (SEQ ID NO: 14) and AD-FLAGXhoI (SEQ ID NO: 15) as primers
PCR was performed using PCR. The obtained DNA fragment was digested with the restriction enzyme Sm.
Digest with aI and XhoI and ligate the approximately 500 bp DNA fragment with QIAEXI
Purification was performed using an IDNA isolation kit (Qiagen). Further, plasmid pBKADIF was digested with restriction enzymes SalI and SmaI, and a DNA fragment of about 700 bp was purified using QIAEXII DNA isolation kit (Qiagen). These two DNA fragments were cut with the restriction enzyme XhoI in advance and inserted into the plasmid pCEP4 (Invitrogen), which had been treated with alkaline phosphatase, using the ligation kit ver.2 (Takara Shuzo). Using the DNA solution after the ligation reaction, E. coli DH5
α (Gibco BRL) was transformed. From the obtained ampicillin-resistant transformant, FLAG-tagged ADIF (FL-A
A strain having a plasmid in which the DIF) analog cDNA was inserted in the same direction as the transcription direction by the CMV promoter was selected. The obtained cells were named DH5α / pCEP-ADIF-FL.
ADIF cDNA portion in this plasmid (pCEP-ADIF-FL) and
The DNA sequence of the FLAG tag was determined, and it was confirmed that there was no base incorporation error during the PCR reaction.

Ii) Transient of FL-ADIF cDNA
Using the FL-ADIF analog expression plasmid pCEP-ADIF-FL obtained in Expression Example 15-i),
The FL-ADIF analog was expressed. 2 × 10 ⁵ 293 /
EBNA cells (Invitrogen) were added to each well of a 24-well plate in 10% fetal bovine serum (Gibco BRL).
) And IMGEN medium (Gibco BRL) containing the same, and the next day, a transfection reagent FuGEN
E ^TM 6 according (base - - ringer Mannheim) attached protocol, after mixing the pCEP-ADIF-FL and FuGENE ^TM 6 which had been diluted with advance IMDM medium, the mixture in each well cell Added. PCEP used
-ADIF-FL and FuGENE ^™ were 1 μg and 2 μg, respectively.
μl. After 72 hours, the medium was collected,
-A sample for ADIF analog purification was used.

[0041]

Example 16 Purification of FL-ADIF Analog iii) Purification by Anti-FLAG-M2-Affinity-Gel 400 ml of the culture solution obtained in Example 15-i) was added to 0.1 M
After washing with Glycine-HCl, pH 2.8, 0.1 g
The mixture was applied to a 2 ml Anti-FLAG-M2-Affinity-Gel (Sigma) column equilibrated with 50 mM Tris-HCl, pH 7.4 containing 15 M NaCl and 0.01% polysorbate 80. 50 mM Tris-HCl containing 0.15 M NaCl and 0.01% polysorbate 80,
After washing with pH 7.4, 0.15 M NaCl and 0.01
Glycine containing 0.1% polysorbate 80
-HCl, pH 2.8, elute with 3M Tris
H was adjusted to 8. About 10 ml of the obtained fraction containing the FL-ADIF analog was concentrated using Centriplus 10 (Millipore), and 0.15 M NaCl and 0.01%
10 mM phosphate buffer pH with polysorbate 80
The solvent was replaced with 7.4 to obtain 2.5 ml of an FL-ADIF analog. A part of the obtained FL-ADIF analog was subjected to N-terminal amino acid sequence analysis using a protein sequencer (Procise, Model 492, Perkin Elmer). The results are shown in SEQ ID NO: 16 in the sequence listing.

[0042]

Example 17 Preparation of ADIF Analog -FLAG (+ XhoI) (I) Introduction of XhoI Recognition Sequence and Sequence Encoding FLAG In the amino acid sequence of the ADIF analog described in SEQ ID NO: 13, C
XhoI immediately after the nucleotide sequence encoding Arg at the terminal 302
A recognition sequence and a nucleotide sequence encoding FLAG were introduced. According to this sequence, Leu, Glu, Asp, Ty
10 amino acids of r, Lys, Asp, Asp, Asp, Asp, Lys are added. Primers ADFY1 Hd and ADFL used for introduction
The sequences of SXR are shown in SEQ ID NOs: 17 and 18. PCR is 97
3 minutes at 55 ° C, 1 minute at 72 ° C, 3 minutes at 72 ° C,
Perform three stages of reaction at 25 ° C for 1 minute and at 72 ° C for 3 minutes 25 times.
This was carried out by holding at 72 ° C. for 5 minutes. The PCR reaction solution is shown below. PCR reaction solution for mutation introduction Ex Taq polymerase (Takara Shuzo) 0.5 μl 10 × Ex Taq buffer (Takara Shuzo) 10 μl 2.5 mM dNTP solution 8 μl Template plasmid vector (pCEPADIF 40 ng / μl) 1 μl 0.1 M primer (ADFY1 Hd) 1 μl 0.1 M primer (ADFLSXR) 1 μl sterile distilled water 78.5 μl DNA obtained by PCR is separated by electrophoresis using 1% agarose gel, and then QIAEXII Gel Extraction kit
(QIAGEN) and extracted with 20 μl of sterile distilled water.
10 μl of the extracted DNA solution was digested with restriction enzymes HindIII (Takara Shuzo) and SalI (Takara Shuzo). The DNA solution was subjected to electrophoresis on a 1.5% agarose gel, and the target DNA fragment was extracted with 20 μl of sterile distilled water using a QIAEXII Gel Extraction kit (DNA solution 1).

(II) ADIF Analog-FLAG (+ XhoI) Expression Vector
After cutting with the restriction over the construction pCEP4 enzymes HindIII and XhoI (Takara Shuzo), subjected to electrophoresis in 1.0% agarose gel, about
A 10 kbp DNA fragment was extracted using a QIAEXII Gel Extraction kit (DNA solution 2). 3μl of DNA solution 1 and 1μ
4 μl of DNA ligation kit Ve
r.2 Solution I (Takara Shuzo) was added, and the mixture was kept at 16 ° C. for 30 minutes to carry out a ligation reaction. Escherichia coli DH5α was transformed using the solution after the reaction. The DNA structure of the obtained ampicillin-resistant transformed E. coli was analyzed, and a strain having the desired plasmid was selected. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. The resulting expression vector was pCEP4-ADIF / v
It was named -FLAG (+ XhoI).

[0044]

Example 18 Preparation of C-terminal deletion mutant (I) Introduction of C-terminal deletion mutation
From Gly at 243 to Arg at 302, from Ser at 213 to Arg at 302, Glu at 183
To 302 Arg, from 153 Thr to 302 Arg
Mutants were prepared. No. 273
Mutants deleted from Arg to Arg 302 were converted to ADIF / vR
1, ADIF / v-R2 is a mutant deleted from Gly at position 243 to Arg at position 302, and ADIF / v-R3 and 183 is a mutant deleted from Ser at position 213 to Arg at position 302. A mutant lacking Glu to Arg at position 302 was transformed with ADIF / v-R4 and Th at position 153.
A mutant deleted from r to Arg at position 302 was transformed into ADIF / v-R5
I named each. Mutagenesis for mutant production is performed by PCR
Performed by the method. The sequences of the primers used in the mutagenesis reaction are shown in Table 4 and SEQ ID NOs: 19 to 23. PCR
After reacting at 97 ° C for 3 minutes, 55 ° C for 1 minute and 72 ° C for 3 minutes,
Min, 55 ° C for 1 minute, 72 ° C for 3 minutes 25 times,
Thereafter, the temperature was maintained at 72 ° C. for 5 minutes. The PCR reaction solution is shown below. PCR reaction solution for introduction of C-terminal deletion mutation Ex Taq polymerase (Takara Shuzo) 0.5 μl 10 × Ex Taq buffer (Takara Shuzo) 10 μl 2.5 mM dNTP solution 8 μl Template plasmid vector (pCEPADIF 40 ng / μl) 1 μl 0.1 M primer (ADFY1 Hd) 1 μl 0.1 M Mutagenesis primer 1 μl Sterile distilled water 78.5 μl DNA obtained by PCR is separated by electrophoresis using 1% agarose gel, and then QIAEXII Gel Extraction kit
And extracted with 20 μl of sterile distilled water. Extracted DN
A solution (10 μl) was digested with restriction enzymes HindIII and XhoI. The DNA solution was subjected to electrophoresis on a 1.5% agarose gel, and the target DNA fragment was extracted with 20 μl of sterilized distilled water using a QIAEXII Gel Extraction kit (DNA solution 3).

[0045]

[Table 4]

(II) Construction of Mutant Expression Vector After digesting pCEP4-ADIF / v-FLAG (+ Xhol) with restriction enzymes HindIII and XhoI, electrophoresis was performed on a 1.0% agarose gel, and a DNA fragment of about 10.4 kbp was obtained. The QIAEXII Gel Extraction k
It was extracted using it (DNA solution 4). To 3 μl of the DNA solution 3 and 1 μl of the DNA solution 4, 4 μl of the DNA ligation kit Ver. 2 I solution was added, and the mixture was kept at 16 ° C. for 30 minutes to carry out a ligation reaction. Escherichia coli DH5α was transformed using the solution after the reaction. The DNA structure of the obtained ampicillin-resistant transformed E. coli was analyzed, and a strain having the desired plasmid was selected. The DNA structure was analyzed by measuring the length of the fragment obtained by restriction enzyme digestion and determining the nucleotide sequence. ADIF / v-R1, ADIF / v-R2, ADIF / v-R3,
ADIF / v-R4 and ADIF / v-R5 expression vectors were converted to pCEP4-ADIF / v-R1, pCEP4-ADIF / v-R2, pCEP4-ADIF / v, respectively.
-R3, pCEP4-ADIF / v-R4, and pCEP4-ADIF / v-R5.

(III) Preparation of Mutant Expression Vector Escherichia coli (5 types in total) having the mutant expression vector
After growing on LB medium, use the QIAGEN Plasmid Midi kit (QIGEN
(AGEN). Each expression vector was precipitated using ethanol, dissolved in 50 μl of sterile distilled water, and used for the following procedure.

[0048](IV) ADIF / v-FLAG (+ XhoI) and mutant cDNA
Transient expression 2 × 10⁶293-EBNA cells in 10% fetal calf serum IMDM
75 cm suspended in 20 ml of medium ^TwoSeed into flask and allow CO_Twoink
The cells were cultured at 37 ° C. for 24 hours in an incubator. 100 μl FuGENE6
 (Roche Diagnostics) 3.5ml bloodless
After adding to the clear IMDM medium and leaving it at room temperature for 5 minutes, the mixture
In a solution containing 35 μg of ADIF / v-FLAG (+ XhoI).
For 15 minutes at room temperature. 700 μl of the mixture
5 pieces of 75cm^TwoBy adding dropwise to the cells in the flask,
The expression vector was introduced into the cells. Perform the same operation for each
A heterologous expression vector was also performed. Expression vector
Entered cells with CO_TwoIncubate at 37 ° C for 24 hours in an incubator
Nourished. Collect the culture solution after culturing and add a fresh 20 ml serum-free
48 hours CO with IMDM_TwoCulture at 37 ° C in an incubator
Was. Collect the culture solution after culturing and combine it with the previously collected amount.
200 ml of culture was obtained.

(IV) Purification of ADIF / v-FLAG (+ XhoI) and mutant ADIF from 200 ml of culture obtained by transient expression
/ v-FLAG (+ XhoI) and each mutant were purified. Purification was performed by specifically adsorbing ADIF / v-FLAG (+ XhoI) and each mutant in a culture solution to a gel carrier having an anti-FLAG antibody (M2; Sigma) as a ligand, and eluting it. . TBS / T (50mM Tris-HCl [pH7.4], 150mM NaCl,
1m of gel suspended in 0.01% Tween80 (Sigma)
was dispensed into eight centrifuge tubes (volume: 50 ml), and the collected culture was added in 25 ml portions and slowly rotated at 4 ° C. for 24 hours to obtain ADIF / v-FLAG (+ XhoI) and each mutation. The body was adsorbed to the antibody. The gel precipitated by centrifugation was collected and collected in one centrifuge tube (capacity: 15 ml). The recovered gel was suspended in 10 ml of TBS / T and then centrifuged, and the gel was recovered by washing the gel. After three washes,
The gel was washed with 5 ml of elution buffer (0.1 M Glycin-HCl [pH
2.8], 150 mM NaCl, 0.01% Tween80), and centrifuged to collect the eluate. This operation was repeated to obtain a total eluate of 10 ml. The eluate was neutralized by adding 100 μl of 3M Tris.
The eluate was prepared using sodium phosphate buffer (10 mM sodium phosphate [p
H7.0], 0.3M NaCl) and the concentration was 32μg /
The volume was adjusted with a sodium phosphate buffer so that the volume became ml.

[0050]

Example 19 SDS-polyacrylamide electrophoresis (SDS-P
AGE) The purified and purified ADIF / v-FLAG (+ XhoI) and each mutant were subjected to SDS-PAGE. Electrophoresis was performed in a reduced state using PhastSystem (Amersham Pharmacia Biotech). Each 60 ng of purified ADIF / v-FLAG (+ XhoI) and each mutant were 10-15% Gradien
Electrophoresis was performed using t gel (Amersham Pharmacia Biotech). As a result, the molecular weights of ADIF / v-FLAG (+ XhoI) and each mutant were almost identical to those expected from the nucleotide sequence. The purity was 90% or more.

[0051]

Example 20: Activity measurement 5 × 10 ⁴ cells / m with DMEM (DMEM-FCS) containing 10% fetal bovine serum
3T3-L1 cells suspended in a 96-well plate are seeded at 100 μl / well in a 96-well plate and placed in a CO ₂ incubator for 96 hours.
The cells were cultured at 37 ° C. In order to differentiate cells that have reached confluence into adipocytes, the medium in the 96-well plate is 5 × 10 ^−4.
M 1-methyl-3-isobutylxanthine and 1 × 10 ^-6
Replace with DMEM-FCS containing M dexamethasone for 48 hours with CO ₂
The cells were cultured at 37 ° C. in an incubator. At this time, AD
IF / v-FLAG (+ XhoI) or each mutant for 20
It was diluted with DMEM-FCS to a concentration of 0 ng / ml to 6.4 μg / ml and added. DMEM containing the same ADIF / v-FLAG (+ XhoI) or each mutant as the previous concentration except the medium after 48 hours of culture
-FCS was newly added in an amount of 100 µl each, and cultured at 37 ° C in a CO ₂ incubator for 5 days to differentiate into adipocytes. After the culture, the culture solution was discarded, and the well-dried 96-well plate was examined for adipocyte differentiation inhibitory activity (ADIF activity) using Triglyceride-G Test Wako (Wako Pure Chemical Industries, Ltd.). ADIF activity was defined as the decrease from the absorbance at 510 nm of the well containing no mutant. FIG. 7 shows the results. The activity of each sample was measured in duplicate, and the average value was shown. Mutants ADIF / v-R1, ADIF / v-R2 and ADIF / v-R3 are ADIF / vF
It showed almost the same specific activity as LAG (+ XhoI). The specific activity of ADIF / v-R4 was less than 1/30 that of ADIF / v-FLAG (+ XhoI). AD
IF / v-R5 did not show ADIF activity even at 6.4 μg / ml.

[0052]

Industrial Applicability According to the present invention, a novel protein having an activity of inhibiting differentiation and / or maturation of an adipocyte, an adipogenesis inhibitory factor (Adipogenesis Inhibitory Factor;
ADIF), a method for producing the protein, characterized in that the protein is purified from a culture obtained by culturing human fibroblasts by ion exchange column, affinity column, and reverse phase column chromatography, and a carrier for adherent cells. The present invention provides an efficient method for producing the protein by culturing cells using alumina ceramic pieces. Furthermore, the present invention
A DNA encoding the protein and a method for producing the protein using the DNA are provided. The protein of the present invention is useful as a pharmaceutical composition for preventing and / or treating obesity, or as an antigen for establishing an immunological diagnosis.

[0053]

[Sequence List] SEQUENCE LISTING <110> Snow Brand Milk Products Co., Ltd. <120> New protein and its production method <130> YTP99001 <160> 13 <210> 1 <211> 20 <212> PRT <213> Human <400> 1 Xaa Xaa Gln Gln Xaa Gly Arg Leu Xaa Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Xaa Leu 20 <210> 2 <211> 40 <212> PRT <213> Human < 400> 2 Xaa Ser Gln Gln Lys Gly Arg Leu Ser Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Cys Leu Val Asn Ala Gly Asp Val Gly Cys Gly Val Phe Glu 20 25 30 Cys Phe Glu Asn Asn Xaa Xaa Glu 35 40 <210> 3 <211> 40 <212> PRT <213> Human <400> 3 Ser Ser Gln Gln Lys Gly Arg Leu Ser Leu Gln Asn Thr Ala Glu Ile 1 5 10 15 Gln His Cys Leu Val Asn Ala Gly Asp Val Gly Cys Gly Val Phe Glu 20 25 30 Cys Phe Glu Asn Asn Ser Cys Glu 35 40 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 4 AATTAACCCT CACTAAAGGG 20 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 5 GTAATACGAC TCACTATAGG GC <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 6 AAGAGGGGAG CACAAAGGAT < 210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 7 GTCCCTGCAG AATACAGCGG <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 8 CAAGGACTTG CTGCTGCACG <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 9 TGGACGGCGT GGAGGAAAGA <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 10 GCTCAAGATG GAGCACAGGC <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 11 GTTGTGCAGA AAAGTCATGC <210> 12 <211> 909 <212> DNA <213> Human <400> 12 ATGTGTGCCG AGCGGCTGGG CCAGTTCATG ACCCTGGCTT TGGTGTTGGC CACCTTTGAC 60 CCGGCGCGGG GGACCGACGC CACCAACCCA CCCGAGGGTC CCCAAGACAG GAGCTCCCAG 120 CAGAAAGGCC GCCTGTCCCT GCAGAATACA GCGGAGATCC AGCACTGTTT GGTCAACGCT 180 GGCGATGTGG GGTGTGGCGT GTTTGAATGT TTCGAGAACA ACTCTTGTGA GATTCGGGGC 240 TTACATGGGA TTTGCATGAC TTTTCTGCAC AACGCTGGAA AATTTGATGC CCAGGGCAAG 300 TCATTCATCA AAGACGCCTT GAAATGTAAG GCCCACGCTC TGCGGCACAG GTTCGGCTGC 360 ATAAGCCGGA AGTGCCCGGC CATCAGGGAA ATGGTGTCCC AGTTGCAGCG GGAATGCTAC 420 CTCAAGCACG ACCTGTGCGC GGCTGCCCAG GAGAACACCC GGGTGATAGT GGAGATGATC 480 CATTTCAAGG ACTTGCTGCT GCACGAACCC TACGTGGACC TCGTGAACTT GCTGCTGACC 540 TGTGGGGAGG AGGTGAAGGA GGCCATCACC CACAGCGTGC AGGTTCAGTG TGAGCAGAAC 600 TGGGGAAGCC TGTGCTCCAT CTTGAGCTTC TGCACCTCGG CCATCCAGAA GCCTCCCACG 660 GCGCCCCCCG AGCGCCAGCC CCAGGTGGAC AGAACCAAGC TCTCCAGGGC CCACCACGGG 720 GAAGCAGGAC ATCACCTCCC AGAGCCCAGC AGTAGGGAGA CTGGCCGAGG TGCCAAGGGT 780 GAGCGAGGTA GCAAGAGCCA CCCAAACGCC CATGCCCGAG GCAGAGTCGG GGGCCTTGGG 840 GCTCAGGGAC CTTCCGGAAG CAGCGAGTGG GAAGACGAAC AGTCTGAGTA TTCTGATATC 900 CGGAGGTGA 909 <210> 13 <211> 302 <212> PRT <213> Human <220> <221> SIGNAL <222> 1 ... 24 <223> The 25th of "Thr" is N -terminal amino acid. <400> 13 Met Cys Ala Glu Arg Leu Gly Gln Phe Met Thr Leu Ala Leu Val Leu 1 5 10 15 Ala Thr Phe Asp Pro Ala Arg Gly Thr Asp Ala Thr Asn Pro Pro Glu 20 25 30 Gly Pro Gln Asp Arg Ser Ser Gln Gln Lys Gly Arg Leu Ser Leu Gln 35 40 45 Asn Thr Ala Glu Ile Gln His Cys Leu Val Asn Ala Gly Asp Val Gly 50 55 60 Cys Gly Val Phe Glu Cys Phe Glu Asn Asn Ser Cys Glu Ile Arg Gly 65 70 75 80 Leu His Gly Ile Cys Met Thr Phe Leu His Asn Ala Gly Lys Phe Asp 85 90 95 Ala Gln Gly Lys Ser Phe Ile Lys Asp Ala Leu Lys Cys Lys Ala His 100 105 110 Ala Leu Arg His Arg Phe Gly Cys Ile Ser Arg Lys Cys Pro Ala Ile 115 120 125 Arg Glu Met Val Ser Gln Leu Gln Arg Glu Cys Tyr Leu Lys His Asp 130 135 140 Leu Cys Ala Ala Ala Gla Gln Glu Asn Thr Arg Val Ile Val Glu Met Ile 145 150 155 160 His Phe Lys Asp Leu Leu Leu His Glu Pro Tyr V al Asp Leu Val Asn 165 170 175 Leu Leu Leu Thr Cys Gly Glu Glu Val Lys Glu Ala Ile Thr His Ser 180 185 190 Val Gln Val Gln Cys Glu Gln Asn Trp Gly Ser Leu Cys Ser Ile Leu 195 200 205 Ser Phe Cys Thr Ser Ala Ile Gln Lys Pro Pro Thr Ala Pro Pro Glu 210 215 220 Arg Gln Pro Gln Val Asp Arg Thr Lys Leu Ser Arg Ala His His Gly 225 230 235 240 Glu Ala Gly His His Leu Pro Glu Pro Ser Ser Arg Glu Thr Gly Arg 245 250 255 Gly Ala Lys Gly Glu Arg Gly Ser Lys Ser His Pro Asn Ala His Ala 260 265 270 Arg Gly Arg Val Gly Gly Leu Gly Ala Gln Gly Pro Ser Gly Ser Ser 275 280 285 Glu Trp Glu Asp Glu Gln Ser Glu Tyr Ser Asp Ile Arg Arg 290 295 300 <210> 14 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 14 GTCCCTGCAG AATACAGCGG <210> 15 < 211> 54 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 15 GGCTCGAGCT ACTTGTCATC GTCGTCCTTG TAATCCCTCC GGATATCAGA ATAC <210> 16 <211> 15 <212> PRT <213> Human <400> 16 Thr Asp Ala Thr Asn Pro Pro Glu Gly Pro Gln Asp Arg Ser Ser 1 5 10 15 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 17 GGGGGAAGCT TGCGGCGAAG GAGGAAGAGG <210> 18 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 18 GGGTCGACCT ACTTGTCATC GTCGTCCTTG TAATCCTCGA GCCTCCGGAT ATCAGAATAC <210> 19 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 19 GGCTCGAGGG CATGGGCGTT TGGGTGG <210> 20 <211> 27 < 212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 20 GGCTCGAGTG CTTCCCCGTG GTGGGCC <210> 21 <211> 27 <212> DNA <213> Artificial Sequence <220> <223 > Description of Artificial Sequence: Synthesized DNA <400> 21 GGCTCGAGGG TGCAGAAGCT CAAGATG <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Description of Arti ficial Sequence: Synthesized DNA <400> 22 GGCTCGAGCC CACAGGTCAG CAGCAAG <210> 23 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthesized DNA <400> 23 GGGGGCTCGA GGTTCTCCTG GGCAGCCGCG CACAG

[Brief description of the drawings]

FIG. 1 shows Superose in Example 3-vii).
The 12-fraction roughly purified product (Sample 7) was
4 shows an elution profile when loaded on a W column.

FIG. 2 Heparin in Example 3-viii)
5 shows an elution profile when a 5PW roughly purified product (Sample 8) was loaded on a Polycat A column.

FIG. 3 shows the polycat A elution fraction No. in Example 3-ix). 43 shows an elution profile obtained by fractionation of No. 43 by a reverse phase column.

FIG. 4 ADI of peak 5 eluted from a reversed-phase column
Shows F activity.

FIG. 5 shows the results of SDS-PAGE of a final purified sample under reducing and non-reducing conditions.

[Explanation of symbols]

Lane 1: Molecular weight marker (SDS-PAGE: under non-reducing conditions) Lane 2: ADIF (SDS-PAGE: under non-reducing conditions) Lane 3: Molecular weight marker (SDS-PAGE: under reducing conditions) Lane 4: ADIF (SDS- PAGE: under reducing conditions)

FIG. 6 shows the results of SDS-PAGE after N-glycanase treatment of the final purified sample.

[Explanation of symbols]

Lane 1: molecular weight marker Lane 2: untreated ADIF Lane 3: ADIF from which N-linked sugar chains have been removed by N-glycanase treatment

FIG. 7 shows the activity of ADIF analogs to suppress adipocyte differentiation (AD
IF activity).

 ────────────────────────────────────────────────── ─── Continued on the front page (72) Inventor Koji Yamaguchi 702-12 Shimacho, Omiya-shi, Saitama 1-524 Lions Garden Higashi-Omiya F-term (reference) 4B024 AA01 AA11 BA80 CA04 CA07 CA09 CA20 DA03 DA06 EA03 GA11 GA18 GA19 GA25 GA27 HA03 HA04 HA09 HA13 HA14 4B064 AG01 CA10 CA19 CC01 CC24 CE03 CE06 CE13 DA01 DA13 4C084 AA06 AA07 BA01 BA22 CA53 DB61 ZA702 4H045 AA10 AA20 BA41 BA53 CA40 EA20 EA50 FA74 GA10 GA26

Claims (9)

[Claims] 1. A protein comprising at least the 25th to 302nd amino acids in the amino acid sequence described in SEQ ID NO: 13 in the sequence listing. 2. A DNA encoding a protein comprising at least the 25th to 302nd amino acids in the amino acid sequence of SEQ ID NO: 13 in the sequence listing. 3. A DNA represented by the nucleotide sequence of SEQ ID NO: 12 in the Sequence Listing. 4. A protein obtained by expressing the DNA according to claim 2 or 3 by genetic engineering. 5. An amino acid sequence represented by SEQ ID NO: 13 in the Sequence Listing or an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence.
The protein according to claim 4. 6. It has an activity of inhibiting adipocyte differentiation and / or maturation obtained by expressing a DNA encoding an amino acid sequence having 80% or more homology with the amino acid sequence described in SEQ ID NO: 13 in the sequence listing. protein. 7. Use of the DNA according to claim 2 or 3,
A method for producing a protein having the following properties by genetic engineering. (a) activity: having an activity of suppressing the differentiation and / or maturation of adipocytes. (b) Affinity: has affinity for heparin. (c) It contains a sugar chain. 8. A protein comprising at least the 25th to 182nd amino acid sequence in the amino acid sequence set forth in SEQ ID NO: 13 in the Sequence Listing. 9. A DNA encoding a protein comprising at least the 25th to 182nd amino acid sequence in the amino acid sequence set forth in SEQ ID NO: 13 in the sequence listing.