JPH07102151B2 - Novel polypeptide production method - Google Patents

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a novel polypeptide, particularly a specific stimulatory factor, namely a colony stimulating factor (hereinafter abbreviated as "CSF"), which is required mainly for colony formation of human granulocyte cells. ) A method for producing an active polypeptide.

[0002]

2. Description of the Related Art In a two-layer soft agar culture method, when bone marrow cells as target cells are placed in the upper layer and kidney cells or fetal cells are placed in the lower layer, a part of the upper layer cells proliferate and differentiate, resulting in neutrophil lineage. It has been known that a factor that promotes colony formation is present in the living body because colonies composed of granulocytes (hereinafter referred to as “granulocyte”) and monocyte macrophages are formed (Pluznik and Sac).
h; Cell. Comp. Physiol, 66 , 319 (1965), Bradley and Metcal.
f; Aust. J. Exp. Biol. Med. Sc
i. 44 , 287 (1966)).

It is known that this factor, which is collectively referred to as "CSF", is produced by cells that are normally and widely distributed in the body, such as T cells, monocyte macrophages, fibroblasts and endothelial cells. CSF-granulocyte-monocyte that acts on stem cells of granulocyte / monocyte macrophage, stimulates its proliferation and induces differentiation to form colonies composed of granulocytes and monocyte macrophages in soft agar Globular macrophage CSF (abbreviated as GM-CSF), monocyte macrophage CSF (abbreviated as M-CSF) mainly having an action of forming colonies of monocyte macrophages, and pluripotency acting on more undifferentiated pluripotent stem cells Sex CS
There are subclasses such as F (abbreviated as multi-CSF) or, as in the present invention, granulocyte CSF (abbreviated as G-CSF) mainly having an action of forming granulocyte colonies, and are targeted by each subclass. It has come to be considered that the differentiation stages of cells are also different [Asano; Metabolism-Metabolism and
Disease, 22 : 249 (1985), Yun.
is et al .; “Growth and Texture”
n Factors "edited by Gurof
f, John Wiley & Sons, NY, Volume 1 , 2
09 (1983)].

Therefore, it is extremely important to purify individual subclasses and examine their chemical and biological properties in more detail in order to analyze the hematopoietic mechanism and the pathological conditions of various hematological diseases. Among them, as the biological action of G-CSF, the induction of differentiation of myeloid leukemia cells and the enhancement of the function of mature granulocytes have been attracting attention, and in particular, G-CSF for the treatment and prevention of leukemia.
The clinical utility of CSF was highly expected.

[0005]

An attempt that has hitherto been made for isolating and purifying G-CSF is a method of isolating G-CSF from the culture supernatant using a cell culture method.
Due to the difficulty that G-CSF is produced only at a low concentration, and that a complicated purification process is required to obtain a minute amount of G-CSF from a large amount of culture solution, a large amount of uniform G-CS is still available.
I didn't get an F. Therefore, recombinant DNA
There has been a long-felt desire to mass-produce G-CSF using technology.

[0006]

The present invention uses a DNA comprising a nucleotide sequence encoding a polypeptide containing the amino acid sequence described in claim 1 to produce a polymorph having human granulocyte colony stimulating factor activity by recombinant DNA technology. A method for producing a peptide is provided. A particularly important constituent element for the present invention is a gene encoding a polypeptide having human G-CSF activity, which is specifically obtained as a 15 to 17S fraction by sucrose density gradient centrifugation.
DNA complementary to messenger RNA (mRNA) encoding a polypeptide having human G-CSF activity
(CDNA), more specifically, having a gene encoding the polypeptide of SEQ ID NO: 5 or 6, or a part thereof, and more specifically, from the 5′-end of the base sequence of SEQ ID NO: 32 to Sequence from ATG at 34 nucleotides to CCC at 650-652 nucleotides, 1
22nd to 124th ACC to 650th to 652nd CCC
Up to the sequence described in SEQ ID NO: 4 or a part thereof.

The gene of the present invention has a double-stranded gene prepared by a known method after preparing mRNA encoding G-CSF from mammalian cells or the like having the ability to produce a polypeptide having G-CSF activity. Obtained by converting to cDNA.

In the present invention, the above-mentioned mammalian cell which is a source of mRNA is a human oropharyngeal carcinoma-derived cell line CH.
U-2 (Collection Nationale
DeCultures De Microorgani
Smes (CNM) deposit number I-483), but not limited to tumor cell lines, cells that can be isolated from mammals or other established cell lines may be used.

[0009] In addition, the preparation of mRNA has already been performed when cloning genes of several other physiologically active proteins, for example, treatment with a surfactant or phenol in the presence of a ribonuclease inhibitor such as vanadium complex. Do (Berger and Birkenmeier
Biochemistry 18 : 5143 (19)
79)) or after treatment with guanidine thiocyanate,
Perform CsCl density gradient centrifugation (Chirgwin et al; B
Iochemistry 18 : 5294 (1979)
After obtaining total RNA, the oligo (d
T) -Cellulose or poly (A ⁺ ) RNA (mRNA) by an affinity-column method or a batch method using poly U-sepharose with Sepharose 2B as a carrier
Can be obtained. Further, the poly (A ⁺ ) RNA can be further fractionated by a sucrose density gradient centrifugation method or the like.

The mRNA obtained as described above is G-
In order to confirm that it encodes a polypeptide having CSF activity, a method such as translating mRNA into a protein and examining the physiological activity or identifying the protein using an anti-G-CSF antibody is used. Just go. For example, Xenopus laevi
Injecting mRNA into the oocyte of (s) for translation (G
Urdon et al .; Nature, 233 : 177 (19).
72)), or rabbit reticulocytes (Reti
cultivated system and wheat germ (Wheat ger)
m) system is used for translation reaction (Schle
if and Wensink; “Practical Met
hods in Molecular Biolog
y ", Springer-Verlag, NY, (19
81)).

The assay of G-CSF activity can be carried out by applying the soft agar culture method using bone marrow cells. There is a review of these methods (Metcalf; “Hemopoi”).
etic Colonies ", Springer-V
erlag, Berlin, Heidiberg, N
Y (1977)).

A single-stranded cDNA is synthesized using the mRNA obtained by the above-mentioned method as a template, and then a double-stranded cDNA is synthesized from this single-stranded cDNA to prepare a recombinant plasmid with an appropriate vector DNA. . With this, E. coli (Esch
E. coli) and the like are transformed to obtain a DNA group (cDNA library) of the transformed strain.

To obtain a double-stranded cDNA from mRNA,
For example, oligo (dT) complementary to the poly A-chain at the 3'-end of mRNA is used as a primer for treatment with reverse transcriptase, or an oligonucleotide corresponding to a part of the amino acid sequence of G-CSF protein is synthesized. Then, this is used as a primer to treat with reverse transcriptase to synthesize cDNA complementary to mRNA. The double-stranded cDNA was obtained by decomposing and removing the mRNA by alkaline treatment and then obtaining the single-stranded cDNA.
A is reverse transcriptase or DNA polymerase I (eg Kl
or a direct RNase H and DNA polymerase (eg E. coli DNA polymerase I).
Etc.) and the like (eg, Maniatis et al .; Molecular cl)
oning, Cold Spring Harbor L
Laboratory (1982) and Gubler and Hoffman; Gene 25 : 263 (1983).
See).

The double-stranded cDNA thus obtained is used as an appropriate vector, for example, pSC101, pDF41,
ColE1, pMB9, pBR322, pBR327,
EK-type plasmid vectors typified by pACYC1 and λgt. After being incorporated into a phage vector typified by λc, λgt10, λgtWES, etc., Escherichia coli (X1776; HB101; DH1, C600 strain, etc.) can be transformed to obtain a cDNA library (see, for example, “ Molecular clone
ing ”).

In order to ligate the double-stranded cDNA with the vector, an appropriate chemically synthesized DNA fragment is added to give a ligable end to the DNA terminus, and vector DNA and ATP which are cleaved with a restriction enzyme in advance are present. It can be carried out by treating with T4 phage DNA ligase below. Alternatively, after adding dG, dC-chain (or dA, dT-chain) to each of the vector DNA and the double-stranded cDNA previously cleaved with a restriction enzyme, for example, both D
It can also be carried out by gradually cooling the solution containing NA (see Molecular cloning above).

Transformation of a host cell with the thus-obtained recombinant DNA is carried out, for example, when the host cell is E. coli.
anahan described in detail (J. Mo.
l. Biol. 166 , 557 (1983)), that is, by adding the recombinant DNA to a competent cell prepared in the coexistence of CaCl ₂ , MgCl ₂ or RbCl.

To search for cells carrying the gene of interest, the plus-minus method used in the cloning of interferon cDNA (Taniguchi et al .; P
roc. Jpn. Acad. Volume 55 Ser. B, 464
Page (1979)) and hybridization-translation assay (Nagata et al .; Nature).
e 284 , 316 (1980)), or a colony or plaque hybridization method using an oligonucleotide probe chemically synthesized based on the amino acid sequence of the protein (Wallace et al .; Nuc).
leic Acids Res. Volume 9 , Pages 879 (198
1)) or the like may be used.

Human G-cloned in this way
The fragment containing the gene encoding the polypeptide having CSF activity can be reintegrated into an appropriate vector DNA to transform other prokaryotic or eukaryotic host cells. Furthermore, it is possible to express the gene in each host cell by introducing an appropriate promoter and a sequence for expression in these vectors.

Examples of prokaryotic host cells include Esc
herichia coli, Bacillus su
btilis, Bacillus thermophi
lus and the like. To express a gene of interest in these host cells, the host cells may be transformed with a replicon derived from a species compatible with the host, that is, a plasmid vector containing an origin of replication and regulatory sequences. Further, the vector preferably has a sequence capable of imparting phenotypic (phenotypic) selectivity to the transformed cell.

For example, E. E. coli can be transformed with pBR322, which is a vector using it as a host (see Boliver et al .; Gene 2:95 (1975)). pBR322 contains genes for ampicillin and tetracycline resistance, and transformed cells can be identified by utilizing either resistance. The promoter required for gene expression in a prokaryotic host includes a β-lactamase gene promoter (Chang et al .; Nature 275 : 615).
Page (1978)), lactose promoter (Goe
ddel et al .; Nature 281 , 544 (197).
See 9). ) And tryptophan promoter (G
oeddel et al .; Nucleic Acid Re
s. 8 ), page 4057 (1980)) and the like, and any promoter can be used for production of the polypeptide having human G-CSF activity of the present invention.

In order to obtain a polypeptide having human G-CSF activity using the host-vector system as described above, recombinant D having the gene integrated at an appropriate site of the above vector.
After transforming a host cell with NA form, the obtained transformant may be cultured. Further, in order to separate and purify the polypeptide from the inside of the cell or from the culture medium, known means can be used.

Generally, eukaryotic genes have polymorphism (po), as is known for human interferon genes and the like.
lymorphysm) (eg Ni
shi et al .; Biochem. Volume 97 Pages 153 (19
85)), one or more amino acids may be replaced by this polymorphism, or the amino acids may not be changed at all even if the base sequence is changed.

Also, a polypeptide lacking or added one or more amino acids in the amino acid sequence of SEQ ID NO: 5 or 6, or one or more amino acids in one or more amino acids Even the substituted polypeptide may have G-CSF activity. For example, a polypeptide obtained by converting a base sequence corresponding to cysteine of human interleukin 2 (IL-2) gene into a base sequence corresponding to serine is interleukin 2
It is already known to retain activity (Wan
g et al., Science, 224 , 1431 (198).
4)). Therefore, all genes encoding such naturally occurring or artificially synthesized polypeptides are included in the present invention as long as they have human G-CSF activity.

A method for obtaining a polypeptide having human-G-CSF activity of the present invention, a recombinant vector having a gene encoding the same, a transformant having the same, and an expression human-G-CSF composition. A brief description will be given below.

(1) Preparation of probe The amino acid sequence of the homogenous human CSF protein obtained by purifying from the culture supernatant of the tumor cell line CHU-2 was determined from the N-terminal, and further fragmented by bromocyan degradation, trypsin treatment, etc. After that, the amino acid sequence of the fragment was also determined [Example 3 (i), (ii), (iii)].
Two kinds of nucleotide probes (A) and probes (IWQ) corresponding to the sequences shown in SEQ ID NOS: 1 and 2 were synthesized from the amino acid sequence (Example 4).

The probe (A) is a mixed probe consisting of 14 consecutive nucleotides. Probe (IW
Q) is as used in the cloning of the human cholecystokinin gene (Takahashi et al .; Proc. Na.
tl. Acad. Sci. , USA, 82 : 1931 (1985)) 30 consecutive nucleotides using deoxyinosine.

The chemical synthesis of nucleotides can be carried out by applying the improved phosphotriester method to the solid phase method.
described in the Lang review (Tetrahedr
39, pp. 3-22 (1983)). The probe used may be based on the amino acid sequence at a position other than the probe used in the present invention.

(2) Construction of cDNA library CHU-2 cells were homogenized by adding a guanidine thiocyanate solution, and total R was analyzed by CsCl density gradient centrifugation.
Get NA. After selecting poly (A ⁺ ) RNA from this total RNA with an oligo (dT) cellulose column, single-stranded cDNA was synthesized with reverse transcriptase, and RNase H and E. Double-stranded cDNA was obtained using E. coli DNA polymerase I. PBR in which dC chain was added to the obtained double-stranded cDNA and dG chain was added to the PstI cleavage site
Escherichia coli X1776 strain was transformed by ligating with 322 vector to construct pBR322 cDNA library (Examples 5 and 6). Similarly, the double-stranded cDNA was ligated to the λgt10 vector using the EcoRI linker to construct a λ phage cDNA library (Example 7).

(3) Screening The recombinant derived from the pBR322 cDNA library was immobilized on Whatman 541 filter paper, and colony hybridization was carried out using a probe (IWQ) radiolabeled with ³² P. A clone could be selected. This clone was cloned using the Southern blotting method (Southe
rn; J. Mol. Biol. Volume 98 Pages 503 (197
When examined in more detail using 5)), it also hybridized with the probe (A). The nucleotide sequence of this clone was determined by the dideoxy method (Sanger; Science 21).
4 , 1205 (1981)).

The nucleotide sequence of the obtained cDNA insert is shown in SEQ ID NO: 3. As shown in SEQ ID NO: 3, this c
The DNA insert consists of 308 base pairs containing the probe (IWQ) and the probe (A), and has an open reading frame encoding 83 amino acids containing the amino acid sequence shown in Example 3 (iii). all right. The plasmid derived from pBR322 containing this 308 base pair is abbreviated as pHCS-1 (Example 8).

A DNA fragment containing 308 base pairs obtained from pHCS-1 was nick-translated (see above,
Radiolabeling with Molecular Cloning), and using this as a probe, λgt10-derived cDNA
Plaque hybridization (B
enton and Davis; Science 196 vol 18
Five clones were obtained by screening on page 0 (1977), and the nucleotide sequence of the clone suspected of containing cDNA was determined by the same method as described above (SEQ ID NO: 4). As shown in SEQ ID NO: 4, this cDNA insert has one large open reading frame. The amino acid sequence encoded by this cDNA can be deduced as shown in SEQ ID NO: 4.

G-CSF shown in Example 3 (i)
By comparison with the N-terminal amino acid sequence of the protein, this cDNA
NA is AT at a position of 32 to 34 nucleotides from the 5'-end
A signal peptide encoded by 90 base pairs beginning with the G sequence and ending with the GCC sequence at positions 119 to 121 and 650 beginning with the ACC sequence at positions 122 to 124
It was found to contain a base sequence corresponding to the mature G-CSF polypeptide encoded by a 531 base pair ending in the CCC sequence at position -652. Therefore, SEQ ID NO: 5
The polypeptide having the amino acid sequence shown in 1) consists of 207 amino acids, and its molecular weight was calculated to be 22292.67 Daltons. Similarly, the polypeptide having the amino acid sequence shown in SEQ ID NO: 6 was 18986.74 Dalton (Example 9).

However, regarding the start site of the protein, 3
ATGs at positions 2-34 or 68-70 can be considered in the same manner. This cDNA (+ VSE) at the EcoR1 cleavage site
The E. coli strain X1776 carrying pBR322 with the inserted gene has been deposited at the Institute for Microbial Technology, Institute of Industrial Science (FERM BP-95).
4). The restriction enzyme cleavage site of the obtained gene is shown in FIG.

(4) Construction of Recombinant Vector A cDNA fragment of the G-CSF polypeptide was excised from the thus obtained pBRV2 plasmid (Example 9) with a restriction enzyme, and pKK223-3 containing the cDNA fragment and the tac promoter was excised. linking fragment and annealed synthetic linker was prepared from (Pharmacia) (ligation) and three prepared from pPL-lambda (manufactured by Pharmacia) containing one (example 10) P _L promoter to construct a recombinant vector The recombinant vector is constructed by ligating the fragment of Example 1 to the annealed synthetic linker and re-conditioning to construct a recombinant vector (Example 11), or by ligating the fragment prepared from the trP promoter-containing pOYI plasmid to the annealed synthetic linker. Construct (Example 12).

(5) Preparation, culture and expression of transformants. Next, using the above three types of recombinant vectors, Mole described above is used.
by the calcium chloride method or the rubidium chloride method described in Clarular Cloning, respectively. coli
DH1 strain, E. coli N4830 strain or E. coli. co
The liJM105 strain was transformed (Examples 10, 11,
12). The obtained transformant is first cultured in an ampicillin-containing Luria medium, and then, if necessary,
Induction was appropriately performed, and culture was performed to express the trait (Example 13).

(6) Recovery and purification of G-CSF polypeptide from Escherichia coli and amino acid analysis The culture solution of the transformant is centrifuged to collect the cells, which is then lysozyme-treated, and freeze-thaw is repeatedly lysed. Then, after treatment with guanidine hydrochloride, centrifugation is performed to obtain a supernatant. Ul this
Gel filtration was performed using a trogel ACA54 column (manufactured by LKB), and the active fraction was concentrated using an ultrafilter.

Next, a trifluoroacetic acid aqueous solution containing n-propanol was added, and the mixture was allowed to stand in ice and centrifuged to give a reverse phase C1.
Adsorption and elution operations are performed on 8 columns. After elution, the activity of each fraction was examined, and the activity peaks were collected and freeze-dried. The lyophilized powder was dissolved, subjected to high performance liquid chromatography, and again subjected to the same purification operation as above to obtain the obtained polypeptide in SD.
A single band indicating the desired G-CSF polypeptide was confirmed by S-polyacrylamide gel electrophoresis (Example 14). The polypeptide thus obtained showed human G-CSF activity (Example 15). Furthermore, in the amino acid analysis of the obtained G-CSF polypeptide, the amino acid composition was analyzed by a special amino acid analysis method using a Hitachi 835 amino acid automatic analyzer (manufactured by Hitachi, Ltd.).
Further, N-terminal amino acid analysis is carried out by Edman degradation using a gas phase sequencer, high performance liquid chromatography and
It was performed using an Ultrasphere-ODS column (Example 16).

[0038]

EXAMPLES The present invention will be described in detail below with reference to examples, but before that, a method for measuring CSF activity will be described with reference to examples.

<Reference Example> Method for measuring CSF activity The method for measuring CSF activity (hereinafter abbreviated as CSA) used in the present invention is as follows. "Method for measuring CSA" (a) When using human bone marrow cells: Bradley
T. R. Metcalf D.M. Etc. (Aust.
J. Exp. Biol. Med. Sci. Volume 44 287
Pp. 300, 1966) according to the monolayer soft agar culture method. That is, 0.2 ml of fetal bovine serum, 0.1 ml of test sample, 0.1 ml of human bone marrow non-adherent cell suspension (1 to 1
2 × 10 ⁵ nucleated cells), modified McCoy's 5A culture solution 0.2 ml, modified McCoy's ⁵ containing 0.75% agar
After mixing 0.4 ml of A culture solution and putting it in a tissue culture plastic dish with a diameter of 35 mm to solidify, 37
Culturing was carried out under the conditions of ℃, 5% carbon dioxide gas / 95% air, 100% humidity, and the number of colonies formed after 10 days (a colony consisting of 50 or more cells is regarded as 1 colony) was counted,
CSA was determined with the activity of forming one colony as one unit (Unit).

(B) When using mouse bone marrow cells: horse serum 0.4 ml, test sample 0.1 ml, C3H / He (female) mouse bone marrow cell suspension 0.1 ml (0.5 to 1 × 1)
0 ⁵ nucleated cells), modified McCo containing agar 0.75%
After mixing 0.4 ml of y's5A culture solution and putting it in a plastic dish for tissue culture with a diameter of 35 mm to solidify, it is cultured for 5 days at 37 ° C., 5% carbon dioxide / 95% air, 100% humidity. The number of colonies formed (5
A colony consisting of 0 or more cells is counted, and the activity of forming one colony is counted as 1 unit (Uni
CSA was determined as t).

The "modified McCoy's 5A culture medium used in the above methods (a) and (b) and the human bone marrow non-adherent cell suspension used in (a) were prepared as follows. Modified McCoy's 5A culture solution (double concentration) "M
cCoy's 5A culture solution (GIBCO) 12 g, M
EM amino acid vitamin medium (Nissui Pharmaceutical Co., Ltd.) 2.55
g, sodium bicarbonate 2.18 g, and penicillin G potassium 50000 units twice dissolved in distilled water 500 ml, and then dissolved.
Filter sterilization was performed with a 22 μm Millipore filter.

"Human bone marrow non-adherent cell suspension" A bone marrow fluid obtained by puncture of the sternum of a healthy person was diluted 5 times with RPMI 1640 culture solution, and overlaid with Ficol-Paque solution (Pharmacia) to prepare 400 layers. The cell layer at the interface (specific gravity <1.077) is collected by centrifugation at 25 ° C for 30 minutes at xg. After washing the cells, the cells were washed with RPMI 1640 containing 20% fetal calf serum at 5 × 10 ⁶ cells / ml.
After adjusting the concentration to 25 cm ^{2 in} a tissue culture plastic flask and incubating in a carbon dioxide incubator for 30 minutes, the non-adherent cells in the supernatant are recovered and
After the cells were placed in a cm ² plastic flask and incubated for 2 hours and 30 minutes, the supernatant non-adherent cells were collected and used.

Example 1 Establishment of "CHU-2" Tumors of a cancer of the floor of the mouth with a marked increase in neutrophils were transplanted into nu / nu mice. About 10 days after transplantation, a marked increase in tumor and an increase in neutrophils were observed in this tumor. The tumor was aseptically excised 12 days after the transplantation, cut into 1-2 mm ³ squares, and cultured as follows. 10 to 15 pieces of the above-mentioned minced tumor mass were put into a 50 ml plastic centrifuge tube, and 5 ml of trypsin solution (trypsin 0.25%, E
DTA (containing 0.02%) was added, and 1
After shaking for 0 minutes, the supernatant was discarded, 5 ml of the same trypsin solution was added again, and trypsin digestion was carried out at 37 ° C. for 15 minutes while stirring. The supernatant cell suspension was collected, 1 ml of fetal bovine serum was added to stop the action of trypsin, and the cells were stored in ice.

The above operation is repeated to collect the cell suspension, and the total amount is 1,500 r. p. m. Cell pellets were obtained by centrifugation for 10 minutes. This cell pellet was washed twice with F-10 containing 10% fetal bovine serum and then seeded in a 25 cm ² plastic culture flask at a cell concentration of 5 × 10 ⁶ cells / flask.
Using F-10 culture medium containing 10% fetal bovine serum,
Carbon dioxide incubator (carbon dioxide concentration 5%, humidity 1
(00%) overnight, the supernatant was removed together with the non-adherent cells, and a new culture medium was added to continue the culture.

On the 6th day after the start of the culture, the cells had grown to the full extent, so the culture medium was replaced with a new one at this point. The next day, the culture solution was discarded, 2 ml of anti-mouse erythrocyte antibody (manufactured by Cappel) diluted 5 times with RPMI 1640 and 2 ml of guinea pig complement (manufactured by Far East Pharmaceutical Co., Ltd.) diluted 2.5 times with RPMI 1640 were added, and the mixture was added at 37 ° C. Incubated for 20 minutes. After the completion of the incubation, the cells were washed twice with F-10 containing 10% fetal bovine serum to remove the fibroblasts derived from nu / nu mice, and then F-10 culture medium containing 10% fetal bovine serum was added for another 2 days. After culturing, a part of the cells was taken out and cloned by the limiting dilution method. The CSF activity of the 11 clones obtained was examined and found to be about 10
A clone (CHU-2) showing double the activity was obtained.

Example 2 Isolation of CSF The cells established as described above grew completely densely 1
The cells were collected from two 50 cm ² culture flasks, and 500 ml of F-10 culture solution containing 10% fetal calf serum.
After being suspended in a glass roller bottle of 1580 cm ² (manufactured by Belco), 0.5 r.p.m. p. m. The rotation culture was performed at the speed of. At the time when the cells are completely and densely grown on the inner wall of the roller bottle, the culture medium is serum-free R
After changing to PMI1640 and culturing for 4 days, the culture supernatant is recovered, and F-10 containing 10% fetal bovine serum is added to continue the culturing.

Serum-free RP again after 3 days of culture
The liquid was changed to MI 1640, and the culture supernatant was collected 4 days later. By repeating the same procedure below, 500 ml of serum-free culture supernatant can be obtained from 1 bottle each week, and this method can maintain cells and recover the culture supernatant for a considerably long period of time. there were. 5 l of the obtained culture supernatant was used as 1 batch, and 0.01%
After adding Tween 20, Hollow FiberDC-
4 and Amicon PM-10 (manufactured by Amicon), after being concentrated about 1000 times by an ultrafiltration method,
It was purified in the following order.

(I) Ul having a diameter of 4.6 cm and a length of 90 cm
0.15 M NaCl and 0.01% Tween 20 using a trogel AcA 54 column (manufactured by LKB).
5 ml of the above concentrated culture supernatant was subjected to gel filtration at a flow rate of about 50 ml / hour using 0.01 M Tris-HCl buffer (pH 7.4) containing (Hanui Chemical Co., Ltd.). The column was previously calibrated with bovine serum albumin (molecular weight 67,000), ovalbumin (molecular weight 45,000), and cytochrome C (molecular weight 12,400).
After completion of gel filtration, 0.1 ml was collected from each fraction and diluted 10-fold, and the active fraction was examined by the above-mentioned "CSA measurement method (b)". As a result, first, it was found that the fraction of Ve = 400 to 700 ml showed CSA in which macrophages were dominant, and the fraction of Ve = 800 to 1200 ml showed CSA in which granulocytes were dominant. Therefore, the latter fraction was collected and PM- It was concentrated to about 5 ml by an ultrafilter using 10 (manufactured by Amicon).

(Ii) To the above concentrated fraction was added 0.1% trifluoroacetic acid aqueous solution containing 30% of n-propanol (manufactured by Tokyo Kasei Co., Ltd., for amino acid sequence determination), and the mixture was kept in ice for 15 minutes.
After leaving for about 1 minute, 15,000 r.p.m. p. m. The precipitate was removed by centrifugation for 10 minutes. Then, after being adsorbed on a μ Bondapak C 18 column (manufactured by Waters, semi-preparative, 8 mm × 30 cm) equilibrated with an aqueous solution containing the above n-propanol and trifluoroacetic acid, 30 to 60
Elution was carried out sequentially with a 0.1% trifluoroacetic acid aqueous solution containing a linear concentration gradient of n-propanol. The high performance liquid chromatograph is Hitachi 685-50, and the detection is Hitachi 638-4.
220n using a type 1 detector (both manufactured by Hitachi)
The absorption at m and 280 nm was measured simultaneously.

After elution, 10 μl of each fraction was collected 100
After doubling the dilution, active fractions were examined by the above-mentioned "CSA assay method (b)". As a result, activity was observed in the peak eluted at 40% of n-propanol. Therefore, when this peak was collected and re-chromatographed again under the same conditions and CSA was examined in the same manner as above, it was found that n-propanol 40 was also obtained. Since activity was observed in the peak at the position of%, this peak was collected (4 fractions = 4 ml) and lyophilized.

(Iii) A 0.1% trifluoroacetic acid aqueous solution containing 40% of n-propanol as the lyophilized powder 2
It was dissolved in 00 μl and subjected to high performance liquid chromatography (HPLC) using a TSK-G3000SW column (Toyo Soda Co., Ltd., 7.5 mm × 60 cm). Elution was performed with the same aqueous solution at a flow rate of 0.4 ml / min, and 0.4 was obtained with a fraction collector FRAC-100 (Pharmacia).
It was divided into ml. As a result of examining CSA for each of the collected fractions in the same manner as above, activity was observed in the fraction having a retention time of 37 to 38 minutes (equivalent to a molecular weight of about 20,000). Therefore, this fraction was collected. , For analysis μ Bondapak C1
After purification by 8 columns (4.6 mm × 30 cm), the main peak was collected and freeze-dried. The obtained preparation was assayed by the above-mentioned “CSA measurement method (a)” and found to have human G-CSF activity.

Example 3 Determination of amino acid sequence (i) Determination of N-terminal amino acid sequence A sample was subjected to Edman degradation using a gas phase sequencer (manufactured by Applied Biosystems), and the obtained PTH amino acid was used as a high-performance liquid. Chromatography device (Beckman Instruments) and Ultr
The analysis was carried out by an ordinary method using an sphere-ODS column (manufactured by Beckman Instruments). After equilibrating the column (5 μm, diameter 4.6 mm, length 250 mm) with a starting buffer solution (15 mM sodium acetate buffer pH 4.5, aqueous solution containing 40% acetonitrile), the sample (20 μl starting buffer solution) (Lysis) was injected and the separation was performed by isocratic elution with starting buffer. The flow rate was kept at 1.4 ml / min and the column temperature was kept at 40 ° C.

Detection of PTH amino acids was detected at 269 nm and 32
UV absorption at 0 nm was used. Standard PTH in advance
2 nmol of each amino acid (manufactured by Sigma) was separated in the same system to determine the retention time, and identification was carried out from the retention time of the test sample. As a result, the amino acid sequence from the N-terminal to the 21st residue was determined as follows.

H ₂ N-Thr-Pro-Leu-Gly
-Pro-Ala-Ser-Ser-Leu-Pro-
Gln-Ser-Phe-Leu-Leu-Lys-
(Cys) -Leu-Glu-X-Val-

(Ii) Bromcian decomposition A sample was dissolved in 70% formic acid, 200 equivalents of sublimated and purified bromcian was added, and the mixture was reacted at 37 ° C. overnight. Next, the reaction product was freeze-dried and then fractionated by HPLC using a TSK G3000SW column (manufactured by Toyo Soda Co., Ltd.) to obtain four peaks. The peaks are arranged in descending order of molecular weight, CN-1, CN-
2, CN-3, CN-4 with good yield of CN-
The amino acid sequences of 1 and CN-2 were analyzed under the same conditions as (i) using an automatic gas phase sequencer (manufactured by Applied Biosystems). As a result, it was found that CN-1 is a peptide from the N terminus of G-CSF protein. Furthermore, CN-2 had the following amino acid sequence.

Pro-Ala-Phe-Ala-Ser
-Ala-Phe-Gln-Arg-Arg-Ala-
Gly-Gly-Val-Leu-Val-Ala-S
er-His-Leu-Gln-

(Iii) Tryptic digestion The sample was treated with 0.1 M Tris-HCl buffer containing 8 M urea (pH
It was dissolved in 7.4), and 0.1 M Tris-hydrochloric acid buffer solution (pH 7.4) containing 0.1% 2-mercaptoethanol was added to adjust to finally obtain 2 M urea. Then, TPCK-treated trypsin (trade name, manufactured by Sigma) was added so that the sample and the enzyme became 50: 1, and after reacting at 25 ° C for 4 hours, the same amount of TPCK-treated trypsin was further added and again at 25 ° C. The reaction was carried out for 16 hours. After the reaction, the reaction product was subjected to high-speed reverse phase column chromatography using a C8 column (manufactured by Yamamura Chemical Co., Ltd.). Elution was performed using n-propanol containing 0.1% TFA and increasing the n-propanol concentration linearly from 5% to 60%. Among the peaks obtained by measuring the ultraviolet absorption at 280 nm, the main peak was analyzed for amino acid sequence using an automatic gas phase sequencer (manufactured by Applied Biosystems) under the same conditions as in (i). As a result, the main peak was found to be a peptide having the following sequence containing a part of the CN-2 fragment of (ii).

Gln-Leu-Asp-Val-Ala
-Asp-Phe-Ala-Thr-Thr-Ile-
Trp-Gln-Gln-Met-Glu-Glu-L
eu-Gly-Met-Ala-Pro-Ala-Le
u-Gln-Pro-Thr-Gln-Gly-Ala
-Met-Pro-Ala-Phe-Ala-Ser-

Example 4 Preparation of DNA probe (i) Synthesis of probe (IWQ) From the amino acid sequence obtained in Example 3 (iii), Il was selected.
e-Trp-Gln-Gln-Met-Glu-Glu
Based on the sequence of 10 amino acids shown by -Leu-Gly-Met, 30 consecutive nucleotides were obtained (SEQ ID NO: 1). In the sequence of SEQ ID NO: 1, for example, the nucleotide at position 9 from the 5'-end indicates that it is a mixture containing equal amounts of dA and dG. The starting material nucleotides were mainly dimers, and if necessary, mononucleotides were also used. 20 mg of nucleotide resin Ap-d (G) (manufactured by Yamasa Shoyu Co., Ltd.) as a starting material was placed in a column with a glass filter, and the washing was repeated with methylene chloride, and then, with a methylene chloride solution containing 3% trichloroacetic acid,
The 4,4'-dimethoxytrityl group was eliminated and then the column was washed several times with 1 ml of methylene chloride.

After washing with anhydrous pyridine to replace the solvent, nucleotide dimer (DMTr) ApTp (NHR
₃ ) (manufactured by Zeon Corporation; NHR ₃ represents triethylammonium, DMTr represents dimethoxytrityl) and 0.2 ml of pyridine were added, and the column was vacuum dried with a vacuum pump. Then, 20 mg of 2,4,6-trimethylbenzenesulfonyl-3-nitrotriazolide (MSNT, manufactured by Wako Pure Chemical Industries, Ltd.) and 0.2 ml of anhydrous pyridine were added, and then the inside of the column was replaced with nitrogen gas to room temperature. The dimers were condensed with the nucleotide resin by occasional shaking for 45 minutes underneath. After completion of the reaction, the column was washed with pyridine, and then the unreacted OH group was treated with excess 4-acetic anhydride.
After acetylation with a pyridine solution containing dimethylaminopyridine, the column was washed again with pyridine.

Similarly, (DMTr) Ip (NH
R ₃ ), (DMTr) GpGp (NHR ₃ ), (DMT
r) Ip (NHR ₃ ), (DMTr) CpTp (NHR
Equivalent mixture of ₃₎ and _{(DMTr) TpTp (NHR 3)} , and _{(DMTr) ApAp (NHR 3)} (DMTr)
An equal mixture of ApGp (NHR ₃ ), (DMTr) Ap
Gp (NHR ₃ ) and (DMTr) GpGp (NHR ₃ )
An equal mixture of (DMTr) GpAp (NHR ₃ ),
(DMTr) TpGp (NHR ₃ ), (DMTr) Ap
Ap (NHR ₃ ) and (DMTr) GpAp (NHR ₃ ).
An equal mixture of (DMTr) CpAp (NHR ₃ ),
(DMTr) ApAp (NHR ₃ ) and (DMTr) Ap
Equal mixture with Gp (NHR ₃ ), (DMTr) GpC
p (NHR ₃ ), (DMtr) TpGp (NHR ₃ ),
(DMTr) Ip (NHR ₃ ) (DMTr) ApTp
(NHR ₃ ) [(DMTr) Ip (NHR ₃ ) is manufactured by Yamasa Shoyu Co., Ltd., all others are manufactured by Nippon Zeon Co., Ltd.] and condensed by repeating the above operation.

After completion of the reaction in the final step, the resin was washed with pyridine, methylene chloride and ether in this order without acetylation and then dried. The dried resin was mixed with 1 ml of dioxane containing 1 M tetramethylguanidine and 1 M α-picoline aldoxime, 0.5 ml of pyridine, water.
The mixture was suspended in 1.7 ml of a mixed solution of 2 ml, allowed to stand at room temperature overnight, and concentrated under reduced pressure to 100 to 200 μl. After adding a small amount (2 to 3 drops) of pyridine to this concentrated liquid, 2 to 3 ml of concentrated aqueous ammonia was added and the mixture was heated at 55 ° C for 6 hours. Then, ethyl acetate was added for extraction and separation, and the obtained aqueous layer was concentrated under reduced pressure, and then dissolved in a 50 mM triethylammonium acetic acid solution (pH 7.0) to be dissolved in a C-18 column (1.0
It was subjected to column chromatography using (15 cm, manufactured by Waters). Elution was performed with acetonitrile having a linear concentration gradient of 10% to 30% in a 50 mM triethylammonium acetic acid solution (pH 7.0), and the peak fraction eluted at a position where the acetonitrile concentration was around 25% was concentrated under reduced pressure.

80% acetic acid was added to this concentrated liquid, and the mixture was allowed to stand at room temperature for 30 minutes, and then ethyl acetate was added thereto for extraction and separation. The aqueous layer obtained was concentrated under reduced pressure. The concentrate obtained is
C18 column (Senshu Scientific Co., Ltd., SSC-ODS-
Further purification was performed by high performance liquid chromatography using 272,6φ × 200 mm). Elution is 10% in 50 mM triethylammonium acetic acid solution (pH 7.0)
Synthetic DNA was obtained in a yield of 10A ₂₆₀ units or more by using acetonitrile with a linear concentration gradient of -20%. The resulting oligonucleotide is Maxam-G
ilbert method (Meth. Enzym. Vol. 65 , 499)
As a result of examining the nucleotide sequence on page (1980), it was confirmed to have the sequence shown in SEQ ID NO: 1.

(Ii) Synthesis of probe (A) From the amino acid sequences obtained in Example 3 (iii), Me was selected.
14 contiguous nucleotides were obtained based on the sequence of 5 amino acids represented by t-Pro-Ala-Phe-Ala (SEQ ID NO: 2). Synthesis is a probe (IWQ)
Nucleotide resin AP-d (T)
(Yamasa Shoyu Co., Ltd.) (DMTr) CpAp (NH
R ₃ ); (DMTr) GpGp (NHR ₃ ); (DMT
r) CpAp (NHR ₃ ), (DMTr) CpTp (N
HR ₃ ), (DMTr) CpGp (NHR ₃ ) and (DMTr) CpCp (NHR ₃ ), an equal mixture; (D
MTr) ApGp (NHR ₃ ), (DMTr) TpGp
An equal mixture of (NHR ₃ ), (DMTr) GpGp (NHR ₃ ) and (DMTr) CpGp (NHR ₃ );
(DMTr) ApAp (NHR ₃ ); (DMTr) Cp
Ap (NHR ₃ ) and (DMTr) CpGp (NHR ₃ )
An equivalent mixture of (DMTr) Gp (NHR ₃ ) (both manufactured by ZEON CORPORATION) are condensed in this order to give about 10 A ₂₆₀ un.
Synthetic DNA of its was obtained. When the base sequence of the obtained oligonucleotide was examined by the Maxam-Gilbert method, it was confirmed to have the base sequence shown in SEQ ID NO: 2.

Example 5 CHU-2 cell culture and mRN
Purification of A 1) CHU-2 cell culture and cell recovery After the established CHU-2 cells were completely and densely grown in two 150 cm ² culture flasks, they contained 10% fetal bovine serum. After suspending in 500 ml of RPMI 1640 culture solution, it was transferred to a glass roller bottle of 1580 cm ² (manufactured by Belco), and 0.5 r.p.m. p. m. The rotation culture was carried out at the speed of 4 days for 4 days. When the cells were completely and densely grown on the inner wall of the roller bottle, the culture solution was removed from the roller bottle, 100 ml of physiological saline containing 0.02% of EDTA preheated to 37 ° C was added, and the mixture was kept at 37 ° C for 2 hours.
After warming for minutes, the cells were peeled off by pipetting.
The resulting cell suspension was 1500 r.p.m. p. m. A cell pellet is obtained by centrifugation for 10 minutes. The cells were resuspended in 5 ml of saline without EDTA, 1500 r. p. m.
A cell pellet was obtained by centrifugation for 10 minutes (wet weight approximately 0.8
g), the cells thus obtained are frozen and stored at −80 ° C. until RNA extraction operation is performed.

2) Purification of mRNA mRNA from CHU-2 cells obtained as described above
Isolation of "Molecular clone
g "[Maniatis et al., Cold Spring
Harbor, 196 (1982)]. CHU-2 was frozen and stored
The cells (wet weight 3.8 g) were suspended in 20 ml of a 6 M guanidine solution (6 M guanidine thiocyanate, 5 mM sodium citrate (pH 7.0), 0.1 M β-mercaptoethanol, 0.5% sodium sarcosyl sulfate). After mixing well with a Vortex mixer for 2 to 3 minutes, inhalation and discharge were repeated 10 times using a 20 ml syringe equipped with an 18G injection needle.

In a centrifuge tube made of polyallomer that fits the Beckman SW40Ti rotor, 6 ml of 5.7M C
sCl-0.1 M EDTA, (pH 7.5) was added in advance, and about 6 ml of a guanidine solution in which the above-mentioned cells were broken and made viscous so as to fill the tube was overlaid. Four centrifuge tubes prepared in this manner were used for 30,000
0r. p. m. After centrifugation at 20 ° C for 15 hours, the obtained pellet was washed 3 times with a small amount of 70% ethanol. Combine the pellets from each tube with 5
It was dissolved in 50 μl of water and adjusted to a NaCl concentration of 0.2 M, then treated with phenol-chloroform (1: 1) and treated with chloroform, and then 2.5 times volume of ethanol was added to precipitate the total RNA. (About 10.1 mg of total RNA was obtained from 3.8 g of wet cells).

Purification of poly (A ⁺ ) -RNA from total RNA was performed as follows. This method is affinity chromatography which utilizes the fact that mRNA has a poly A chain added to the 3'end. Oligo (dT) -cellulose (Type manufactured by PL Biochemicals, Type)
7), the total RNA was adsorbed using an adsorption buffer (10 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 1 m).
M EDTA, containing 0.1% SDS solution),
After heating at 65 ° C. for 5 minutes, the solution was passed through an oligo (dT) -cellulose column filled with the same solution, and elution was performed with a TE solution (10 mM Tris-hydrochloric acid (pH 7.5),
1 mM EDTA was included). The unadsorbed flow-through solution was passed through the same column again to carry out the same elution operation, and mixed with the first eluate. Using such an operation, 400 μg of poly (A ⁺ ) -RNA was obtained.

The mRNA thus prepared was used for Sch
Leif and Wensink's experimental technical book (Practi
cal Methods in Molecular
Biology, Springer-Verlag, N
ew York, Heiderberg, Berli
n, (1981)) and size fractionation by sucrose density gradient centrifugation. That is, SW40Ti rotor (manufactured by Beckman)
Make a 5% to 25% sucrose density gradient in the working tube. The sucrose solution was 0.1 M NaCl, 10 mM Tris-hydrochloric acid (pH 7.5), 1 mM EDTA, and 0.5% SDS at a ratio of 5% and 25%, respectively, and RNase-free sucrose (Schwarz / Mann). ) Is included.

MRNA prepared by the method as described above
(Poly (A ⁺ ) -RNA) 800 μg to 200 μl-5
It is dissolved in 00 μl of TE solution, heated at 65 ° C. for 5 minutes and then rapidly cooled, and then placed on a sucrose density gradient solution. 300
00r. p. m. Fractions of 0.5 ml each were collected after centrifugation for 20 hours at 260 nm, the absorbance at 260 nm was measured, and the size of the fractionated RNA was determined based on the position of the standard RNA (28S, 18S, 5S ribosomal RNA) which was similarly measured. At the same time, the G-CSF activity of each fraction was examined using an oocyte line of Xenopus laevis.

That is, 1 μg / μl of mRNA of each fraction
After preparing an aqueous solution with a concentration of 10 ng and injecting 50 ng of mRNA into 1 oocyte taken from Xenopus laevis (about 1 year old), 10 oocytes were placed in 1 well of a 96-well microtiter plate. Add 100 μl each
Bath medium (88 mM NaCl, 1 mM KCl,
2.4 mM NaHCO ₃ , 0.82 mM MgS
O ₄ , 0.33 mM Ca (NO ₃ ) ₂ , 0.41 mM
CaCl ₂ , 7.5 mM Tris-hydrochloric acid (pH 7.
6), penicillin 10 mg / l, streptomycin sulfate 10 mg / l) were incubated at room temperature for 48 hours, and then the supernatant was recovered, concentrated and purified to measure G-CSF activity. As a result, G-CSF activity was observed in the 15 to 17S fractions.

Example 6 Synthesis of cDNA (pBR system cD
Construction of NA library) From the poly (A ⁺ )-RNA obtained by the above method, Lan
d etc. [Nucleic Acids Res. ，
9, Vol. 2251 (1981)], based on Gubler
And Hoffman's method [Gene, 25 , 263 (1983)] were added to synthesize cDNA.

1) Synthesis of Single-Stranded cDNA The reagents are put in a 1.5 ml tube manufactured by Eppendorf Co. in the following order. 80 μl of reaction buffer (500 m
M KCl, 50 mM MgCl ₂ , 250 mM Tris-HCl, pH 8.3), 20 μl of 200 mM dithiothreitol, 32 μl of 12.5 mM dNTP (d
12.5 each for ATP, dGTP, dCTP, dTTP
mM), 10 μl of α- ³² P-dCTP (Amersham, PB 10205), ³² μl of oligo (dT).
_12-18 (manufactured by P-L Biochemicals, 50
0 μg / ml), 20 μl of poly (A ⁺ ) -RNA (2.
1 μg / μl) and 206 μl of distilled water in a total of 400 μl of reaction solution are heated at 65 ° C. for 5 minutes and then heated at 42 ° C. for 5 minutes. To this reaction solution, 120 units of reverse transcriptase (Takara Shuzo) was added, and further reacted at 42 ° C. for 2 hours, and then RNase
Inhibitor (Bethesda Research)
Laboratories) 2 μl, 20 μl T
E solution, 16 μl of 100 mM sodium pyrophosphate,
Add 48 units (4 μl) of reverse transcriptase, this time 4
The reaction was allowed to proceed at 6 ° C for 2 hours. 0.5M EDTA 8μl
, 10% SDS (8 μl) was added to stop the reaction, followed by phenol-chloroform treatment and ethanol precipitation (twice) to obtain single-stranded cDNA.

2) Addition of dC-chain to single-stranded cDNA After dissolving the single-stranded cDNA obtained above in 60 μl of distilled water, 60 μl of dC-chain addition buffer (400 mM potassium cacodylate; 50 mM tris- Hydrochloric acid (pH 6.
9); 4 mM dithiothreitol; 1 mM CoC
1 ₂ ; 1 mM dCTP) and heated at 37 ° C. for 5 minutes. Add terminal transferase (2
7 unit / μl, P-L Biochemicals
3 μl) was added and reacted at 37 ° C for 2.5 minutes.
Phenol-chloroform treatment (once) and ethanol precipitation (twice) were carried out and dissolved in 40 μl of a TE solution containing 100 mM NaCl.

3) Synthesis of double-stranded cDNA 4 μl of oligo (dG) was added to 40 μl of the above DNA solution.
_12-18 (200 μg / ml, PL Biochemical
als) and added at 65 ° C. for 5 minutes, then at 42 ° C. for 30 minutes.
After warming for minutes, the reaction was kept at 0 ° C. 80 μl of buffer solution (100 mM Tris-hydrochloric acid, pH 7.
5,20mM MgCl ₂ , 50mM (NH ₄ ) ₂ S
O ₄ , 500 mM KCl), 4 μl of 4 mM dNT
P (dATP, dCTP, dGTP, dTTP 4 each
mM), 60 μl of 1 mM β-NAD and 210
μl of distilled water, 20 μl of E. E. coli DNA polymerase I (Takara Shuzo), 15 μl of E. coli D
NA ligase (Takara Shuzo), 15 μl of E. coli
After adding RNase H (Takara Shuzo Co., Ltd.) and reacting at 12 ° C. for 1 hour, 4 μl of 4 mM dNTP was further added, reacting at 25 ° C. for 1 hour, phenol-chloroform treatment, and ethanol precipitation (once). Thus, about 8 μg of double-stranded cDNA was obtained. This double-stranded cDNA was dissolved in TE solution and subjected to 1.2% agarose gel electrophoresis to obtain about 560 base pairs (bp) to 2 kilo base pairs (Kbp).
Was adsorbed on a portion corresponding to the size of Whatman DE81 (manufactured by Whatman), and was eluted and recovered.
g were recovered.

4) Addition of dC-strand to double-stranded cDNA The double-stranded cDNA obtained as described above was dissolved in 40 μl of TE solution, and 8 μd of the dC-strand addition buffer described in 2) was added.
l was added and heated at 37 ° C. for 2 minutes, then 1 μl of terminal transferase (27 units / μl) was added and reacted at 37 ° C. for 3 minutes. The reaction solution was immediately cooled to 0 ° C., 1 μl of 0.5 M EDTA was added to stop the reaction, followed by phenol-chloroform treatment and ethanol precipitation, and the obtained precipitate was suspended in 10 μl of TE solution.

5) Construction of pBR cDNA library Commercially available oligo (dG) chain-added pBR322 vector (manufactured by Bethesda Research Laboratories, 10 ng / μl)
75 μl of 4 μl and 2 μl of the above dC-chain-added double-stranded cDNA
Annealed in TE solution containing μl 0.1M NaCl. Annealing was performed by heating at 65 ° C. for 5 minutes, then at 40 ° C. for 2 hours, and then leaving it to reach room temperature. On the other hand, Maniatis et al.'S experimental book [Molecu
lar cloning, Cold Spring H
Arbor, p. 249 (1982)] and the like to prepare competent cells from the Escherichia coli X1776 strain, and transform with the annealed plasmid to obtain a transformant (transformant). It was

Example 7 cDNA Synthesis (Construction of λ Phage-Based Library) 1) Synthesis of Single-Stranded cDNA According to the method described in Example 5, 3.8 g of cryopreserved CH
From U-2 cells, 400 μg of poly (A ⁺ ) -RNA was obtained through purification twice using an oligo (dT) cellulose column. TE in which 12 μg of this poly (A ⁺ ) RNA was dissolved
10 μl of the solution was placed in a reaction tube containing 10 μg of actinomycin D (manufactured by Sigma), and then reagents were added in the following order: 20 μl of reverse transcription buffer (250 mM Tris-HCl (pH 8.3), 40 mM) MgCl ₂ , 2
50 mM KCl), 20 μl of 5 mM dNTP (d
ATP, dGTP, dCTP, and dTTP each contained 5 mM), 20 μl of oligo (dT) _12-18 (0.2 μg / m 2)
l P-L Biochemicals), 1 μl
1 M dithiothreitol, 2 μl 30 unit / μ
l Rnasin (Promega Biotech), 10 μl
Reverse transcriptase (10 unit / μl manufactured by Seikagaku Corporation),
1 μl of α- [ ³² p] dATP (10 μCi manufactured by Amersham) and 16 μl of water give a total reaction volume of 100 μl. The reaction solution was kept at 42 ° C. for 2 hours, and then 5 μl of 0.
The reaction was stopped by adding 5 M EDTA and 1 μl of 20% SDS. Phenol-chloroform (100 μl)
Treatment and ethanol precipitation (twice) were performed to obtain about 4 μg of single-stranded cDNA.

2) Synthesis of double-stranded cDNA The cDNA obtained as described above was dissolved in 29 μl of TE solution, and reagents were added in the following order to prepare a reaction solution; 25 μl
l of polymerase buffer (400 mM Hepes (p
H7.6); 16 mM MgCl ₂ ; 63 mM β-mercaptoethanol; 270 mM KCl); 10 μl
5 mM dNTPs; 1.0 μl of 15 mM β-NA
D; 1.0 μl of α- [ ³² p] dATP (10 μCi /
.mu.l); 0.2 .mu.l E. coli DNA ligase (60
unit / μl manufactured by Takara Shuzo); 5.0 μl of E. col
iDNA Polymerase I (New England B)
iolabs, 10 unit / μl); 0.1 μl of RNase H (60 unit / μl manufactured by Takara Shuzo); 2
8.7 μl distilled water.

The reaction solution was incubated at 14 ° C. for 1 hour, then returned to room temperature and further incubated for 1 hour. Then 5 μl 0.5 M EDTA and 1 μl 2
The reaction was stopped by adding 0% SDS, followed by phenol-chloroform treatment and ethanol precipitation. DN obtained
Dissolve A in 20 μl of 0.5 mM EDTA, and add 3 μ
l Klenow buffer (500 mM Tris-HCl (p
H8.0), 50 mM MgCl ₂ ), 3 μl of 5 mM
After preparing a reaction solution by adding dNTP and 4 μl of water,
1 μl of DNA polymerase (Klenow fragment) (Takara Shuzo) was added and incubated at 30 ° C. for 15 minutes.
The reaction solution was diluted by adding 70 μl of TE solution, and further the reaction was stopped by adding 5 μl of 0.5 M EDTA and 1 μl of 20% SDS. The reaction solution was treated with phenol-chloroform and precipitated with ethanol to obtain about 8 μg of double-stranded cDNA.

3) Methylation of double-stranded cDNA 30 μl of aqueous solution of double-stranded cDNA synthesized in 2)
Methylation buffer (500 mM Tris-hydrochloric acid (pH 8.
0), 50 mM EDTA) 40 μl, SAM solution (8
00 μM S-adenosyl-L-methylmethionine (S
AM), 50 mM β-mercaptoethanol) 20 μl
, EcoRI methylase (New England Biolabs, 20)
unit / μl) Add 15 μl to make the total reaction mixture 200 μl
and incubated at 37 ° C. for 2 hours. After phenol treatment and ether treatment, ethanol precipitation was performed to collect DNA.

4) Addition of EcoRI linker About 1.2 μg of the above-mentioned methylated double-stranded DNA was added to a ligase buffer solution (250 mM Tris-hydrochloric acid (pH 7.5), 1).
00 mM MgCl ₂ ) 1.5 μl, pre-phosphorylated EcoRI linker 0.5 μl (10 mer,
Takara Shuzo), 1.5 μl of 10 mM ATP, 100 m
1.5 μl of M dithiothreitol and 2 μl of H ₂ O were added to make the reaction solution 15 μl of T4 DNA ligase (3.
(4 u / μl, manufactured by Takara Shuzo Co., Ltd.) 0.7 μl was added and the reaction was carried out at 4 ° C. overnight, followed by heating at 65 ° C. for 10 minutes to inactivate the ligase.

This reaction solution was further added with 100 mM Tris-hydrochloric acid (pH 7.5), 5 mM MgCl ₂ , 50 mM N ₂ .
aCl, gelatin concentration of 100 μg / ml, total liquid volume is 5
After making up to 0 μl, EcoRI (10un
it / μl) 3.5 μl was added and reacted at 37 ° C. for 2 hours. Then 2.5 μl of 0.5 M EDTA, 20% SD
After adding 0.5 μl of S, phenol-chloroform treatment was performed, and DNA was recovered by ethanol precipitation. After this, unreacted E was obtained by gel filtration of Ultrogel AcA34 (manufactured by LKB) or agarose gel electrophoresis.
coRI linker removed, linker-added double-stranded cDNA
About 0.5 to 0.7 μg of A was recovered.

5) Ligation of double-stranded cDNA and λgt10 vector The above-mentioned linker-added double-stranded cDNA was treated with 2.4 μg of EcoRI in advance by EcoRI to prepare a λgt10 vector (Vector Cloning System), ligase buffer (250).
mM Tris-HCl, 100 mM MgCl ₂ ) 1.4 μl
After adding 6.5 μl of distilled water and treating at 42 ° C. for 15 minutes, 1 μl of 10 mM ATP, 1 μl of 0.1M dithiothreitol and 0.5 μl of T ₄ DNA ligase were added to adjust the total amount to 15 μl. Allowed to react overnight.

6) In vitro packaging About 1/3 of the recombinant DNA obtained in the above 5) is in vitro packaging kit (Promega Biotech).
Were used to obtain phage plaques.

Example 8 pB with probe (IWQ)
Screening of R system library Whatman 541 filter paper was placed on the agar medium on which colonies were grown, and left at 37 ° C. for 2 hours. Below, Taub and Th
Ompson method [Anal. Biochem. 12
6 , page 222 (1982)].
That is, after the colonies were transferred to 541 filter paper, they were transferred to an agar medium containing chloramphenicol (250 μg / μl) and left at 37 ° C. overnight. After taking out the 541 filter paper, it was left at room temperature on the filter paper soaked with 0.5N NaOH solution for 3 minutes, and this was repeated twice. The same procedure is repeated twice using a 0.5 M Tris-hydrochloric acid (pH 8) solution for 3 minutes each, and then 0.05 M Tris-at 4 ° C.
Hydrochloric acid (pH 8) solution for 3 minutes, 1.5 mg / ml lysozyme solution (0.05M Tris-hydrochloric acid (pH 8), containing 25% sucrose) for 10 minutes, and then 1 × SSC at 37 ° C.
(0.15M NaCl and 0.015M sodium citrate) solution for 2 minutes, 200 μg / ml Protease K
30 minutes with 1 × SSC solution containing 1 × SS at room temperature again
The C solution was used for 2 minutes and the 95% ethanol solution was used for 2 minutes twice, and then the 541 filter paper was dried.

The dried 541 filter paper thus obtained was placed at room temperature in the form of phenol: chloroform: isoamyl alcohol (25: 2).
4: 1,100 mM Tris-hydrochloric acid (pH 8.5), 10
The solution was equilibrated with 0 mM NaCl, 10 mM EDTA) and soaked for 30 minutes. Repeat the same operation for 5 × SS
The solution C was used for 3 minutes and 3 times, and then 95% ethanol solution was used for 3 minutes and twice, and then the filter paper was dried.

The probe (IWQ) was prepared by a conventional method (Molecu).
Radiolabeling with ³² P according to Lar cloning), followed by the method of Wallace et al. (Nucle.
icAcids Res. 9 : 879 (1981))
Colony hybridization was performed according to. 6
X NET [0.9 M NaCl, 0.09 M Tris-HCl (pH 7.5), 6 mM EDTA], 5 x Denh
Ardt solution, 0.1% SDS, 0.1 mg / ml modified DN
After prehybridization at 65 ° C. for 4 hours in a hybridization buffer containing A (calf thymus DNA), radiolabeled probe (IWQ) 1 × 1
Hybridization was carried out at 56 ° C. overnight using the hybridization buffer containing 0 ⁶ cpm / ml. After completion of the reaction, filter 541 at room temperature with 6 × SSC solution containing 0.1% SDS for 30 minutes, twice and at 56 ° C. for 1.5 minutes.
After washing for a minute, autoradiography was performed. After separating the plasmid from the clones that gave a signal, Southern blotting was performed using a probe (IWQ). Hybridization and autoradiography were performed under the same conditions as described above.

Similarly, Southern blotting was carried out using the probe (A). Hybridization was carried out using the above-mentioned hybridization buffer at 49 ° C. for 1 hour, gradually cooled to 39 ° C., and further carried out at 39 ° C. for 1 hour. After the reaction is completed, add a nitrocellulose filter to 0.1.
After washing with 6 × SSC containing% SDS twice at room temperature for 30 minutes and then at 39 ° C. for 3 minutes, autoradiography was performed. As a result, one clone was obtained as a positive clone, and when the nucleotide sequence was determined by the dideoxy method, as shown in SEQ ID NO: 3, a DNA consisting of 308 base pairs containing the probe (IWQ) and probe (A) portions. The plasmid derived from pBR322 containing this insert was named pHCS-1.

Example 9 Screening of λ Phage-Based Library with pHCS-1-Derived DNA Probe Method of Benton and Davis [Science 1
96 , page 180, (1977)], and plaque hybridization was performed. P obtained in Example 8
HCS-1 was treated with Sau3A and EcoRI to obtain a DNA fragment of about 600 base pairs, and this DNA fragment was radiolabeled by nick translation according to a conventional method.
Place the nitrocellulose filter paper (S & S Co.) on the agar medium on which the phage plaque was generated, and transfer the phage to 0.5M.
The DNA was denatured with NaOH and the filter paper was treated in the following order. 20 seconds with 0.1 M NaOH, 1.5 M NaCl followed by 0.5 M Tris-HCl (pH 7.5),
20 seconds twice with 1.5M NaCl, and finally 120 mM
NaCl, 15 mM sodium citrate, 13 mM KH
_It was treated with ₂ PO ₄ , 1 mM EDTA, pH 7.2 for 20 seconds.

Then, the filter paper was dried and heated at 80 ° C. for 2 hours to fix the DNA. 5 x SSC, 5 x Denha
Prehybridization overnight at 42 ° C in a hybridization buffer containing rdt solution, 50 mM phosphate buffer, 50% formamide, 0.25 mg / ml denatured DNA (salmon testis DNA), and 0.1% SDS. And radiolabeled p by nick translation.
Hybridization buffer (5 x SSC, 5 x Denhard) containing 4 x 10 ⁵ cpm / ml of HCS-1 probe.
t solution, 20 mM phosphate buffer (pH 6.0), 50%
Hybridization was performed at 42 ° C. for 20 hours with a mixture of formamide, 0.1% SDS, 10% dextran sulfate, and 0.1 mg / ml denatured DNA (salmon testis DNA).

Add nitrocellulose filter paper to 0.1% at room temperature.
Rinse with 2 × SSC with SDS for 20 minutes, then 44
After washing with 0.1 × SSC containing 0.1% SDS for 30 minutes at 0.1 ° C. and further with 0.1 × SSC for 10 minutes at room temperature, detection was carried out by autoradiography. As a result, 5 positive clones (G1-5) were obtained. Then, among the obtained clones, the DNA base sequence of the clone which seems to contain the full-length cDNA was examined by the dideoxy method, and the base sequence as shown in SEQ ID NO: 4 was obtained. Therefore, this cDNA was excised from the λgt10 vector and ligated with pBR327 [Soberon et al .; Gene 9 : 287 (1980)] at the EcoRI site.
It was prepared in large quantities as a plasmid. This plasmid is pB
It is called RG4.

Example 10 [Example using vector containing tac promoter] 1) Construction of recombinant vector Preparation of vector 5 μg of vector pKK223-3 containing tac promoter (Pharmacia) was added to 30 μl of a reaction solution (50 mM).
Tris-HCl, 7 mM MgCl ₂ , 100 mM
NaCl, 7 mM 2-mercaptoethanol)
Medium, 8 units of EcoRI (manufactured by Takara Shuzo) were treated at 37 ° C for 2 hours. Then, 3 μl of alkaline phosphatase (Takara Shuzo) was added and treated at 60 ° C. for 30 minutes, followed by phenol treatment 3 times, ether treatment and ethanol precipitation according to a conventional method to recover a DNA fragment. 50 mM of this DNA
Tris-HCl, 5 mM MgCl ₂ , 10 mMD
TT, 1 mM dATP, dCTP, dGTP, TTP
Was dissolved in a mixed solution of 50 .mu.l, and 3 .mu.l of E. coli DNA polymerase I-Klenow fragment (manufactured by Takara Shuzo) was added and reacted at 14.degree. C. for 2 hours to make the ends bluntend.

Preparation of Synthetic Linker Synthetic Linker, CGAATGACCCCCCTGGGC
3 μg of oligonucleotide having the sequences of C and CAGGGGGGGTCATTCG was added to 50 mM Tris-HC.
40 μl of reaction solution consisting of 1, 10 mM MgCl ₂ , 10 mM 2-mercaptotoethanol, 1 mM ATP
In the presence of 4 units of T ₄ polynucleotide nucleoside kinase,
The mixture was reacted at 60 ° C. for 60 minutes and phosphorylated. Next, 0.2 μg of each of the phosphorylated oligonucleotides was added to 100 mM.
TE containing NaCl (10 mM Tris-HCl,
Dissolve in 20 μl of pH 8.0, 1 mM EDTA,
After treatment at 5 ° C. for 10 minutes, annealing was performed by gradually cooling to room temperature.

Preparation of cDNA fragment of G-CSF p containing the cDNA shown in SEQ ID NO: 4 obtained in Example 9
60 μg of BRG4 was added to 6 mM Tris-HCl, 6 m
In a reaction solution (200 μl) containing M MgCl ₂ and 6 mM 2-mercaptoethanol, the restriction enzyme ApaI (Ne
w England Biolabs) 100 units, DraI (Takara Shuzo) 50 units at 37 ° C. for 3 hours, and 1.2% agarose gel electrophoresis for about 590.
About 2 μg of the ApaI-DraI fragment of bp was recovered.

Ligation of each of the above fragments, about 0.1 μg of each fragment was taken, and 20 μl of the ligation reaction solution (66 mM Tris-HCl, 6.6 mM was used.
It was dissolved in MgCl ₂ , 10 mM DTT, 1 mM ATP), added with 175 units of T ₄ DNA ligase, and reacted at 4 ° C. overnight to obtain a recombinant vector.

2) Transformation 20 μl of reaction solution containing the recombinant vector obtained above
Rubidium chloride method (T. Maniatis, supra)
Etc. "Molecular cloning" P252
(1982)). E. coli JM105 strain was transformed. The obtained transformant was isolated from the ampicillin-resistant colony culture medium, and the plasmid was separated and treated with the restriction enzymes BamHI, AccII, and ApaI, whereby it was confirmed that the transformant was the target transformant.

Example 11 [Example using vector containing PL promoter] 1) Preparation of recombinant vector vector pPL-lambda containing PL promoter
(Pharmacia) 100 μg with BamH restriction enzyme
I, 50 units of reaction solution (10 mM Tris-HCl,
pH 7.6, 7 mM MgCl ₂ , 100 mM NaC
1, 10 mM DTT) in 100 μl at 37 ° C. overnight. From this, in 1% agarose gel electrophoresis,
About 49 μg of about 4 Kbp fragment and about 11 μg of about 1.2 Kbp fragment were recovered.

Of the above fragments, about 4 Kbp was first dissolved in 100 μl of the TE buffer described above, and then dephosphorylated by reacting with 5 μl of alkaline phosphatase (Takara Shuzo) at 60 ° C. for 60 minutes. The remaining fragment of about 1.2 Kbp is buffer (10 mM Tris-HCl, 10 mM
MgCl ₂ , 6 mM KCl, 1 mM DTT) 20μ
1 and dissolved in the restriction enzyme MboII (New Englan
d Biolabs) 20 units at 37 ° C. overnight. Then, a BamHI-MboII fragment of about 200 bp of about 0.9 was obtained by 4% polyacrylamide gel electrophoresis.
μg and about 310 bp MboII-BamHI fragment about 1.
9 μg was recovered.

Preparation of Synthetic Linkers The synthetic linkers TAAGGAGAATTCCATGAT and TCGATGAATTCTCCCTTAG were prepared in Example 1.
Phosphorylated and annealed in the same manner as in 0.
/ D linker was obtained.

Preparation of Expression Vector 0.1 μg of the approximately 4 Kbp fragment prepared above and O _L P
40 μl of 0.1 μg of synthetic S / D linker annealed with 0.05 μg of BamHI-MboII fragment having _L region and MboII-BamHI fragment having tL ₁ region, respectively
Reaction solution (66 mM Tris-HCl, 6.6 mM
MgCl ₂ , 10 mM DTT, 1 mM ATP), T
_{4 In the} presence of 175 units of DNA ligase (Takara Shuzo) 12
The reaction was allowed to proceed overnight at ℃. Using 20 μl of this reaction solution,
E. E. coli N99cI ⁺ strain (manufactured by Pharmacia) was subjected to the CaCl ₂ method (the above-mentioned "Molecular clone").
ing ”)).

The transformant was cultured, the plasmid was separated from the culture solution of the apicillin-resistant colonies, and treated with the restriction enzymes EcoRI, BamHI, and SmaI, and it was confirmed that the plasmid was the desired plasmid. Next, 2 μg of this plasmid was taken and 20 μl of buffer solution (10 m
M Tris-HCl, 6 mM MgCl ₂ , 50 mM
Restriction enzyme ClaI (New Engl) in
and Biolabs) were reacted at 37 ° C. for 2 hours and then deactivated at 65 ° C. for 10 minutes. Furthermore, the reaction solution 1
20 μl of the ligation reaction solution and 175 units of T ₄ DNA ligase (Takara Shuzo) were reacted at 12 ° C. overnight and then E. coli N99cI ⁺
The strain (Pharmacia) was transformed again. The plasmid was isolated from the ampicillin-resistant colony culture medium, and E. C
It was treated with oRI and BamHI, and the target plasmid was confirmed.

Preparation of Recombinant Vector for G-CSF Expression and Transformant The expression plasmid obtained in the above was treated with restriction enzyme ClaI to make the ends blunt-ended, and then G-CSF was prepared in the same manner as in Example 10. A cDNA fragment was incorporated to obtain a recombinant vector. Using this, the above-mentioned Molecular
By the CaCl ₂ method described in Cloning.
E. coli N4830 strain (Pharmacia) was transformed. The target transformant was also confirmed in the same manner as in Example 10.

Example 12 [Example using vector containing trp promoter] 1) Construction of recombinant vector Preparation of vector pOY1 plasmid (10 μg) prepared by inserting an HpaII-TaqI fragment of about 330 bp containing a tryptophan promoter into the ClaI site of pBR322. To 10 mM
Tris-HCl, 6 mM MgCl ₂ , 50 mM N
Restriction enzyme ClaI 7 units P in 30 μl of aCl reaction solution
It was treated with 8 units of vuII for 3 hours at 37 ° C. Then, add 2 μl of alkaline phosphatase (Takara Shuzo) to 60
The reaction was allowed to proceed at ℃ for 1 hour. From this, about 2.5 μg of a fragment of about 2.6 Kbp was recovered by 1% agarose gel electrophoresis.

Preparation of Synthetic Linker Synthetic Linker CGCGAATGACCCCCCTGGG
CC and CAGGGGGGGTCATTCG Example 10
Phosphorylated and annealed in the same manner as above.

Preparation of Recombinant Vector About 1 μg of the above-prepared vector fragment, and about 1 μg of the synthetic linker thereof, and G-CS prepared in Example 10 were used.
Approximately 1 μg of the F cDNA fragment, 20 μl of the above ligation reaction solution
Medium, T ₄ DNA ligase (Takara Shuzo) 175 units and 1
The mixture was reacted overnight at 2 ° C to obtain a recombinant vector.

2) Transformation 20 μl of the above reaction solution was added to the above-mentioned “Molecular”.
by the rubidium chloride method of "cloning". coliD
HI strain was transformed. In the same manner as in Example 10, a plasmid was taken from an ampicillin-resistant colony, and it was confirmed that the target transformant was obtained with the restriction enzymes ApaI, DraI, NruI and PstI.

Example 13 Cultivation of transformant strain 1) Cultivation of transformant strain (containing Tac) obtained in Example 10 100 μl of Luria medium containing 25 μg / ml or 50 μg / ml of ampicillin at 37 ° C. 1 ml of a culture solution of the transformed strain that was cultured overnight was added, and the mixture was cultured at 37 ° C for 2 to 3 hours. Then, isopropyl-β-D-thiogalactoside was adjusted to 2 mM and incubated at 37 ° C. for 2 to 4 hours.

2) Cultivation of the transformant strain (containing P _L ) obtained in Example 11 1 ml of a culture solution of the transformant strain cultured overnight at 28 ° C. in 100 ml of Luria medium containing 25 μg / ml or 50 μg / ml of ampicillin. In addition, the cells were cultured at 28 ° C for about 4 hours. afterwards,
This was brought to 42 ° C. and cultured for 2 to 4 hours.

3) The transformant (trp) obtained in Example 12
Culture) 0.5% glucose, 0.5% casamino acid (manufactured by Difco), ampicillin 25 μg / ml or 5
To 100 ml of M9 medium containing 0 μg / ml, 1 ml of the culture solution of the transformant which has been cultured overnight at 37 ° C. is added, and the mixture is cultured at 37 ° C. for 4 to 6 hours. Next, 50 μg / ml of 3-β-indoleacrylic acid (IAA) was added and the mixture was cultured at 37 ° C. for 4 to 8 hours.

Example 14 Recovery / Purification of G-CSF Polypeptide from Escherichia coli 1) Recovery The transformant strain cultured in Example 13 was subjected to the following recovery operation. 100 ml of the culture solution was centrifuged to collect the cells, and 20 mM Tris-HCl (pH 7.
5), suspended in 5 ml of 30 mM NaCl mixture. Next, 0.2M phenylmethylsulfonylfluoride was added to each of 1 mM, 10 mM and 0.2 μg / ml,
0.2M EDTA and lysozyme were added, and the mixture was left at 0 ° C. for 30 minutes. Next, freeze-thawing was repeated three times for lysis. Then, using 8M guanidine hydrochloride, finally 6M
After making it to guanidine hydrochloride, it centrifuged at 30,000 rpm for 5 hours, and obtained the supernatant liquid.

2) Purification (i) The supernatant obtained in 1) was used to have a diameter of 4.6 cm and a length of 90 cm.
UltrogelAcA54 column (manufactured by LKB) of 0.15M NaCl and 0.01% Tween 2
Gel filtration was performed using 0.01 M Tris-HCl buffer (pH 7.4) containing 0 (manufactured by Hanai Chemical Co., Ltd.) at a flow rate of about 50 ml / hour. Then, a fraction showing activity was collected by the above-mentioned “method for measuring CSA (b)” and pM-10 (manufactured by Amicon)
Concentrated to about 5 ml by ultrafiltration using a.

(Ii) A 0.1% trifluoroacetic acid aqueous solution containing 30% of n-propanol (manufactured by Tokyo Kasei Co., Ltd., for amino acid sequence determination) was added to the above concentrated fraction, and the mixture was placed in ice to 15
After leaving for about 1 minute, 15,000 r.p.m. p. m. The precipitate was removed by centrifugation for 10 minutes. Then, after adsorption to a μ Bondapak C18 column (manufactured by Waters, semi-preparative, 8 mm × 30 cm) equilibrated with an aqueous solution containing the above n-propanol and trifluoroacetic acid, 30 to 60
Elution was carried out sequentially with a 0.1% trifluoroacetic acid aqueous solution containing a linear concentration gradient of n-propanol. The high performance liquid chromatograph is Hitachi 685-50, and the detection is Hitachi 638-4.
220n using a type 1 detector (both manufactured by Hitachi)
The absorption at m and 280 nm was measured simultaneously. After elution, 10 μl of each fraction was collected and diluted 100-fold, and the active fraction was examined by the above-mentioned “CSA assay method (b)”. As a result, activity was observed in the peak eluted with 40% of n-propanol, so this peak was collected and re-chromatographed again under the same conditions to perform CSA in the same manner as above.
As a result, the activity was recognized in the peak at the position of 40% of n-propanol. Therefore, the peaks were collected (4 fractions = 4 ml) and lyophilized.

(Iii) The above freeze-dried powder was used as a 0.1% trifluoroacetic acid aqueous solution containing 40% of n-propanol 2
It was dissolved in 00 μl and subjected to high performance liquid chromatography (HPLC) using a TSK-G3000SW column (Toyo Soda Co., Ltd., 7.5 mm × 60 cm). Elution was performed with the same aqueous solution at a flow rate of 0.4 ml / min, and 0.4 was obtained with a fraction collector FRAC-100 (Pharmacia).
It was divided into ml. The CSA of each of the fractions collected was examined in the same manner as above, and the active fraction was collected.
After purification with an ondapak C18 column (4.6 mm × 30 cm), the main peak was collected and freeze-dried. The obtained protein was treated with 2-mercaptoethanol to give an SDS-polyacrylamide gel (15.0
%) Electrophoresis (15 mV, 6 hours) and staining with Coomassie blue confirmed that the desired G-CSF polypeptide was a single band.

Example 15 Assay of G-CSF Activity of Expressed Substance The CSF sample obtained in Example 14 was treated with the above-mentioned <Reference Example> CSF.
The assay was performed according to the activity measurement method (a). The results are shown in Table 1.

[0116]

[Table 1]

Example 16 Amino Acid Analysis 1) Analysis of Amino Acid Composition The CSF sample purified in Example 14 was hydrolyzed by a conventional method, and the amino acid composition of the protein portion was analyzed by a Hitachi 835 amino acid automatic analyzer (manufactured by Hitachi Ltd.). It was analyzed by a special amino acid analysis method. The results are shown in Table 2.
The hydrolysis conditions are as follows. 6N HCl, 110 ° C., 24 hours, in vacuum 4N methanesulfonic acid + 0.2% 3- (2-aminoethyl) indole, 110 ° C., 24 hours, 48 hours, 72 hours, in vacuum Sample: 40% n- After being dissolved in a solution containing propanol and 0.1% trifluoroacetic acid (1.5 ml), each solution was adjusted to 0.
After taking 1 ml and drying with dry nitrogen gas, or the reagent of (1) was added and the tube was sealed in a vacuum and subjected to hydrolysis.

In the table, the actually measured value is 2 with the 24-hour value.
It is an average value of 4, 48, 72 hour values a total of 4 times. However, Thr, Ser, 1 / 2Cys, Met, Val,
Ile and Trp were calculated by the following method. (See Biochemistry Laboratory, Protein Chemistry II (Tokyo Kagaku Dojin Shuppan)) ・ Thr, Ser, 1 / 2Cys, Met are 24,
Extrapolated to zero time after 48-72 hours change.・ Val and Ile are 72-hour values.・ Trp is the average of 24, 48 and 72 hours.

[0119]

[Table 2]

2) N-terminal amino acid analysis The sample was subjected to Edman decomposition using a gas phase sequencer (manufactured by Applied Biosystems), and the obtained PTH amino acid was analyzed by a high performance liquid chromatography device (manufactured by Beckman Instruments). ) And Ultr
The analysis was carried out by an ordinary method using an sphere-ODS column (manufactured by Beckman Instruments). Start the column (5 μm, diameter 4.6 mm, length 250 mm) with starting buffer (15 mM sodium acetate buffer pH 4.5, 40%)
After equilibration with an aqueous solution containing acetonitrile), a sample (dissolved in 20 μl of a starting buffer solution) was injected and separation was performed by isocratic elution with the starting buffer solution.
The flow rate was kept at 1.4 ml / min and the column temperature was kept at 40 ° C.
Detection of PTH amino acids utilized ultraviolet absorption at 269 nm and 320 nm. 2 nmol of each standard PTH amino acid (manufactured by Sigma) was separated in advance in the same system to determine the retention time, and the retention time of the test sample was identified. As a result, PTH-methionine and PTH-threonine were detected.

<Experimental Example> Infection protection effect of human G-CSF 1. Pseudomonas Aeruginosa (Pseudom
onas aeruginosa) Protective effect against infection 8-9 week-old (body weight 35.3 ± 1.38 g) ICR mice (male) and endoxan (Shionogi, trade name)
After intraperitoneal administration of 200 mg / kg, divided into 3 groups, 2
Human G-CSF (25000u / mouse or 500
00u / mouse containing solvent (1% propanol, 5%
(W / V) mouse serum albumin), and another group with solvent only, 0.1 ml every 24 hours.
It was administered subcutaneously once. Three hours after the fourth administration, Pseudomonas aeruginosa (Pseudomon) was added to each group.
as aeruginosa) GNB-139 (3.9
X10 ⁴ CFU / mouse) was subcutaneously administered to infect.
21 hours after infection, human G-CSF (2
A solvent containing 5000 u / mouse or 50,000 u / mouse) or only the solvent was subcutaneously administered to the corresponding group. The protective effect against infection was examined by the number of surviving mice up to 10 days after infection. (Table 3)

(Preparation of Bacterial Solution) Pseudomonas aeruginosa GNB-139 was shake-cultured overnight at 37 ° C. using a heart infusion agar plate (manufactured by Difco, trade name). The culture solution was prepared by suspending it in physiological saline.

[0123]

[Table 3] As shown in Table 3, it was confirmed that the human G-CSF of the present invention has a remarkable infection protective effect.

[0124]

INDUSTRIAL APPLICABILITY As described above, according to the present invention, it has become possible to provide human G-CSF, which has been extremely difficult to obtain in the past, in large quantities and with high quality by using recombinant vector technology. Have contributed greatly to the analysis of the hematopoietic mechanism and the pathological conditions of various hematological diseases, and also to the differentiation of myeloid leukemia cells and the enhancement of the function of mature granulocytes. It can be used for treatment and prevention using biochemical action. Therefore, it should be administered to patients with malignant tumors that have lost their resistance due to decreased function of bone marrow tissues or decreased white blood cells due to irradiation or administration of anticancer drugs, or patients with severe infections that cannot be treated with antibiotics. Is highly expected.

[0125]

[Sequence Listing] SEQ ID NO: 1 Sequence length: 30 Sequence form: Nucleic acid Number of strands: Single strand Troposite: Linear Sequence type: Synthetic DNA sequence ATNTGGCARC ARATGGARGA RYTNGGNATG 30

SEQ ID NO: 2 Sequence length: 14 Sequence form: Nucleic acid Number of strands: Single strand Troposity: Linear Sequence type: Synthetic DNA Sequence GCRAANGCNG GCAT 14

SEQ ID NO: 3 Sequence length: 308 Sequence form: Nucleic acid Number of strands: Double-stranded tropology: Linear Sequence type: cDNA sequence CC CTG GAA GGG ATC TCC CCC GAG TTG GGT CCC ACC TTG GAC ACA CTG 47 CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA 95 GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG 143 GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG GT CTG GTT GCC 191 TCC CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC 239 CTT GCC CAG CCC TGA GCC AAG CCC TCC CCA TCC CAT GTA TTT ATC TCT 287 ATT TAA TAT TTA TGT CTA TTT 308

SEQ ID NO: 4 Sequence length: 1531 Sequence form: Nucleic acid Number of strands: Double-stranded tropology: Linear Sequence type: cDNA sequence CGGAGCCTGC AGCCCAGCCC CACCCAGACC C ATG GCT GGA CCT GCC ACC CAG 52 Met Ala Gly Pro Ala Thr Gln -30 -25 AGC CCC ATG AAG CTG ATG GCC CTG CAG CTG CTG CTG TGG CAC AGT GCA 100 Ser Pro Met Lys Leu Met Ala Leu Gln Leu Leu Leu Trp His Ser Ala -20 -15 -10 CTC TGG ACA GTG CAG GAA GCC ACC CCC CTG GGC CCT GCC AGC TCC CTG 148 Leu Trp Thr Val Gln Glu Ala Thr Pro Leu Gly Pro Ala Ser Ser Leu -5 -1 1 5 CCC CAG AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG 196 Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln 10 15 20 25 GGC GAT GGC GCA GCG CTC CAG GAG AAG CTG GTG AGT GAG TGT GCC ACC 244 Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Val Ser Glu Cys Ala Thr 30 35 40 TAC AAG CTG TGC CAC CCC GAG GAG CTG GTG CTG CTC GGA CAC TCT CTG 292 Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu 45 50 55 GGC ATC CCC TGG GCT CCC CTG AGC AGC TGC CCC AGC CAG GCC CTG CAG 340 Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln 60 65 70 CTG GCA GGC TGC TTG AGC CAA CTC CAT AGC GGC CTT TTC CTC TAC CAG 388 Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln 75 80 85 GGG CTC CTG CAG GCC CTG GAA GGG ATC TCC CCC GAG TTG GGT CCC ACC 436 Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr 90 95 100 105 TTG GAC ACA CTG CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG 484 Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp 110 115 120 CAG CAG ATG GAA GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG 532 Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln 125 130 135 GGT GCC ATG CCG GCC TTC GCC TCT GCT TTC CAG CGC CGG GCA GGA GGG 580 Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly 140 145 150 GTC CTG GTT GCC TCC CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC 628 Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg 155 160 165 GTT CTA CGC CAC CTT GCC CAG CCC TGA GCCAAGCCCT CCCCATCCCA 675 Val Leu Arg His Leu Ala Gln Pro End 170 175 TGTATTTATC TCTATTTAAT ATTTATGTCT ATTTAAGCCT CATATTTAAA GACAGGGAAG 735 AGCAGAACGG AGCCCCAGGC CTCTGTGTCC TTCCCTGCAT TTCTGAGTTT CATTCTCCTG 795 CCTGTAGCAG TGAGAAAAAG CTCCTGTCCT CCCATCCCCT GGACTGGGAG GTAGATAGGT 855 AAATACCAAG TATTTATTAC TATGACTGCT CCCCAGCCCT GGCTCTGCAA TGGGCACTGG 915 GATGAGCCGC TGTGAGCCCC TGGTCCTGAG GGTCCCCACC TGGGACCCTT GAGAGTATCA 975 GGTCTCCCAC GTGGGAGACA AGAAATCCCT GTTTAATATT TAAACAGCAG TGTTCCCCAT 1035 CTGGGTCCTT GCACCCCTCA CTCTGGCCTC AGCCGACTGC ACAGCGGCCC CTGCATCCCC 1095 TTGGCTGTGA GGCCCCTGGA CAAGCAGAGG TGGCCAGAGC TGGGAGGCAT GGCCCTGGGG 1155 TCCCACGAAT TTGCTGGGGA ATCTCGTTTT TCTTCTTAAG ACTTTTGGGA CATGGTTTGA 1215 CTCCCGAACA TCACCGACGC GTCTCCTGTT TTTCTGGGTG GCCTCGGGAC ACCTGCCCTG 1275 CCCCCACGAG GGTCAGGACT GTGACTCTTT TTAGGGCCAG GCAGGTGCCT GGACATTTGC 1335 CTTGCTGGAC GGGGACTGGG GATGTGGGAG GGAGCAGACA GGAGGAATCA TGTCAGGCCT 1395 GTGTGTGAGA GGAAGCTCCA CTGTCACCCT CCACCTCTTC ACCCCCC ACT CACCAGTGTC 1455 CCCTCCACTG TCACATTGTA ACTGAACTTC AGGATAATAA AGTGTTTGCC TCCAAAAAAA 1515 AAAAAAAAAA AAAAAA 1531

SEQ ID NO: 5 Sequence length: 207 Sequence form: Amino acid Number of chains: Tropoposity: Linear Sequence type: Peptide sequence Met Ala Gly Pro Ala Thr Gln Ser Pro Met Lys Leu Met Ala Leu Gln 16 Leu Leu Leu Trp His Ser Ala Leu Trp Thr Val Gln Glu Ala Thr Pro 32 Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu 48 Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys 64 Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu 80 Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser 96 Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His 112 Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile 128 Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala 144 Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala 160 Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala 176 Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser 192 Phe Leu Glu Val Se r Tyr Arg Val Leu Arg His Leu Ala Gln Pro 207

SEQ ID NO: 6 Sequence length: 177 Sequence form: Amino acid Number of chains: Tropoposity: Linear Sequence type: Peptide Sequence Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys 16 Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln 32 Glu Lys Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu 48 Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu 64 Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln 80 Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu 96 Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp 112 Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly 128 Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala 144 Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu 160 Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln 176 Pro 177

[Brief description of drawings]

FIG. 1 shows restriction enzyme cleavage sites of pBRG4-derived human G-CSF cDNA.

FIG. 2 shows a part of the process for preparing a tac promoter-containing vector.

FIG. 3 shows a process for preparing a synthetic P _L promoter-containing vector.

FIG. 4 shows a process for preparing a trp promoter-containing vector.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI technical display location C12R 1:19) (72) Inventor Yasuo Sekimori 3-41-8 Takada, Toshima-ku, Tokyo Chugai Pharmaceuticals Co., Ltd. (72) Inventor Shigekazu Nagata Room No. 305, Building 2-34-262, Tamagawa, Ota-ku, Tokyo

Claims (2)

[Claims] 1. A polypeptide having human granulocyte colony stimulating factor activity, which is transformed with a recombinant expression vector containing a DNA comprising a nucleotide sequence encoding a polypeptide containing the following amino acid sequence in its amino acid sequence. A method for producing a polypeptide having human granulocyte colony stimulating factor activity, comprising culturing the bacterium, and separating the produced polypeptide having human granulocyte colony stimulating factor activity. (Met) n Thr Pro Leu Gly Pro Ala Ser Ser Leu Pro Gln Ser Phe Leu Leu Lys Cys Leu Glu Gln Val Arg Lys Ile Gln Gly Asp Gly Ala Ala Leu Gln Glu Lys Leu Val Ser Glu Cys Ala Thr Tyr Lys Leu Cys His Pro Glu Glu Leu Val Leu Leu Gly His Ser Leu Gly Ile Pro Trp Ala Pro Leu Ser Ser Cys Pro Ser Gln Ala Leu Gln Leu Ala Gly Cys Leu Ser Gln Leu His Ser Gly Leu Phe Leu Tyr Gln Gly Leu Leu Gln Ala Leu Glu Gly Ile Ser Pro Glu Leu Gly Pro Thr Leu Asp Thr Leu Gln Leu Asp Val Ala Asp Phe Ala Thr Thr Ile Trp Gln Gln Met Glu Glu Leu Gly Met Ala Pro Ala Leu Gln Pro Thr Gln Gly Ala Met Pro Ala Phe Ala Ser Ala Phe Gln Arg Arg Ala Gly Gly Val Leu Val Ala Ser His Leu Gln Ser Phe Leu Glu Val Ser Tyr Arg Val Leu Arg His Leu Ala Gln Pro (where n is 0 or 1) 2. The method for producing the polypeptide according to claim 1, wherein the base sequence is the following base sequence. (ATG) n ACC CCC CTG GGC CCT GCC AGC TCC CTG CCC CAG AGC TTC CTG CTC AAG TGC TTA GAG CAA GTG AGG AAG ATC CAG GGC GAT GGC GCA GCG CTC CAG GAG AAG CTG GTG AGT GAG TGT GCC ACC TAC AAG CTG TGC CAC CCC GAG GAG CTG GTG CTG CTC GGA CAC TCT CTG GGC ATC CCC TGG GCT CCC CTG AGC AGC TGC CCC AGC CAG GCC CTG CAG CTG GCA GGC TGC TTG AGC CAA CTC CAT AGC GGC CTT TTC CTC TAC CAG GGG CTC CTG CAG GCC CTG GGG ATC TCC CCC GAG TTG GGT CCC ACC TTG GAC ACA CTG CAG CTG GAC GTC GCC GAC TTT GCC ACC ACC ATC TGG CAG CAG ATG GAA GAA CTG GGA ATG GCC CCT GCC CTG CAG CCC ACC CAG GGT GCC ATG CCG GCC GTC TTC GCC TCT GCC TTC CAG CGC CGG GCA GGA GGG GTC CTG GTT GCC TCC CAT CTG CAG AGC TTC CTG GAG GTG TCG TAC CGC GTT CTA CGC CAC CTT GCC CAG CCC (where n is 0 or 1)