JPH08173185A - Production of stimulating factor for human granulocytic macrophage colony - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain the subject substance useful for treatment of leukopenia caused from radiation therapy or chemotherapy of cancer and quick hypercytosis after bone marrow transplantation, or the like, at a sufficient manifestation level by culturing a transformant obtained by transforming Escherichia coli with a specific plasmid.

SOLUTION: This human granular macrophage colony stimulation factor is obtained by binding a DNA chain containing either one of base sequences of formula I and formula II encoding a polypeptide added with Met on an N-end of an amino acid sequence of hGM-CSF (Mature part) to a plasmid in a state capable of manifesting its genetic information, followed by introducing into Escherichia coli to highly manifest.

COPYRIGHT: (C)1996,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a technology for producing human granulocyte macrophage colony stimulating factor (hereinafter referred to as “hGM-CSF”) by gene recombination.

[0002]

2. Description of the Related Art A colony stimulating factor (hereinafter referred to as "CSF") is a physiologically active substance that stimulates proliferation and differentiation of hematopoietic stem cells in soft agar and promotes colony formation of blood cells of a specific type. hGM-CSF is a factor that stimulates granulocyte-macrophage stem cells (CFU-GM) and promotes the formation of granulocyte-macrophage, and is expected to be used for the following applications.

That is, it is considered to be useful for treating leukopenia by radiotherapy or chemotherapy for cancer, for promptly proliferating leukocytes after bone marrow transplantation, and so on.

Some reports have already been published on the production technology of hGM-CSF by gene recombination (PCT Publication Gazette W.
O86 / 00639, WO86 / 03225, EPO
Published gazette 0183350 and others), hGM-CS
Both the base sequence of the gene encoding F and the amino acid sequence of the polypeptide produced thereby are known. Note that hGM-CSF is considered to be a glycoprotein containing a polypeptide consisting of 144 amino acids (PC
(See T Publication WO 86/00639).

[0005]

Generally, in the production of a substance by a gene recombination technique, the productivity of the substance is usually higher when Escherichia coli is used as a host than when an animal cell or yeast is used.

[0006] As far as we know, there are two reports regarding the expression of the gene encoding hGM-CSF by E. coli. Clark et al. In Escherichia coli encode a gene encoding an analog of a polypeptide constituting hGM-CSF (a polypeptide having an amino acid sequence different from that of hGM-CSF at three positions and having methionine at the N-terminus). Although it is expressed (see PCT Publication WO86 / 00639), the expression level of the gene at this time is extremely low at most 5% of cellular (Escherichia coli) protein and whether or not the expression product has hGM-CSF activity. It was completely unknown. In addition, Burgess et al. Expressed a cDNA encoding hGM-CSF obtained from the human monocyte cell line U937 in Escherichia coli, and the expression level at this time was 8 to 10% of E. coli protein (Blood, 6).
9 (1), 43 (1987)), which was not sufficient.

[0007] Therefore, it has been desired to establish a more efficient production technique for a polypeptide or glycoprotein having hGM-CSF activity.

[0008]

The present invention has been made to solve the above problems, and provides a means for efficiently producing a polypeptide having hGM-CSF activity in Escherichia coli. is there. That is, the present invention comprises: (1) preparing a DNA chain containing the nucleotide sequence shown in FIG. 1 or FIG. 2 and preparing a plasmid containing this DNA chain in a state in which its genetic information can be expressed; (E. coli) and the resulting transformants were cultivated to produce hGM in the culture.
HGM-CSF characterized by producing -CSF
Is a manufacturing method of.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION DNA Strand of the Present Invention The DNA strand of the present invention contains the nucleotide sequence shown in FIG. 1 or 2. This nucleotide sequence is known as hGM-CSF.
It encodes a polypeptide in which Met is added to the N-terminus of the amino acid sequence of (Mature part), and is designed by the present inventors in consideration of the following points so that it can be highly expressed in E. coli. is there.

(1) Use codons preferentially used in E. coli. The codon used preferentially in E. coli has been reported by Ikemura (Ikemura, Jpn. J. Genet. 56 p533-555).
(1981)).

(2) The 5'-terminal nucleotide sequence was brought close to the nucleotide sequence common to the translation initiation sites of various genes in Escherichia coli (Scherer et al., Nucl. Acid).
s. Res. 8 p3895-3905 (198
0)).

[0012] The DNA chain of the present invention is for ligating it to an appropriate vector and introducing it into E. coli for high expression. To realize high expression of this DNA chain, the above-mentioned bases are used. The S. D. Contains an array.

[0013]

Embedded image

The base sequence of (Scherer. Et a
1, Nucl. Acids Res. 8 p3895-3
905 (1980)), and may have two or more stop codons downstream of the 3'end of the above base sequence. Further, it is preferable to add appropriate restriction enzyme sites to the 5'end and 3'end of this gene to facilitate the gene recombination operation.

Production of DNA Strand of the Present Invention The DNA strand of the present invention can be produced by any purposeful method, and an example thereof is as follows.

(1) Production of DNA chain containing the nucleotide sequence shown in FIG. 1 A DNA chain is divided into 3 to 4 blocks each having about 100 base pairs and synthesized, and each block is sequentially subjected to an appropriate cloning plasmid (for example, pUC19, Messing et al Gen
obtaining a DNA strand of interest by inserting linked to e 33 p110~115 (1985)).

The synthesis of each block is carried out by dividing it into oligonucleotides of about 25 to 35 bases and synthesizing them. When the blocks are divided into oligonucleotides, it is necessary to prevent self-complementary sequences from appearing in one oligonucleotide so that each oligonucleotide does not self-associate. It is also necessary to prevent the repeating sequence from appearing within one oligonucleotide. FIG. 3 shows an example of the nucleotide sequences of the segmented oligonucleotides, FIG. 4 shows an example of the configuration of each block from these oligonucleotides, and FIG. 5 shows an example of the nucleotide sequences of each block and restriction enzyme sites.

Each oligonucleotide can be synthesized by a known synthesis method [for example, a solid-phase synthesis method by the phosphoamidite method (Beaucage et al. Tetrahhe.
dron Letters 22 1859-1862
(1981)]. It is also possible to use an automatic synthesizer based on this synthesis method. Each block was constructed by phosphorylating the 5'end of each oligonucleotide with a polynucleotide kinase, if necessary, and then annealing,
It is carried out by forming double-stranded DNA with ligase.
The procedure for connecting the blocks is as follows.

The C block of FIG. 5 is replaced with Hin of pUC19.
It is ligated with the fragment cleaved with dIII and PstI to obtain the plasmid pYC8, which is used to transform Escherichia coli JM109. Then, pYC8 was isolated, the nucleotide sequence was confirmed by the dideoxy method, and pYC8 was digested with NheI and AvaI and then the B block was ligated to obtain pSC.
B86 is obtained, which transforms JM109. pSC
After confirming the base sequence of B86, Escherichia coli dam ^- strain GM33 is transformed with pSCB86, and pSCB86 is isolated again. pSCB86 was digested with BclI and SacI, and then an A block was ligated to pSCBA86.
7, which transforms JM109. Similarly pS
Confirmed the base sequence of CBA867, and further confirmed pSCBA86
7 was digested with NcoI and EcoRI and the D block was ligated to obtain pSCBAD8676,
JM109 is transformed and the nucleotide sequence is confirmed in the same manner.

PSCBA867 thus obtained
To XbaI (SacI) and BamHI (Hind II
By digesting with I), the DNA strand of the present invention containing the nucleotide sequence shown in FIG. 1 can be obtained (FIG. 6A).
pSCBAD8676 with ClaI (EcoRI) and B
By digesting with amHI (Hind III), the DNA chain of the present invention containing the SD sequence can be obtained (FIG. 6B).

(2) Production of DNA Chain Containing Base Sequence of FIG. 2 It can be produced by a procedure according to the above-mentioned “Production of DNA Chain Containing Base Sequence of FIG. 1”.

In this case, however, the following C-13 and C- were used instead of the above oligonucleoside C-3.
The following C-14 is used instead of 9.

[0023]

[Chemical 4]

The DNA chain of the present invention thus obtained is shown in FIG.

Preparation of Transformant The DNA chain of the present invention prepared as described above is hGM.
-Since it contains genetic information for producing CSF protein, this DNA chain is introduced into E. coli by bioengineering as a plasmid containing this DNA in a state in which the genetic information can be expressed and transformed. Then, the hGM-CSF protein can be produced by culturing the obtained transformant.

Generation of transformants (and thereby hG
The procedure or method itself for the production of M-CSF) may be one that is commonly used in the fields of molecular biology, biotechnology or genetic engineering. It may be carried out according to a conventional technique.

In order to express the gene of the DNA chain of the present invention in Escherichia coli, it is first necessary to ligate this gene into a plasmid vector stably existing in E. coli.
As the plasmid vector, any arbitrary vector such as pBR322 can be used.

On the other hand, in order to express the gene of the DNA chain of the present invention in E. coli, it is necessary to transcribe the DNA into mRNA. For that purpose, a promoter which is a signal for transcription may be incorporated upstream of the 5'side of the DNA chain of the present invention. For this promoter, trp,
Various types of lac, PL, OmpF and the like are known, and any of these can be used in the present invention.

Further, it is preferable to incorporate a terminator for terminating the transcription at the 3'side downstream of the DNA chain of the present invention. When a strong promoter is used, the stability of the plasmid can be increased by inserting a terminator. As the terminator, trpa, r
rnc, λtop, etc. can be used.

In the step of translating mRNA into protein, the sequence necessary for the ribosome, which is the site of protein synthesis, to bind to the tip of the translation initiation site (called the SD sequence) is used as the initiation signal for protein synthesis. It has to be put before a certain ATG. For more efficient expression, this S.
D. The sequence and this S. D. It is preferable that the base sequence between the sequence and ATG, which is the initiation signal for protein synthesis, be close to the base sequence common to the translation start site in E. coli.
For example

[0031]

Embedded image

It is preferable that

Transformation of Escherichia coli with the plasmid thus prepared can be carried out by any purposeful method commonly used in the field of genetic engineering or biotechnology. Suitable general texts or reviews, such as Maniatis et al.
ural Cloning-A Laboratory
Manual ", Cold Spring Harb
or Laboratory (1982).

The transformant is a new trait (that is, hGM-) due to the genetic information introduced by the DNA chain of the present invention.
CSF productivity) and traits derived from the used vector and, in some cases, the corresponding genotype, except for the loss of the corresponding trait due to the loss of some genetic information from the used vector at the time of gene recombination. It is the same as the E. coli used in phenotype or mycological properties.

Production of hGM-CSF The clone of the transformant obtained as described above produces the hGM-CSF protein in the cells when it is cultured.

The culture and growth conditions of the transformant are essentially the same as those for the E. coli used. In addition, any desired method for the purpose of recovering the produced protein from the culture, that is, the bacterial cells and / or the culture medium (see, for example, Example 6 described later).
Described method). hGM-C
Since SF is produced in E. coli in an aggregated form,
After recovering this protein, it is preferable to dissolve it with a denaturing agent (eg, guanidine hydrochloride, urea), cleave the disulfide bond with a reducing agent (eg, dithiothreitol), and then purify by a conventional method.

[0037]

EXAMPLE 1 Synthesis of Oligonucleotides The oligonucleotides shown in FIG. 3 and the above-mentioned oligonucleotides C-13 and C-14 were prepared by the phosphoamidite method (Beaucage et al., Tetrahedron L
etters 22 1859-1962 (198)
1)) automatic DNA synthesizer (Applied Biosystems model 380A, M. Hunkapiller)
Et al., Nature 310 105-111 (198).
4)) was used.

After completion of the synthesis, the mixture was concentrated with aqueous ammonia at 60 ° C. for 5
After treatment for a period of time, the base protecting group was removed and the oligonucleotide was cleaved from the carrier. The obtained oligonucleotide was purified using high performance liquid chromatography (HPLC). That is, a column (φ4.1 × 1) of reverse phase neutral rigid polystyrene gel (PRP-1, Hamilton Co.)
50 mm) and purified by a linear gradient method of acetonitrile in 0.1 M triethylamine acetate buffer (pH 7.0). Collect the target peaks, 8
Electrophoresis was performed using 12% polyacrylamide containing M urea. TLC containing fluorescent dye under gel after electrophoresis
The plate was placed and the presence of the band of interest was confirmed using a UV lamp. A gel piece containing the desired oligonucleotide was enclosed in a dialysis tube, and the DNA was electroeluted from the gel. The solution in the dialysis tube was treated with a Sephadex G25 (Pharmacia) gel filtration column
1.5 × 43 cm) and eluted with 0.05 M triethylamine bicarbonate buffer (pH 7.5) for desalting. The eluate containing the target oligonucleotide was concentrated under reduced pressure to obtain a pure oligonucleotide.

Example 2 Ligation of oligonucleotides
The 34 chemically synthesized oligonucleotides were ligated according to the block preparation plan in FIG. Details are given below. The remaining 26 oligonucleotides (0.
8 nmol) to 20 μl of phosphorylation reaction solution [50 mM
Tris-HCl, pH 7.4, 10 mM MgC
l ₂ , 10 mM DTT, 30 μCi [α- ³² P] A
TP (3000 Ci / mmol), 15 units of T4
Polynucleotide kinase (Boehringer Mannheim) was reacted at 37 ° C. for 30 minutes to phosphorylate the 5 ′ end for labeling. Then 1 μl of 100 mM ATP
In addition, the reaction was carried out at 37 ° C for 30 minutes to completely phosphorylate the 5'end, and the reaction was stopped by heating at 100 ° C for 5 minutes.

Then, according to the ligation plan of FIG. 4, each oligonucleotide was mixed, heated at 95 ° C. for 5 minutes, returned to room temperature for 2 hours, and annealed. 200 μl of this ligation reaction solution [50 m
M Tris-HCl, pH 7.4, 10 mM MgC
l ₂ , 15 mM DTT, 1 mM ATP, T4 DN
The reaction was carried out in 30 units of A ligase (Bethesda Research Laboratories) at 15 ° C. for 14 hours. Part of the reaction solution was subjected to 8% polyacrylamide gel electrophoresis,
It was confirmed by radio autograph that each block was obtained as a result of ligation reaction. Next, ethanol was added to the above reaction solution to precipitate the DNA, which was similarly separated by 8% polyacrylamide gel electrophoresis. A gel piece containing each block was placed in a dialysis tube and electrophoresed in a running buffer to elute each. Next, the solution in each dialysis tube was applied to a NACS column (Bezesda Research Laboratory), and ethanol was added to the eluate to precipitate DNA, respectively.

Example 3 Claw of hGM-CSF gene
Training in the cloning vector of E. coli plasmid pUC1
9 (Pharmacia) was used. 1 μg of pUC19D
30 μl of NA reaction solution [10 mM Tris-HC
1, pH 7.5, 10 mM MgCl ₂ , 1 mM DT
T, 50 mM NaCl, 12 units of Hind III
(Takara Shuzo) 10 units of PstI (Takara Shuzo)]
After reacting at 37 ° C for 2 hours, it was treated at 65 ° C for 20 minutes to inactivate the restriction enzyme. 1μg of C-block DNA 20μ
1 reaction solution [50 mM Tris-HCl, pH 7.
4, 10 mM MgCl ₂ , 5 mM ATP, 10 units of T4 polynucleotide kinase (Takara Shuzo)],
The mixture was reacted at 37 ° C. for 1 hour and phosphorylated. 2 μl of this reaction
To pUC19 DNA and Hind III and Pst I
In addition to 7 μl of reaction solution with
Tris-HCl, pH 7.4, 100 mM MgCl
₂ ] solution, 1 μl of 100 mM DTT, 1 μl of 10
0 mM ATP, 2 μl (2 units) T4 DNA
Ligase (Boehringer Mannheim) and 15 μl of autoclave water were added, and the mixture was reacted at 14 ° C. for 14 hours.

Using this reaction solution, Escherichia coli JM109 strain (C. Yanisch-Perron et al. Gene 33) was used.
p103-119 (1985)) by a known method (D.
Hanahan J. Mol. Biol. p557-5
80 (1980)). At that time, an LB plate was used as a plate, and 50 μg / ml of ampicillin and 0.24 mg / ml of isopropyl-β-D were used.
-Galactopyranoside (IPTG) and 0.04 mg /
ml 5-Bromo-4-chloro-3-indolyl-β-
It contained galactoside (X-gal). Select colorless colonies resistant to ampicillin and select one of them from pY
It was named C8 / JM109.

C block DNA is the Hind of pUC19
C block D if inserted between III and PstI
There is a unique NheI restriction enzyme site with NA. Therefore, plasmid DNA was prepared from the pYC8 / JM109 strain by the alkaline method (T. Maniatis et al., Mo.
regular Cloning p368-369
(1982) Cold Spring Harbor)
It was confirmed that it was digested with NheI (New England Biolabs). Also pYC
8 about the dideoxy method (Hattori et al., Anal. Bioc
hem. 152 p232-238 (1986)) was used to confirm the nucleotide sequence.

After pYC8 was digested with NheI and AvaI, B block DNA was ligated and inserted by the same method as above to transform Escherichia coli JM109 strain. The insertion of B block was confirmed by the disappearance of the SalI restriction enzyme, and the nucleotide sequence was confirmed by the above method. The obtained strain was designated as pSCB86 / JM109.
And E. coli dam with this plasmid pSCB86 ^-
The strain GM33 strain (obtained from Toyobo Co., Ltd.) was transformed. This strain was designated as pSCB86 / GM33.

A plasmid was isolated from this strain, and Bcl
After digestion with I and SacI, A block DNA was inserted and E. coli JM109 strain was transformed. The insertion of the A block was confirmed by the presence of the NcoI restriction enzyme site. The nucleotide sequence was confirmed by the method described above. The obtained strain was designated as pSCBA867 / JM109. This allows
The DNA strand of the present invention shown in FIG. 6A was prepared.

Furthermore, S. D. HGM-CSF containing sequences
To prepare the gene, the plasmid pSCBA867 was digested with NcoI and EcoRI to obtain D-blocked DNA.
Was inserted and Escherichia coli JM109 strain was transformed. The insertion of the A block was confirmed by the presence of the ClaI restriction enzyme site. The nucleotide sequence was confirmed by the method described above. The obtained strain was designated as pTCBAD8676 / JM109. As a result, the S. D. DN of the invention comprising a sequence
The A chain was prepared.

The outline of the above cloning process is shown in FIG.
It was shown to. Further, the DNA chain of the present invention shown in FIGS. 7A and 7B was produced by the same procedure as described above. However, C-13 was used instead of the oligonucleotide C-3, and C-14 was used instead of C-9.

Example 4 Construction of Expression Vector Regarding the nucleotide sequences of the promoter and operator regions of the Escherichia coli tryptophan operon, Bennett has already been described.
(J. Mol. Biol. 12).
1 p113-p137 (1978)). Based on a sequence from the transcription start point to 94 bases 5 ′ upstream to 21 bases 3 ′ downstream +21, immediately after the transcription start point, the ClaI site was added to the S. D. An XbaI site is provided immediately after the sequence,
Further, 131 base pairs having an EcoRI site at the 5'end and a HindIII site at the 3'end were chemically synthesized (Fig. 9). The 131-base tryptophan promoter is used for EcoRI and HindI of pBR322.
It was inserted into the DNA digested with II to give an expression vector containing a tryptophan promoter, and named pST8.

The terminator for the termination of transcription of the tryptophan operon has been reported by Christie et al. In terms of its nucleotide sequence and its termination strength (Proc.
Natl. Acad. Sci. USA 78 (7) p
4180-4184 (1981)). BamHI site at the 5'end of the Trpa terminator and S at the 3'end
An oligonucleotide provided with a phl site was chemically synthesized (Fig. 10). An oligonucleotide containing this terminator was inserted into BSTHI-digested and SphI-digested DNA of pST8 to obtain plasmid pST81.

A plasmid derived from pBR322 and having a high copy number in Escherichia coli, pAT153 was produced by Twigg et al. (Nature 283 p216-).
p218 (1980)). Therefore, D containing the tryptophan promoter and Trpa terminator of pST81
The NA fragment was obtained by digesting with EcoRI and SphI, p
AT153 (Amersham) EcoRI and Sph
It was inserted into the I digested fragment to obtain the plasmid pST811 (see FIG. 11).

Example 5 Plus for expression of hGM-CSF
Construction of mid (1) Plasmid for expression by plasmid pST811 The pSCBAD8676 was digested with ClaI and BamHI, and then 423 by 0.8% agarose electrophoresis.
The base-paired DNA fragment was purified. The expression vector p
After digesting ST811 with ClaI and BamHI, the S. D. The hGM-CSF gene containing the sequence (FIG. 6B) was ligated with T4 ligase and inserted, and Escherichia coli RR1 strain was transformed. A plasmid was isolated from an ampicillin-resistant colony, digested with PstI, and the digestion pattern and disappearance of the XbaI restriction enzyme site resulted in S. D. It was confirmed that the hGM-CSF gene containing the sequence was inserted. The obtained strain was designated pST6
311 / RR1 (see FIG. 12A).

(2) Plasmid for expression by plasmid pCFM526 Expression vector pCF containing λ phage PL promoter
Using M526 (Tokutei Sho 50-501988, PCT publication WO 85/00829, ATCC 39932),
S. D. The hGM-CSF gene containing the sequence (Figure 6B) was inserted. That is, the S. D. The sequence containing the hGM-CSF gene, pCFM526 and Cla.
And insert were ligated with T4 ligase into the digested DNA at I and BamHI, and the E. coli AM7 with plasmid pMW1 (ATCCNo.39933) containing RamudaCI ₈₅₇ genes were transformed. A plasmid was isolated from a colony resistant to ampicillin and kanamycin,
It was digested with PstI and S. D. It was confirmed that the hGM-CSF gene containing the sequence was inserted. The obtained strain was designated as pTO614 / AM7 (Fig. 1
2B).

Example 6 Method for producing hGM-CSF (see FIG. 1)
(1) Expression of plasmid pST6311 / RR1 The pST6311 / RR1 was shake-cultured overnight at 37 ° C. in an L medium containing ampicillin. 2m of this culture
1 to 100 ml of M9 medium (0.8% glucose, 0.
4% casamino acid, 10 μg / ml thiamine, 50 μg /
ml ampicillin) and shaken at 37 ° C. for 3 hours. Final concentration of indole acrylic acid 40μg /
It was added to make up to ml. The culture was shaken for another 5 hours. A part of the obtained Escherichia coli was boiled in the sample buffer (5 minutes), and the boiled solution was subjected to SDS-polyacrylamide electrophoresis to examine the content of hGM-CSF. Under these conditions, hGM-CSF was 30% or more of the E. coli cell protein.

(2) Plasmid pTO614 / AM7
Expression of pTO614 / AM7 was cultured with shaking in L medium containing ampicillin and kanamycin at 29 ° C. overnight.
10 ml of this culture broth was added to 500 ml of L medium containing ampicillin and kanamycin, and cultivated with shaking at 29 ° C. for 3 hours. L-medium 500 pre-heated to 54 ° C
ml was added to each culture, and the mixture was heated to 42 ° C. and further cultured for 3 hours. As a result of examining the content of hGM-CSF in the same manner as above, it was 30% or more of the E. coli cell protein.

Further, the culture solution was centrifuged to give a total of about 10 g.
The bacterial cells of Distilled water was added and dispersed using a homogenizer at 3 ° C. until completely dispersed. This suspension was run on a French press three times. During this time, the suspension is 18
This homogeneous liquid, which was kept at ℃ or below, was diluted to 125 ml with distilled water, and the obtained mixed liquid was centrifuged at 30 ℃. The supernatant was decanted and the residue was resuspended with distilled water to a final volume of 80 ml. The obtained mixed solution was centrifuged at 3 ° C., the supernatant was decanted, and the residue was suspended in distilled water to a final volume of 12 ml. 4 ml of 1M Tris buffer (pH
8.5) and 64 ml of 10 M urea were added. The obtained mixed solution was centrifuged at 14 ° C., and the supernatant was collected. 533 mg of reduced glutathione and 107 were added to the supernatant.
720 ml of 20 mM containing mg of oxidized glutathione
Tris buffer (pH 8.5) was added. This mixture was left at 5 ° C. for 20 hours. The mixture was concentrated by passing it through a membrane having a molecular weight of 10,000 at 5 ° C. Add at least 400 ml of this concentrated solution at 5 ° C.
Molecular weight 1 with 0 mM Tris buffer (pH 8.5)
The buffer was exchanged by passing it through a membrane of 10,000 (using a Pellicon lab cassette manufactured by Millipore). The pH of the concentrate was adjusted to pH 5.3 with 50% acetic acid. The mixture was centrifuged at 3 ° C. The diluted supernatant was applied to a CM-Sepharose column and 45 mM NaCl-
Elution was performed with a 20 mM sodium acetate (pH 5.4) buffer solution. The pH of the eluate was 1M Tris buffer (pH 8.5)
Was used to adjust the pH to 7.7. The eluate from the CM-Sepharose column was applied to a C4 column at 5 ° C., 20%
Elution was performed with ethanol-50 mM Tris buffer (pH 7.7), and then with 40% ethanol-50 mM Tris buffer. hGM-CF is 40% ethanol-
Elution was performed with a gradient from 50 mM Tris buffer (pH 7.7) to 60% ethanol-50 mM Tris buffer (pH 7.7). Collect the eluate and DEAE at 5 ℃
Apply to a Sepharose column and apply 20 mM Tris buffer (p
H7.7), then 10 mM phosphate buffer (pH 7.
5), and eluted with 15 mM NaCl-10 mM phosphate buffer. hGM-CSF is 60 mM NaC
Purification was carried out by column elution with 1-10 mM phosphate buffer (pH 7.5).

Example 7 Confirmation of hGM-CSF activity The activity of hGM-CSF was confirmed by assaying its ability to promote the growth of human bone marrow cell-derived colonies in agar.
Bone marrow cells of a healthy person were diluted with McCoy's 5A medium (Gibco) and overlaid on a Ficoll-Plaque (Pharmacia) solution. 500 xg for 20 minutes at room temperature
Centrifuge at 20 ° C and collect the intermediate layer to obtain 20 times the amount of McCoy '.
It was washed with s5A medium. The suspension is then kept at room temperature for 10 minutes 2
It was washed by centrifugation at 50 × g. Next, the cells were suspended in McCoy's 5A medium containing 10% fetal bovine serum and cultured in a plastic petri dish for 2 hours at 37 ° C. and 5% carbon dioxide. After culturing, collect the supernatant and 2 x 10 ⁶ cells
/ Ml of cell suspension was prepared.

In the assay, bone marrow cells were added to a medium having the following composition so that the final concentration was 2 × 10 ⁵ cells / ml. a) 1.84 × McCoy's 5A medium-2
mM sodium pyruvate-0.8xMEM amino acid (Gibco) -0.08xMEM non-essential amino acid (Gibco) -0.09% sodium bicarbonate-0.8xMEM
Vitamin solution (Gibco) -2.4 mM L-glutamine-3.2 mg / ml L-serine-1.68 mg / ml
5 parts of a solution containing L-asparagine, and b) 0.6%
Noble agar (Difco) 3 parts, c) fetal bovine serum 1 part. HGM-CSF purified in Example 6 (2)
The solution was added. The cultured cells were kept warm at 37 ° C. in the presence of 5% CO ₂ . After culturing for 7 to 14 days, granulocytes and macrophages were confirmed by double staining with esterase. As a result, it was found to have an activity of 0.5 × 10 ⁷ units / mg or more. The result was that the average number of colonies obtained from 2 × 10 ⁵ bone marrow cells was about 270. The definition of the unit was reported by Metcalfe et al. [Blood 67 (1) 37-4.
5 (1986)].

As a positive control experiment, human GCT culture supernatant (Gibco) was added to the above medium so that the concentration was 10%, and similar results were obtained.

Deposition of microorganisms Expression vector pCF containing the λ phage PL promoter
E. coli harboring M526. coli AM7, ie E. coli. coli AM7 (pCFM526) is an American Type Culture Collection (AT) at Rockville Park Lawn Drive, Maryland, USA.
(CC) has been internationally deposited and has obtained a deposit number of ATCC 39932.

Plasmid pMW containing the λCI ₈₅₇ gene
1 is an E. E. coli JM103, that is E. coli JM103 (pMW1), as ATC
It has been internationally deposited with C and has obtained a deposit number of ATCC 39933.

[Brief description of drawings]

FIG. 1 is a diagram showing an amino acid sequence of hGM-CSF (n = 101, Thr) and a base sequence encoding the amino acid sequence.

FIG. 2 is a diagram showing an amino acid sequence of hGM-CSF (n = 101, Ile) and a base sequence encoding the same.

FIG. 3A is a diagram showing a nucleotide sequence of an oligonucleotide.

FIG. 3B is a diagram showing a nucleotide sequence of an oligonucleotide.

FIG. 4 shows a ligation scheme of oligonucleotides to form each block.

FIG. 5A is a diagram showing a nucleotide sequence of each block and a restriction enzyme site.

FIG. 5B is a diagram showing a nucleotide sequence of each block and a restriction enzyme site.

FIG. 5C is a diagram showing a nucleotide sequence of each block and a restriction enzyme site.

FIG. 5D is a diagram showing a nucleotide sequence of each block and a restriction enzyme site.

FIG. 6A shows the DNA strand of the present invention encoding hGM-CSF (n = 101, Thr).

FIG. 6B is an S.E. encoding hGM-CSF (n = 101, Thr). D. FIG. 3 shows a DNA strand of the present invention containing a sequence.

FIG. 7A shows a DNA strand of the present invention encoding hGM-CSF (n = 101, Ile).

FIG. 7B is an S.E. encoding hGM-CSF (n = 101, Ile). D. FIG. 3 shows a DNA strand of the present invention containing a sequence.

FIG. 8A: hGM-CSF (n = 10) on pUC19.
FIG. 1 is a diagram showing cloning of the 1, Thr) gene.

FIG. 8B: hGM-CSF on pUC19 (n = 10)
FIG. 1 is a diagram showing cloning of the 1, Thr) gene.

FIG. 9 is a diagram showing a nucleotide sequence of a synthetic trp promoter and a restriction enzyme site.

FIG. 10 is a diagram showing the base sequence and restriction enzyme site of a synthetic trpa terminator.

FIG. 11 is a diagram showing the construction of an expression vector.

FIG. 12A shows the construction of pST6311.

FIG. 12B shows the construction of pTO614.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI Technical indication C12R 1:19) (C12N 1/21 C12R 1:19) (72) Inventor Tadashi Ozawa Maebashi Gunma Prefecture 1-2-2 Sorin-sha, Soja-machi, Kirin Brewery Co., Ltd., Pharmaceutical Development Laboratory (72) Inventor Yukio Tamai 1-2-2, Soja-sha, Maebashi City, Gunma Kirin Brewery Co., Ltd.

Claims (1)

[Claims] 1. A DNA chain containing the nucleotide sequence shown in either (a) or (b) below is prepared, and a plasmid containing this DNA chain in a state in which its genetic information can be expressed is prepared. Human granulocyte macrophage, characterized in that it produces a human granulocyte macrophage colony stimulating factor in the culture, which is subjected to a step consisting of transformation of E. coli with the plasmid and culturing of the resulting transformant. Method for producing colony stimulating factor. Embedded image Embedded image