GB2249100A - Expression of M-CSF deletion mutant polypeptides - Google Patents

Abstract

A recombinant vector capable of expressing M-CSF active polypeptide containing an M-CSF-delete DNA (DNA containing DNA encoding the M-CSF amino acid sequence starting from the N-terminal to the 179 amino acid of a polypeptide which contains M1-CSF signal peptide and not containing a part or whole of the DNA encoding amino acids from the 180 amino acid to the C-terminal) downstream of a promoter and upstream of a dhfr marker DNA. The promoter is preferably SV40 early promoter. Also disclosed are animal cells, eg. CHO cells, transformed with the above vector. The transformants provide for a sustained, high level production of M-CSF active polypeptide.

Description

TITLE OF THE INVENTION STABLE PRODUCTION OF M-CSF ACTIVE POLYPEPTIDE BACKGROUND OF THE INVENTION Field of the Invention: This invention relates to a recombinant vector having a capability of high and stable expression of human M-CSF active polypeptide, a transformed animal cell having a capability of high and stable production of M-CSF active polypeptide which is prepared by introducing said recombinant vector into animal cells and selecting a high methotrexate resistant cell, and a process of producing M CSF active polypeptide by the use of said transformed animal cell.

Description of the Backcrround Art: M-CSF active polypeptide is a factor which acts on monocytic colony-forming cells, which are monocyte precursors in the human bone marrow, thus differentiating and maturing the monocytic colony-forming cells into macrophages via monoblasts or premonocytes. Conventionally, two genetically cloned genes which have involvement in the expression of M-CSF active polypeptide are known; the one is a gene corresponding to 256 amino acid residues, containing a signal peptide consisting of 32 amino acids in the Nterminal [Science, 230, 291-296 (1985)], and the other is a gene corresponding to 554 amino acid residues [Science, 235, 1504-1508 (1987)].

In the formation of a matured type M-CSF active polypeptide which is produced by the gene corresponding to 256 amino acids (such a polypeptide is hereinafter referred to as M1-CSF), a signal peptide consisting of 32 amino acids is released during the early stage of its expression. The remaining portions of the polypeptide form a homodimer, which moves in the Golgi body. The homodimer is considered to be cut and release its C-terminal after the translation and to grow into the matured type M-CSF active polypeptide.

The cutting site is identified in M1-CSF expressed by CV-1 cell as a host cell tJ. Biotechnology, 8, 45-88 (1988)].

According to the report, the matured type M1-CSF is confirmed to be a polypeptide consisting of 158 amino acids.

The M-CSF active polypeptide which is produced by the gene corresponding to the 554 amino acid residues (such a polypeptide is hereinafter referred to as M2-CSF) can be extracted from human urine and purified. Thus, M2-CSF can be produced in amounts good for the supply to the research and clinical evaluations.

On the other hand, there are no processes for producing sufficient amounts of Ml-CSF to be served for the research and clinical evaluation, even though, as hereinafter mentioned, several processes are under trial stages. Nor, there have been any reports dealing with the production of other polypeptides resembling to M1-CSF polypeptide and possessing M-CSF activity.

J. Biotechnology, 8, 45-88 (1988) reports the production of M1-CSF polypeptide by a transformed CV-1 host cell transformed by plasmid pcCSF17 which has been obtained by introducing a naturally'occurring gene involving the production of M1-CSF (such a gene may hereinafter referred to simply as natural-type M1-CSF gene) into an expression vector possessing SV40 early gene promoter. Since the method uses SV40 virus in order to promote the production of M1-CSF polypeptide, CV-1 cell is only alive for 5-6 days.

In addition, the transformed CV-1 cell can produce M1-CSF active polypeptide in a limited concentration, i.e., approximately 10,000 U/ml. If no SV40 virus is used, the amount of M1-CSF polypeptide produced is small even more.

WO 88/03173 discloses a method of transforming COS cell by introducing a recombinant vector prepared in the same manner as the aforementioned pcCSF17 vector, but by using, instead of M1-CSF gene, a modified gene prepared by inserting termination codons at various sites of the M1-CSF gene. The transformed cell prepared by this method can maintained its activity only for about 1 week. It can produce M1-CSF polypeptide in a low concentration of about 20,000 U/ml.

The present inventors previously reported a process for transforming COS host cell by plasmid pSVL-M1-del produced by introducing a gene modified by removing specific sites of Ml-CSF gene into an expression vector possessing SV40 later gene promoter [Proceedings of the 6th Meeting, Symposium on the Next Generation Basic Industrial Technology; 258-259 (1988)]. The transformed cell prepared by this method, however, could maintain its activity for only about 1 week, while producing M-CSF active polypeptide in a very low concentration of about 2,000 U/ml.

Among the above processes for the production of M-CSF active polypeptide, processes in which a modified M1-CSF gene is used achieved a higher production activity as compared to a process using non-modified M1-CSF gene. The amounts produced by the former processes, however, were not sufficient; nor the processes could continue a stable production of M-CSF active polypeptide. Thus, no process has existed heretofore which can produce M-CSF active polypeptide in a stable manner and in a high concentration.

Development of a process which can produce M-CSF active polypeptide in a stable manner in a large scale has therefore been desired.

In view of this situation, the present inventors have undertaken extensive studies and found that the use of a recombinant vector constructed so as to contain M-CSF-delete DNA, which is a DNA containing, in the downstream of a promoter, a portion of a DNA encoding an amino acid sequence starting from the N-terminal to the 179 amino acid of a polypeptide which contains M1-CSF signal peptide and not containing a part or whole of the DNA encoding amino acids from the 180 amino acid to the C-terminal, and to possess a dhfr marker DNA in the downstream of the M-CSF-delete DNA, in a transformation process and a cell selection process could produce M-CSF active polypeptide at a high productivity and in a stable manner. This finding has led to the completion of the present invention.

SUMMARY OF THE INVENTION Accordingly, an object of the present invention is to provide a recombinant vector capable of expressing M-CSF active polypeptide, containing an M-CSF-delete DNA and a dhfr marker DNA constructed in the downstream of said M-CSF-delete DNA, wherein said M-CSF-delete DNA is a DNA containing a portion of a DNA encoding an amino acid sequence starting from the N-terminal to the 179 amino acid of a polypeptide which contains M1-CSF signal peptide and not containing a part or whole of the DNA encoding amino acids from the 180 amino acid to the C-terminal, and said M-CSF-delete DNA is introduced by substitution into a dhfr marker DNA site of an expression vector possessing a promoter and the dhfr marker DNA.

Another object of the present invention is to provide a transformed animal cell transformed by introducing said recombinant vector into an animal cell and capable of stably expressing M-CSF active polypeptide.

Still another object of the present invention is to provide a process for the preparation of M-CSF active polypeptide comprising cultivating said transformed animal cell in a medium containing methotrexate to select a methotrexate resistant transformed animal cell possessing a capability of a high and stable expression of M-CSF active polypeptide and cultivating said selected methotrexate resistant transformed animal cell in a medium.

Other objects, features and advantages of the invention will hereinafter become more readily apparent from the following description.

BRIEF DESCRIPTION OF THE DRAWING Figure 1 diagrammatically shows recombinant vector pSV2-dhfr-M1-del of the present invention capable of expressing M-CSF active polypeptide.

Figure 2 is a schematic illustration of a method for constructing recombinant vector pSV2-dhfr-Ml-del.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS M-CSF-delete DNA in the present invention means a DNA containing a portion of a DNA encoding an amino acid sequence starting from the N-terminal to the 179 amino acid of a polypeptide which contains M1-CSF signal peptide, and not containing a part or whole of the DNA encoding amino acids from the 180 amino acid to the C-terminal. The amino acid and base sequences of the polypeptide containing M1-CSF signal peptide have already been clarified. This is a gene encoding 256 amino acid residues consisting of a signal peptide of 32 amino acid residues and a polypeptide of 224 amino acid residues Science, 230, 291-296 (1985)].

Deletion of the whole or a part of the DNA encoding the above amino acid residues existing in the C-terminal side can be effected by means of appropriate gene manipulation techniques, e.g., by cutting restriction endonuclease sites of the DNA encoding the amino acid sequence by using a suitable restriction endonuclease, optionally by linking DNA fragments thus obtained with a small fragment of naturally occurring or synthesized oligonucleotide or by creating a termination codon at a suitable site by the point mutation technique or the like. M-CSF-delete DNA thus obtained preferably possesses a termination codon at its C-terminal.

The codon encoding the C-terminal amino acid of matured type M1-CSF which is constituted by 158 amino acid corresponds to the 190th codon of the natural-type M1-CSF gene. However, the present inventors confirmed that M-CSF activity is exhibited if there are codons up to 179. A typical example of M-CSF-delete DNA is a modified gene prepared by digesting M1-CSF gene with restriction endonuleases BamH I and Stu I, and linking the appropriate fragments with Klenow enzyme.

Such a modified gene is that having a base sequence corresponding to the 1-189 codons of the natural-type M1-CSF gene and further having, immediately following such a base sequence, a base sequence corresponding codons from 217 codon to 256 codon which is the last codon (such modified gene herein may be referred to as M1-del). The amino acid sequence coded by the M1-del gene is shown by SEQ ID No. 1.

A specific base sequence is given as SEQ ID No. 1.

In the present invention, "dhfr marker DNA" means dehydrofolate reductase gene. A dhfr marker DNA derived from mouse [Nature, 275, 617-624 (1978)] can be conveniently used for the purpose of the present invention. Cells containing a dhfr marker can grow in culture media containing methotrexate (herein may be abbreviated as MTX) and are suitable for the selection of transformed cells as a drug resistant marker.

There are no specific restrictions as to a promoter which is present in the expression vector possessing a dhfr marker and which is introduced by substitution in the upstream of M-CFS-delete DNA of the recombinant vector of the present invention. Examples of such promoters are SV40 early gene promoter, metallothionein gene promoter, thymidine kinase gene promoter, etc., with an especially preferable promoter being SV40 early gene promoter.

Construction of the recombinant vector can be carried out according to the present invention, for example, by replacing the dhfr marker DNA site of an expression vector possessing the promoter and the dhfr marker DNA with M-CSFdelete DNA, and introducing the dhfr marker DNA into the downstream of M-CSF-delete DNA. Here, there are no specific restrictions as to the expression vector possessing the promoter and the dhfr marker DNA, so long as the same contains a dhfr marker DNA. A preferable vector is that having a suitable restriction endonuclease site which can easily be processed by a gene manipulation technique. A typical example is pSV2-dhfr. This type of vector is particularly preferable because it has a promoter of the type mentioned above.

The aforementioned substitution of dhfr marker DNA with M-CSF-delete DNA can be carried out by the utilization of restriction endonuclease sites, e.g., Hind III and Bgl II, existing in the downstream of these promoters. The introduction of the dhfr marker DNA can be carried out by the utilization of restriction endonuclease sites, e.g., Pvu II, Bam HI, and Eco RI existing in the upstream of the dhfr marker DNA and containing these promoters, e.g. SV40 early promoter, or, optionally, by converting them to other restriction endonulease sites. Other appropriate promoters, e.g., metallothionein gene promoter, thymidine kinase gene promoter, etc., can also be used. In this manner, it is preferable that both the M-CSF-delete DNA and the dhfr marker DNA be designed so as to exist in the downstream of different promoters.

A method of constructing a recombinant vector of the present invention, which has a dhfr marker DNA and a SV40 early gene promoter in its upstream, is described with respect to pSV2-dhfr as an example. A schematic illustration of the method is given in Figure 2.

Both ends of the dhfr marker DNA of pSV2-dhfr vector DNA are cut by suitable restriction endonucleases to obtain purified vector DNA from which the dhfr marker DNA has been removed. The terminals of the vector DNA can be made blunt by selecting a suitable restriction endonuclease.

Dephosphorylation of the terminals by alkaline phosphatase or the like is also preferable.

Hind III and Bgl II are given as preferable restriction endonucleases for cutting the both ends of the dhfr marker DNA. Making the terminals blunt can be carried out, for example, by rendering the cutting end blunt with Klenow fragment enzyme in the presence of dATP, dTTP, dGTP, and dCTP.

For the production of the M-CSF-delete DNA fragment, cDNA involving the production of M1-CSF is collected from cells capable of producing M1-CSF (e.g. MIA-PaCa cells, TRC-29R cells), inserted into a suitable vector by a conventional method, if necessary, and proliferated by the cultivation to obtain M1-CSF gene. A process of deleting a part or whole of the DNA encoding amino acids from the 180 amino acid to the C-terminal of the M1-CSF gene is then carried out. This is done, for example, by digesting the M1-CSF gene with a suitable restriction endonuclease, optionally by linking DNA fragments thus obtained with a small fragment of oligonucleotide or by creating a termination codon by the point mutation technique or the like. When these processes are effected after insertion of the M1-CSF gene into a vector, the M-CSF-delete DNA fragment is obtained by digesting with a suitable restriction endonuclease. The terminals of the M-CSF-delete DNA fragment may be made blunt depending on the restriction endonuclease used. Preferably, the terminals are dephosphorylated by alkaline phosphatase or the like.

A vector DNA can be prepared from the vector DNA fragment from which the dhfr marker DNA has been removed and the M-CSF-delete DNA fragment thus obtained are ligated, introduced into a suitable host cell to transform the latter, and the transformant is cultivated to produce a vector DNA. This vector containing the M-CSF-delete DNA is digested with a suitable restriction endonuclease which does not act on the M-CSF-delete DNA to produce a vector DNA fragment. An example of such a restriction endonuclease which does not act the M-CSF-delete DNA is Eco RI. It is desirable that the terminals of this latter vector DNA be dephosphorylated by alkaline phosphatase or the like.

Separately, both ends of dhfr marker DNA of the pSV2dhfr vector DNA, in which the restriction endonuclease Pvu II site has been converted into the restriction endonuclease Eco RI site by a Eco RI linker, are cut by a suitable restriction endonuclease to prepare a dhfr marker DNA fragment. In this instance, it is desirable to cut the DNA fragment broad enough to include a promoter suitable for the expression of the dhfr marker DNA. Preferably, a DNA fragment containing the dhfr marker DNA is prepared by the use of restriction endonuclease Eco RI. Dephosphorylation of the terminals by alkaline phosphatase or the like is preferable also with respect to this DNA fragment.

A recombinant vector can be constructed by the ligation of the DNA fragment containing M-CSF-delete DNA and the DNA fragment containing dhfr marker DNA prepared according to the above procedures, and by introducing it into a host microorganism, e.g., E. coli, to transform the latter.

In the above procedures, restriction endonuleases are used in an amount equivalent to the amount of DNA, preferably 1-2 U for 1 jig of the DNA, to be digested. The amount of other enzymes are determined-depending on the reaction system involved, usually in the range of about 1020 U.

An especially preferable recombinant vector among the vectors of the present invention prepared by the above method is shown in Figure 1, in which M-CSF-delete DNA and containing dhfr marker DNA are located in the downstream of SV40 early gene promoter with a distance of 1.5 kbp. A specific amino acid sequence encoded by the M-CSF-delete DNA contained in the recombinant vector of Figure 1 is shown in SEQ ID No. 1. A base sequence for the M-CSF-delete DNA is shown as SEQ ID No. 1.

Transformed animal cells can be obtained according to the present invention by introducing the recombinant vector into animal cells by the calcium phosphate precipitation method, the DEAE-dextran method, the electric pulse method, or the like. Among these methods, the electric pulse method is particularly preferable.

There are no specific restrictions as to animal cells to be used as a host cells, so long as such cells can be grown by cultivation. Preferable cells are those having dhfr- characteristics. A most preferable example of such cells is CHO cell [dhfr CHO; Proc. Natl. Acad. Sci., USA, 77, 4216-4220 (1980)]. However, TRC-29 cell (FERM BP-2375) which does not possess dhfr- characteristics can also be used.

Transformed animal cells thus obtained are then cultivated in a medium containing MTX (methotrexate) to select high MTX-resistant cells, thus obtaining a transformant with a capability of stable production of M-CSF active polypeptide. Usually, it is preferable to cultivate the MTX-resistant cells by progressively increasing the MTX content, in the range between about 0.1-10 RM. MTXresistant cells exhibiting strong resistance to MTX above about 10 RM thus obtained provide a transformed animal cell with a capability of stable, high production of M-CSF active polypeptide.

Given as a preferable example of such transformed cell is M1-del 14-2 which was deposited with Fermentation Research Institute, Agency of Industrial Science and Technology (FERM BP-3522). The cell was produced by introducing a stable, high-expression recombinant vector containing the M1-del gene into dhfr- CHO cells to transform the latter, and by selecting MTX resistant cells.

In the present invention, "stable" is used to indicate a characteristic of the cell of producing M-CSF active polypeptide without decreasing the yield of the polypeptide during its subculture, and, more specifically, the capability of the cell of producing M-CSF active polypeptide for at least for 2 weeks without lowering the yield in the cultivation under the normal conditions. The above M1-del 14-2 could produce M-CSF active polypeptide in an amount of as much as about 600,000 U/ml at least for about 1 month.

A conventional animal cell cultivation method can be used in the production of M-CSF active-polypeptide by the cultivation of the transformed cells. For example, the transformant is inoculated into a CHO or TRC-29R cell culture medium, such as, for example, into a Dulbecco's- modified Eagle's MEM medium containing 10% FCS, in an incubator or, for simplicity, in a Shale, in an amount of about 104/cm2 and cultured at 30L370C for 2-6 days. The supernatant of the culture broth is collected and subjected to the detection of the target M-CSF active polypeptide activity. The M-CSF active polypeptide is then collected from the supernatant and purified by a suitable means which is conventionally applicable to the protein recovery and purification.For the mass production, cells are attached to micro-carriers and cultivated under suitable conditions, e.g., stirring, pH, and oxygen concentration conditions.

The solution containing M-CSF active polypeptide thus obtained is subjected to one or more of the means selected from vacuum concentration, concentration using membrane, desalting treatment using ammonium sulfate, sodium sulfate, or the like, fractional precipitation using a hydrophilic solvent such as methanol, ethanol, acetone, or the like, and other suitable means, thus obtaining precipitate containing M-CSF active polypeptide. The precipitate is further purified, if required, for example, by dissolving it into water or a buffer solution and by dialyzing the solution to remove low molecular weight impurities, or further by ion exchange chromatography, adsorption chromatography, or gel filtration using adsorbents or gel filtration agents.

Purified M-CSF active polypeptide thus obtained may be stored after lyophilization.

Other features of the invention will become apparent in the course of the following description of the exemplary embodiments which are given for illustration of the invention and are not intended to be limiting thereof.

EXAMPLES Example 1 < Preparation of Poly(A)+RNA > Human cells TRC-29R (FERM BP-2375) were incubated at 370C for 4 days in a 5% CO2 incubator using D-MEM medium (a product of Gibco) containing 5% of FCS. 108 cells were homogenized in 400 ml of 6 M guanidine isothiocyanate and passed through a 18.5 guage injection needle to fragment the high molecular DNA and to decrease its viscosity. The homogenate was multi-layered onto a 1/3-volume of a 5.7 M cesium chloride-0.1 M EDTA solution (pH 7.5) and centrifuged for 18 hours at 250C and at 35,000 rpm. The precipitate was dissolved into 3 ml of water, treated with an equivalent amount of phenol-chloroform. The water layer was transferred to another test tube, followed by an addition of a 1/10 volume of 3 M sodium acetate and a 2.5 volume of ethanol.After centrifugation, the residue was collected and dried in vacuo to produce 5 mg of crude RNA.

The precipitate was dissolved into 1.5 ml of water, incubated at 650C for 5 minutes and quenched, and mixture was adsorbed into a column of 75 mg of oligo(dT)-cellulose (a product of Pharmacia) which had been equilibrated with a buffer; 40 mM Tris-HCl (pH 7.6), 1.0 M NaCl, 1 mM EDTA, and 0.1% SDS. After washing with 10 ml of the same buffer, 5 ml of a buffer; 20 mM Tris-HCl (pH 7.6), 0.1 M Nail, 1 mM EDTA, and 0.1% SDS, was passed'through the column to elute RNAs other than poly(A)+RNA. Poly(A)+RNA was then eluted with a buffer; 10 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.05% SDS.

To 1 ml of a fraction initially discharged were added a 1/10-volume of 3 M sodium acetate and a 2.5-volume of ethanol, and the mixture was allowed to stand overnight at -200C, centrifuged for 30 minutes at 15,000 rpm to collect the precipitate. The precipitate was dried in vacuo to obtain 50 jig of poly(A)+RNA.

Example 2 < Preparation of cDNA Library > Poly(A)+RNA prepared in Example 1 was dissolved into water to the concentration of 1 jig/jil. 5 jil of the solution was transferred to a micro-tube, heated for 5 minutes at 650C, and quenched. To the solution were added a buffer; 50 mM Tris-HCl (pH 8.3), 10 mM MgC12, 140 mM KC1, 10 mM dithiothreitol, and 2 mM dNTPs (a mixture of equivalent amounts of dATP, dGTP, dCTP, and dTTP); 5 jig of oligo (dT) (a product of Pharmacia); and 1.5 unit of a reverse transcriptase (a product of Takara Shuzo Co., Ltd.) to make the total volume to 20 jil, and the mixture was reacted for 1 hour at 42"C. To the reaction mixture were added a buffer; 80 mM Tris-HCl (pH 7.5); 200 mM KC1, 10 mM MgC12, and 25 Rg/ml BSA; 60 units of RNaseH (a product of Takara Shuzo Co., Ltd.); and 5 units of DNA polymerase I (a product of Boehringer Mannheim) to make the total volume 150 jil. After reacting for 1 hour at 120C, for 1 hour at 220C and terminating the reaction by an addition of 20 jil of 0.25 M EDTA and 10 jil of 10% SDS, an equivalent amount of phenolchloroform was added, followed by centrifuge at 10,000 rpm for 5 minutes. To the water layer were added an equivalent amount of 4 M ammonium acetate and a 2-fold volume of ethanol, and the mixture was again centrifuged at 15,000 rpm for 15 minutes to collect the residue.The residue was collected and dried in vacuo, and to this a buffer; 100 mM Tris-HCl (pH 8.0), 10 mM EDTA, 80 MM S-adenosylmethionine, 100 Fg/ml BSA; and 2 units of Eco RI methylase (a product of Promega Corp.) were added to make the total volume 10 jil, and the mixture was incubated at 370C for 1 hour. To the resulting reaction mixture were added 40 jil of water, the mixture was treated with an equivalent amount of phenolchloroform and centrifuged to separate a water layer. After an addition of an equivalent amount of 4 M ammonium acetate and a 2-fold volume of ethanol, the mixture was allowed to stand for 15 minutes at -700C, centrifuged at 15,000 rpm for 15 minutes to collect a residue.To the residue were added a buffer; 67 mM Tris-HCl (pH 8.8), 6.7 mM MgC12, 16.6 mM ammonium sulfate, 10 mM 2-mercaptoethanol, 6.7 RM EDTA, 0.167% BSA; dATP, dGTP, dCTP, and dTTP, each at 750 WM; and 4 units of T4 DNA polymerase (manufacture by Takara Shuzo Co., Ltd.), to make the total volume 12 jil, following which the mixture was reacted at 370C for 1 hour. The resultant mixture was treated with an equivalent amount of phenolchloroform and ethanol to produce precipitate.The precipitate collected by centrifugation was dried in vacuo and to this were added 1 jil of Eco RI linker, a buffer solution; 50 mM Tris-HCl (pH 7.6), 10 mM Mac12, 10 mM dithiothreitol, 0.1 mM spermidine, 0.1 mM EDTA, 1 mM ATP, and 3 units of T4 polynucleotide kinase (manufacture by Takara Shuzo Co., Ltd.), to make the total'volume 10 jil, followed by a reaction at 37 C for 30 minutes. The whole mixture was added to a sample which had been treated with T4 DNA polymerase.After an addition of 60 units of T4 ligase (a product of Pharmacia) and reacting the mixture at 140C overnight, a solution of 100 mM NaCl, 50 mM Tris-HC1 (pH 7.5), 10 mM MgC12, 7 mM 2-mercaptoethanol, 100 pg/ml BSA, and 250 units of Eco RI was added to the resulting reaction mixture to make the total volume 40 1, followed by a further reaction at 370C for 2 hours. The reaction mixture thus obtained was fractionated in 1% low melting point agarose gel to collect a 600-2,000 base DNA-containing gel.

The gel was incubated at 650C for 10 minutes to melt and, after an addition of an equivalent amount of phenol and ice-cooling for 10 minutes, the mixture was centrifuged at 40C for 10 minutes at 15,000 rpm. To the water layer thus obtained was added an equivalent amount of phenol and the same procedure was repeated. The water layer was treated twice with chloroform and a 1/10 volume of 3 M sodium acetate and a 2.5-fold volume of ethanol were added. After having been allowed to stand at -700C, the mixture was centrifuged at 15,000 rpm for 15 minutes to collect the residue. The residue was washed twice with 75% ethanol, dried in vacua, and, after an addition of 1 jig of phage vector, Lamda-gtll (manufactured by Stratagene), reacted at 260C for 10 minutes using a ligation kit (a product of Takara Shuzo Co., Ltd.). After this, the sample was reacted using an in vitro packaging kit (manufactured by Stratagene). Escherichia coli LE392 (a product of Stratagene) was infected with the Lamda phage. The total number of the phage was counted to be 5.0 x 105 pfu (plaque forming unit).

Example 3 < Screening of Clone Containing M-CSF Gene > The TRC-29R cDNA library (5.0 x 105 pfu) thus prepared was distributed on 1.5% LB agar medium (containing 10 g of bacto-tryptone, 5 g of bacto-yeast extract, and 10 g of NaCl for 1 liter). The clone of the M-CSF gene was selected by the plaque hybridization method using Escherichia coli LE392 as an indicator. Furthermore, a 24 base nucleotide: 5' GAGGAGGTGTCGGAGTACTGTAGC, was synthesized based on the known DNA sequence of M-CSF gene [Science, 230, 293-296 (1985)].

The plaque on a LB plate was transferred onto a nylon membrane, and the membrane was treated with 0.5 M NaOH-1.5 M NaCl for 5 minutes, then with 3 M sodium acetate (pH 5.5) for 5 minutes, and dried at 800C for 2 hours under a reduced pressure. The membrane was put into a vinyl pack, and incubated at 370C for 4 hours in 10 ml of a solution of 5fold SSC (1-fold: 150 mM NaCl, 15 mM sodium citrate), 5-fold Denhardt's solution (1-fold: 0.02% phycol, 0.02% polyvinyl pyrrolidone, 0.02% BSA), 50 mM sodium phosphate (pH 6.5), and 0.1% SDS (sodium lauryl sulfate), 250 Rg/ml salmon sperm DNA, and 20% formamide. After removing the liquid, 20 ng of the aforementioned synthesized oligonucleotide probe (108 cpm/ > g) was added and the hybridization was carried out at 370C overnight.The membrane was washed three times with 2xSSC, 0.1% SDS at room temperature at 370C for 10 minutes, dried in the air, and subjected to autoradiograph overnight.

Portions of the medium at which signals appeared was cut out and suspended into 1 ml of an SM solution (0.1 M Nail, 8 mM MgSO4, 50 mM Tris-HCl (pH 7.5), 0.01% gelatin). The suspension was diluted, charged onto a LB plate, and the screening was repeated using the same probe. Plaque was purified to obtain two stocks of the clone.

Example 4 < Preparation of M1-CSF Gene > A host microorganism, Escherichia coli LE392, was infected with the recombinant lamda-phage 1x105 pfu prepared in Example 3 and distributed onto two sheets of LB agar medium plate (13 cm x 9 cm). 15 ml of SM solution was added to each sheet to infiltrate pharge thereinto. The infiltrate together with the upper agar layer was transferred to a test tube and centrifuged at 8,000 rpm for 10 minutes. To the supernatant were added 60 units of DNaseI (a product of Takara Shuzo Co., Ltd.) and 100 jig of RNaseA (a product of Sigma Co.), and the mixture was incubated at 370C for 30 minutes. After an addition of an equivalent amount of 20% polyethylene glycol and 2.5 M Nail, the mixture was ice-cooled for 1 hour, centrifuged at 15,000 rpm for 20 minutes to obtain a residue.The suspension of the residue in 0.5 ml of SM solution was treated with an equivalent amount of chloroform and centrifuged to separate a water layer. Cesium chloride was added to the water layer to make the density of the solution 1.15. This solution was layered onto SM solutions to which cesium chloride was added to make their densities 1.6 (2 ml), 1.5 (3 ml), and 1.4 (3 ml), and centrifuged at 30,000 rpm for 3 hours. A band appeared between densities 1.4 and 1.5 which contained the pharge was taken out and dissolved into a Tris-KC1 buffer (pH 7.5), centrifuged art 40,000 rpm for 1 hour to obtain a residue. The residue was treated with a solution of 20 mM EDTA, 0.5% SDS, 50 pl/mg protease K (a product of Sigma Co.) at 650C for 1 hour.After an addition of an equivalent amount of phenol to the reaction solution, followed by centrifuge to separate a water layer, this water layer was again treated with an equivalent amount of phenol-chloroform and again centrifuged. The water layer obtained was treated with chloroform, and a 1/10 volume of 3 M sodium acetate and a 2.5 volume of ethanol were added to it. After having been allowed for 15 minutes at -700C, the mixture was centrifuged at 15,000 rpm for 15 minutes to collect a residue, which was washed twice with 75% ethanol and dried under reduced pressure.The residue was dissolved into 50 jil of water containing 5 Rg/ml of RNaseA. 10 jil of an aliquote of the solution was completely digested with Eco RI (a product of Toyo Boseki Co., Ltd.) in an H buffer [Maniatis et al., Molecular Cloning, 104, Cold Spring Harbor (1982)], thus obtaining M-CSF clone of which fragment had a maximum length of 1.0 kbp. The fragment of the length of 1.0 kbp obtained by the digestion with Eco RI was recovered by low melting point agarose gel electrophoresis.

Furthermore, after complete digestion of vector pUC118 with Eco RI, 0.5 unit of bacterial alkaline phosphatase (a product of Toyo Boseki Co., Ltd.) was added and reacted in 50 mM Tris-HCl (pH 8.0) at 650C for 1 hour (such a reaction is hereinafter called "BAP treatment). The reaction product was treated twice with phenol-chloroform solution. To the water layer were added a 1/10 volume of 3 M sodium acetate and a 2 volume of ethanol, and the mixture was centrifuged to collect the vector.Insertion fragment recovered from the gel and the vector pUC118 digested with Eco RI were joined using a ligation kit (a product of Takara Shuzo Co., Ltd.) to obtain plasmidr which was named pUC118-M1-CSF. Example 5 < Preparation of M-CSF-delete DNA > 50 pg of pUC118-M1-CSF was digested in 100 jil of a solution containing 100 mM Nail, 50 mM Tris-HCl (pH 7.5), 10 mM MgC12, 7 mM 2-mercaptoethanol, and 100 units of restriction endonuclease Bam HI at 370C for 2 hours.

Water-saturated phenol was added to the reaction solution, and the mixture was stirred and centrifuged. To the water layer were added a 1/10 volume of 3 M sodium acetate (pH 5.5) and a 2.5 volume of ethanol with stirring. After having been allowed to stand still at -800C for 15 minutes, the mixture was centrifuged at 15,000 rpm for 15 minutes to obtain white pellets. The pellets were dissolved into 10 jil of a solution containing 67 mM KPO4, 6.7 mM MgC12, 1 mM 2mercaptoethanol, and 33 RM dATP, dCTP, dGTP, and dTTP.

After an addition of 20 units of Klenow fragment enzyme (manufacture by Takara Shuzo Co., Ltd.), the mixture was reacted at 220C for 1 hour. The reaction mixture was submitted to 1% low melting point agarose gel electrophoresis (a product of BRL Co.) to extract and isolate DNA fragments with a length of about 700 bp and about 3.2 kbp. 20 jig of the DNA fragment with a length of about 3.2 kbp was digested with restriction endonuclease Stu I in the same way as above to isolate a DNA fragment with a length of about 3.1 kbp. 0.5 unit of bacterial alkaline phosphatase was added to 2 jig of the fragment with a length of about 3.1 kbp thus isolated and reacted in 50 mM Tris-HCl (pH 8.0) at 650C for 1 hour.The DNA fragment was collected from the reaction mixture by the phenol extraction and ethanol precipitation. 100 ng of the dephosphorylated DNA fragment with a length of about 3.1 kbp and 300 ng of the DNA fragment with a length of about 700 bp which had been previously obtained were linked using a ligation kit (a product of Takara Shuzo Co., Ltd.), and added to competent cells: Escherichia coli JM109 (a product of Takara Shuzo Co., Ltd.). The mixture was distributed on an LB agar medium containing 50 zg/ml of ampicillin, and cultivated at 370C for 18 hours to obtain a transformant. The plasmid (hereinafter referred to as plasmid pUC118-M1-del) was prepared from the transformant and inserted DNA sequence (hereinafter referred to as Ml-del) was determined to confirm that the sequence is as shown in SEQ ID No. 1.

Example 6 < Preparation of Expression Vector > 10 jig of plasmid pSV2-dhfr (produced by BRL Co.) was digested at 370C for 2 hours with restriction endonucleases Hind III and Bgl II, each in an amount of 20 units. To this was added 20 units of Klenow fragment enzyme and the mixture was reacted at 220C for 1 hour in the presence of 4 kinds of dNTP. The reaction solution was submitted to low melting point agarose gel electrophoresis to isolate DNA fragments with a length of about 3.6 kbp to obtain 5 jig of bacterial alkaline phosphatase-treated DNA fragments.Separately, 10 jig of pUC118-M1-del was digested with 20 units of restriction endonuclease Eco RI at 37OC for 2 hours, and reacted with 20 units of Klenow fragment enzyme at 220C for 1 hour in the presence of 4 kinds of dNTP. The reaction solution was submitted to low melting point agarose gel electrophoresis to isolate DNA fragment with a length of about 700 bp. 300 ng of the DNA fragment with a length of about 700 bp and 100 ng ofthe DNA fragment with a length of about 3.6 kbp which had been previously obtained were linked using a ligation kit to produce a transformant with Escherichia coli JM109 as a host microorganism. Plasmid pSV2-M1-del was obtained.from the transformant.

Separately, 5 jig of plasmid DNA pSV2-dhfr was digested with 30 units of restriction endonucleases Pvu II and linked with 1 jig of phosphorylated Eco RI linker (G-G-A-A-T-T-C-C: produced by Takara Shuzo Co., Ltd.) using a ligation kit.

The reaction solution was digested with 100 units of restriction endonuclease Eco RI at 370C for 2 hours and submitted to low melting point agarose gel electrophoresis to isolate DNA fragment with a length of about 2.4 kbp. 300 ng of this fragment and 5 jig of plasmid pSV2-M1-del which was previously produced were digested with 10 units of restriction endonuclease Eco RI at 370C for 2 hours, and then linked with 100 ng of the bacterial alkaline phosphatase-treated DNA by means of a ligation kit. A transformant was produced by using Escherichia coli JM109 as a host microorganism. Plasmid pSV2-dhfr-M1-del was obtained from the transformant. The restriction endonuclease map of the plasmid is shown in Figure 1, wherein the portion shaded by oblique lines is the base sequence encoding the signal peptide.The same procedure was repeated by using 50 jig of pUC118-Ml-CSF for the construction of an expression vector.

to be used for a control test to produce plasmid pSV2-dhfr M1-CSF.

Example 7 < Cultivation of'M1-CSF-High-Production Cell byCHO-dhfr Cells > Introduction of the gene was carried out by means of the electric pulse boring method by using a high voltage cell processor (a product of Bioelectronics Co.). CHO-dhfr- cells (supplied by Tokyo University Microorganism Institute) cultivated on a plastic plate by using HAM F12 medium containing 5% FCS were peeled with a detachment enzyme, Nagase (a product of Nagase Industries, Ltd.), suspended in PBS(-), and centrifuged to collect cells. The cells were suspended in PBS(-) to make the concentration lx107/ml.

Plasmids pSV2-dhfr-M1-del and pSV2-dhfr-M1-CSF, prepared in Example 6, were added to the suspension to a concentration of 40 jig/ml, each. After stirring, the suspension was transferred to a chamber, where electric pulse was applied 5 times under the conditions of an electrode distance of 3 mm, a voltage of 1200 V, and a pulse interval of 30 jisec. After the application of pulse, the cell suspension was promptly recovered and stored under ice-cooled conditions. Cells were inoculated into a plastic petri into which HAM F12 medium containing 5% FCS was placed in advance.

Cells were cultivated at 370C in a 5% CO2 incubator for 48 hours, whereupon the medium was replaced with a selective medium; cL-MEM medium containing 5% dialized FCS). 10-14 days after the medium replacement, cell colonies proliferating in the selective medium were observed. The colonies were cloned by the filer paper method to measure the M-CSF activity (the activity of one colony was taken as 1 unit) on the cultivation liquid supernatant according to the Metcalf method [J. Biol. Med., 44, 287-300 (1966)].The transformant of which the M-CSF activity was confirmed was cultivated in an MTX-containing medium (a-MEM containing 5% dialyzed FCS and 0.1-10 RM MTX), while increasing the MTX concentration stepwise from 0.1, 0.5, 2, and to 10 zM. The M-CSF activity of the resistant strain with the 10 M MTX concentration was measured and found to be 70,000 units/ml of productivity when an native type M1-CSF gene was used.

The strain with which M1-del gene was used exhibited 600,000 units/ml of productivity, showing that the introduction of a gene of which the membrane-bonding site was removed results in a transformant with a 10 times as much productivity. The transformant was named M-del 14-2 and deposited with Fermentation Research Institute, Agency of Industrial Science and Technology (FERM BP-3522). The transformant M1-del 14-2 exhibited 600,000 units/ml of productivity consistently for 1 month in the a-MEM medium containing 5% FCS.

Example 8 < Cultivation of M1-CSF-High-Production Cell by TRC-29R Cells > Introduction of the gene was carried out by means of the electric pulse boring method by using a high voltage cell processor (a product of Bioelectronics Co.). TRC-29R cells (FERM BP-2375) cultivated on a plastic plate by using RPMI 1640 medium containing 5% FCS were peeled with a detachment enzyme Nagase (Trademark, a product of Nagase Industries, Ltd.), suspended in PBS(-), and centrifuged to collect cells. The cells were suspended in PBS(-) to make the concentration 5x106/ml. Plasmids pSV2-dhfr-M1-del and pSV2-dhfr-M1-CSF were added to the suspension to a concentration of 40 Rg/ml, each.After stirring, the suspension was transferred to a chamber, where an electric pulse was applied 3 times under the conditions of an electrode distance of 3 mm, a voltage of 800 V, and a pulse interval of 300 jisec. After the application of pulse, the cell suspension was promptly recovered and stored under ice-cooled conditions. Cells were inoculated into a plastic petri into which RPMI 1640 medium containing 5% FCS was placed in advance.

Cells were cultivated at 370C in a 5% CO2 incubator for 48 hours, whereupon the medium was replaced with a selective medium (RPMI 1640 medium containing 5% FCS, 200 Rg/ml G418).

10-14 days after the medium replacement, cell colonies proliferating in the selective medium were observed. The colonies were cloned by the filer paper method to measure the M-CSF activity (the activity of one colony was taken as 1 unit) on the cultivation liquid supernatant according to the Metcalf method [J. Biol. Med., 44, 287-300 (1966)]. The transformant of which the M-CSF activity was confirmed was cultivated in an MTX-containing medium (RPMI 1640 medium containing 5% dialyzed FCS.and 0.1-10 gM MTX), while increasing the MTX concentration stepwise from 0.1, 0.5, 2, and to 10 WM. The M-CSF activity of the resistant strain with the 10 pM MTX concentration was measured and found to be 4,000 units/ml of productivity when an native type M1-CSF gene was used.The strain with which M1-del gene was used exhibited 35,000 units/ml of productivity, showing that the introduction of a gene of which the membrane-bonding site was removed results in a transformant with a 10 times as much productivity. The transformant was named delM-15. The transformant delM-15 exhibited consistently 35,000 units/ml of productivity for 1 month in the RPMI 1640 medium containing 5% FCS.

Example 9 < Purification of M1-CSF from CHO Cell M1-del 14-2 Holding M1-del Gene > CITO cells, M1-del 14-2, were cultivated at 370C in a 5% CO2 incubator for two weeks by using D-MEM medium containing 5% FCS. 20 1 of the supernatant of the culture broth was concentrated to 2 1 with a ultrafilter (a product of Millipore Co.). To the concentrate was added NaCl to make its concentration 4 M, and the mixture was adsorbed in Phenyl-Sepharose CL-4B (50x50 cm: trademark, a product of Pharmacia Co.)- which had been equilibrated with a 10 mM sodium phosphate buffer (pH 7.0) containing 4 M NaCl. The column was linear gradiently eluted with a 10 mM sodium phosphate buffer (pH 7.0; hereinafter referred to as Buffer A) at NaCl concentrations between 4 M and 0 M.An M-CSF active fraction was obtained from the fractions eluted at NaCl concentrations between 1.5-1.0 M. The active fraction was concentrated to 10 ml by YM-10 membrane (trademark, a product of Amicon Co.) and desalted with Sephadex G-25 (9.1 ml: trademark, a product of Pharmacia Co.) which had been equilibrated with Buffer A containing 80 mM NaCl, adsorbed in DEAE-Cellulofine (2.5x10 cm: trademark, a product of Seikakaku Kogyo Co.) equilibrated with the same buffer, and linear gradiently eluted at NaCl concentrations between 0.1 M and 0.5 M. An M-CSF active fraction eluted at 0.12-0.15 NaCl concentrations was concentrated with YM-10 membrane to 6 ml, which was submitted å gel filter Ultrogel AcA34 (2.5x92 cm: trademarkr a product of IBF Biotechnic Co.) to obtain an M-CSF active fraction.This active fraction was concentrated to 7 ml, and NaCl was added to make the final concentration 4 M. The mixture was adsorbed in Phenyl Superose (10x10 cm: trademark, a product of Pharmacia Co.) which had been equilibrated with Buffer A containing 4 M NaCl, and was linear gradiently eluted with NaCl concentrations between 4 M to 0 M. An M-CSF active fraction obtained from the fractions eluted at NaCl concentrations between 2-1 M was dialyzed against a 200-volume of Buffer A to produce a final purified product. The molecular weight of this purified product was measured by Superose 12 (1.0x30 cm: trademark, a product of Pharmacia) equilibrated with Buffer A to find that the molecular weight to be about 70,000 dalton. SDS-polyacrylamide electrophoresis revealed a slightly wide band of 28;000+2,000 dalton.The total amount of the purified product determined by W absorption was A280=0.26. The M-CSF activity measured by the soft agar colony method was 1.2x108/A280.

Example 10 < Analysis of C-terminal Amino Acids of M1-SCF Produced by M1-del 14-2 Ceil Introduced M1-del Gene > 250 Rg (9 ml) of the product obtained in Example 9 was placed in a dialysis tube, concentrated to about 1 ml by mixing Sephadex G-100, and dialyzed against 200 ml of a 65 mM Tris buffer solution (pH 6.8) containing 0.1 M ss- mercaptoethanol, 1% SDS, and 10% glycerol to effect the reduction of SH group and modification of proteins.

Monoiode acetate was added to the internal dialysis liquid to a final concentration of 150 mM and the mixture was allowed to stand still at room temperature for 30 minutes to effect the reduction-carboxymethylation of M1-CSF. The mixture was desalted and SDS was removed therefrom by submitting it to a column of ion-exchanged resin AGllA8 (0.6x10 cm: trademark, a product of Biolad Co.) equilibrated with 0.001% PEG and a Sephadex G-50 column (1.0x9.0 cm: trademark, a product of Pharmacia Co.) which were linked together. The reduction-carboxymethylated Ml-CSF fraction identified by the ELISA method was freeze-dried and dissolved in 1 ml of 100 mM sodium phosphate buffer (pH 7.0) containing 10 mM EDTA, 10 mM DTT, 0.1% SDS, and 1% Triton X-100.After the addition of 1 U N-Glycanase (trademark, a product of Genzyme Co.), the solution was incubated at 370C for 24 hours to cut sugar chains of M1-CSF. The M1-CSF from which sugar chains were eliminated was adsorbed in TMC-PACK AP-303 300AODS (trademark, a product of Murayama Chemical Company) and linear gradiently eluted with 0-70% acetonitrile concentrations. The fraction of M1-CSF from which sugar chains were eliminated eluted at 60% acetonitrile concentration was freeze-dried and dissolved in 400 jil of a 10 mM sodium phosphate buffer (pH 7.0). After the addition of 5 jig of endoproteinase Lys-C (trademark, a product of Beringer Manheim), the solution was incubated at 370C for 17 hours.The reaction mixture was submitted to 500 jil of anhydrotripsin agarose (a product of Takara Shuzo Co., Ltd.) equilibrated with a 50 mM acetate buffer (pH 5.0) containing 20 mM CaC12 to collect the fractions passing through the column. C-terminal peptide contained in the fractions thus collected were adsorbed in TSKODS 120T (trademark, a product of Toso Co.) and linear gradiently eluted with acetonitrile with concentrations of 5-60%. Cterminal peptide eluted at 25% acetonitrile concentration was collected and analyzed by PSG-1 amino acid sequencer to find the amino acid sequence is -Asn-X-Asp-Asn-Ser-Phe-Ala Glu-X-Ser-Ser-Gln-Gly-His-Glu-Arg-Gln-Ser-Glu-Gly-Ser-.

As a control, purified M1-CSF was produced from a CITO cell M1-CSF high production strain holding the negative type M1-CSF gene according to the procedures of Examples 9 and 10. The amino acid sequence of the C-terminal peptide produced by the digestion with endoproteinase Lys-C of this purified M1-CSF was found to be -Asn-X-Asp-Asn-Ser-Phe-Ala Glu-X-Ser-Ser-Gln-Gly-His -Glu-Arg-Gln-Ser-Glu-Gly-Ser-, confirming that the M-CSF active polypeptide produced by M1-del 14-2 cells is identical with the negative type M-CSF.

As illustrated above, the recombinant vector of the present invention can express M-CSF active polypeptide at a high level and in a stable manner. The transformant prepared by the use of the recombinant vector ensures mass production of the M-CSF active polypeptide in an industrial scale.

********************************************* Obviously, numerous modifications and variations of the present invention are possible in light of the above teachings. It is therefore to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described herein.

SEOUENCE LISTING 1 1. SEQ ID No. 1 2. Length of the sequence: 693 Base pairs 3. Type of the sequence: Nucleic acid 4. Strandedness: Double-stranded 5. Topology of the sequence: Linear 6. Kind: Other nucleic acid; Synthetic DNA 7. Source: TRC-29R derived from a human cell 8. Name: Mi-del gene 9.Sequence ATG ACC GCG CCG GGC GCC GCC GGG CGC TGC CCT CCC ACG ACA TGG CTG 48 Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu -32 -30 -25 -20 GGC TCC CTG CTG TTG TTG GTC TGT CTC CTG GCG AGC AGG AGT ATC ACC 96 Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser Ile Thr -15 -10 -5 -1 GAG GAG GTG TCG GAG TAC TGT AGC CAC ATG ATT GGG AGT GGA CAC CTG 144 Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu 1 5 10 15 CAG TCT CTG CAG CGG CTG ATT GAC AGT CAG ATG GAG ACC TCG TGC CAA 192 Gln Ser Leu Gin Arg Leu lie Asp Ser Gln Met Glu Thr Ser Cys Gln 20 25 30 ATT ACA TTT GAG TTT GTA GAC CAG GAA CAG TTG AAA GAT CCA GTG TGC 240 Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys 35 40 45 TAC CTT AAG AAG GCA TTT CTC CTG GTA CAA TAC ATA ATG GAG GAC ACC 288 Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Tyr Ile Met Glu Asp Thr 50 55 60 ATG CGC TTC AGA GAT AAC ACC CCC AAT GCC ATC GCC ATT GTG CAG CTG 336 Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu 60 70 75 80 CAG GAA CTC TCT TTG AGG CTG AAG AGC TGC TTC ACC AAG GAT TAT GAA 384 Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu 85 90 95 GAG CAT GAC AAG GCC TGC GTC CGA ACT TTC TAT GAG ACA CCT CTC CAG 432 Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln 100 105 110 TTG CTG GAG AAG GTC AAG AAT GTC TTT AAT GAA ACA AAG AAT CTC CTT 480 Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu 115 120 125 GAC AAG GAC TGG AAT ATT TTC AGC AAG AAC TGC AAC AAC AGC TTT GCT 528 Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala 130 135 140 GAA TGC TCC AGC CAA GGC CAT GAG AGG CAG TCC GAG GGA TCC CTC TTG 576 Glu Cys Ser Ser Gln Gly His Glu Arg Gln Ser Glu Gly Ser Leu Leu 145 150 155 160 TTC TAC AGG TGG AGG CGG CGG AGC CAT CAA GAG CCT CAG AGA GCG GAT 624 Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu Pro Gln Arg Ala Asp 165 170 175 TCT CCC TTG GAG CAA CCA GAG GGC .AGC CCC CTG ACT CAG GAT GAC AGA 672 Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr Gln Asp Asp Arg 180 185 190 CAG GTG GAA CTG CCA GTG TAG 693 Gln Val Glu Leu Pro Val 195 198

Claims (23)

What is Claimed is:

1. A recombinant vector capable of expressing M-CSF active polypeptide, containing an M-CSF-delete DNA introduced by substitution into a dhfr-marker DNA site of an expression vector possessing a promoter and the dhfr marker DNA, and a dhfr marker DNA constructed in the downstream of said M-CSF-delete DNA, wherein said M-CSF-delete DNA is a DNA containing a portion of a DNA encoding an amino acid sequence starting from the N-terminal to the 179 amino acid of a polypeptide which contains M1-CSF signal peptide and not containing a part or whole of the DNA encoding amino acids from the 180 amino'acid to the C-terminal.

2. The recombinant vector according to Claim 1, wherein said promoter is SV40 early promoter.

3. The recombinant vector according to Claim 1, wherein said M-CSF-delete DNA encodes the following amino acid sequence.

Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser lie Thr Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Tyr Ile Met Glu Asp Thr Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala Glu Cys Ser Ser Gln Gly His Glu Arg Gln Ser Glu Gly Ser Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu Pro Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr Gln Asp Asp Arg Gln Val Glu Leu Pro Val

4. The recombinant vector according to Claim 1, wherein said M-CSF-delete DNA has the following base sequence.

ATG ACC GCG CCG GGC GCC GCC GGG CGC TGC CCT CCC ACG ACA TGG CTG GGC TCC CTG CTG TTG TTG GTC TGT CTC CTG GCG AGC AGG AGT ATC ACC GAG GAG GTG TCG GAG TAC TGT AGC CAC ATG ATT GGG AGT GGA CAC CTG CAG TCT CTG CAG CGG CTG ATT GAC AGT CAG ATG GAG ACC TCG TGC CAA ATT ACA TTT GAG TTT GTA GAC CAG GAA CAG TTG AAA GAT CCA GTG TGC TAC CTT AAG AAG GCA TTT CTC CTG GTA CAA TAC ATA ATG GAG GAC ACC ATG CGC TTC AGA GAi AAC ACC CCC AAT GCC ATC GCC ATT GTG CAG CTG CAG GAA CTC TCT TiG AGG CTG AAG AGC TGC TTC ACC AAG GAT TAT GAA GAG CAT GAC AAG GCC TGC GTC CGA ACT TTC TAT GAG ACA CCT CTC CAG TTG CTG GAG AAG GTC AAG AAT GTC TTT AAT GAA ACA AAG AAT CTC CTT GAC AAG GAC TGG AAT ATT TTC AGC AAG AAC TGC AAC AAC AGC TTT GCT GAA TGC TCC AGC CAA GGC CAT GAG AGG CAG TCC GAG GGA TCC CTC TTG TIC TAC AGG TGG AGG CGG CGG AGC CAT CAA GAG CCT CAG AGA GCG GAT TCT CCC TTG GAG CAA CCA GAG GGC AGC CCC CTG ACT CAG GAT GAC AGA CAG GTG GAA CTG CCA GTG TAG

5. The recombinant vector according to Claim 1, having a construction shown in Figure 1.

6. A transformed animal cell capable of stably expressing M-CSF active polypeptide, transformed by introducing into an animal cell a recombinant vector containing an M-CSF-delete DNA introduced by substitution into a dhfr marker DNA site of an expression vector possessing a promoter and the dhfr marker DNA, and a dhfr marker DNA constructed in the downstream of said M-CSFdelete DNA, wherein said M-CSF-delete DNA is a DNA containing a portion of a DNA encoding an amino acid sequence starting from the N-terminal to the 179 amino acid of a polypeptide which contains M1-CSF signal peptide and not containing a part or whole of the DNA encoding amino acids from the 180 amino acid to the C-terminal.

7. The transformed animal cell according to Claim 6, wherein said promoter is SV40 early promoter.

8. The transformed animal cell according to Claim 6, wherein said M-CSF-delete DNA encodes the following amino acid sequence.

Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser Ile Thr Glu Glu Val Ser Glu Tyr Cys Ser His Met lie Gly Ser Gly His Leu Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Tyr Ile Met Glu Asp Thr Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp Tyr Glu Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala Glu Cys Ser Ser Gln Gly His Glu Arg Gln Ser Glu Gly Ser Leu Leu Phe Tyr Arg Trp Arg Arg Arg Ser His Gln Glu Pro Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr Gln Asp Asp Arg Gln Val Glu Leu Pro Val

9. The transformed animal cell according to Claim 6, wherein said M-CSF-delete DNA has the following base sequence.

ATG ACC GCG CCG GGC GCC GCC GGG CGC TGC CCT CCC ACG ACA TGG CTG GGC TCC CTG CTG TTG TTG GTC TGT CIC CTG GCG AGC AGG AGT ATC ACC GAG GAG GTG TCG GAG TAC TGT AGC CAC ATG ATT GGG AGT GGA CAC CTG CAG TCT CTG CAG CGG CTG ATT GAC AGT CAG ATG GAG ACC TCG TGC CAA ATT ACA TTT GAG TTT GTA GAC CAG GAA CAG TTG AAA GAT CCA GTG TGC TAC CTT AAG AAG GCA TTT CIC CTG GTA CAA TAC ATA ATG GAG GAC ACC ATG CGC TTC AGA GAT AAC ACC CCC AAT GCC ATC GCC ATT GTG CAG CTG CAG GAA CTC TCT TTG AGG CTG AAG AGC TGC TTC ACC AAG GAT TAT GAA GAG CAT GAC AAG GCC TGC GTC CGA ACT TTC TAT GAG ACA CCT CTC CAG TTG CTG GAG AAG GTC AAG AAT GTC TTT AAT GAA ACA AAG AAT CTC CTT GAC AAG GAC TGG AAT ATT TTC AGC AAG AAC TGC AAC AAC AGC TTT GCT GAA TGC TCC AGC CAA GGC CAT GAG AGG CAG TCC GAG GGA TCC CIC TTG TTC TAC AGG TGG AGG CGG CGG AGC CAT CAA GAG CCT CAG AGA GCG GAT TCT CCC TTG GAG CAA CCA GAG GGC AGC CCC CTG ACT CAG GAT GAC AGA CAG GTG GAA CTG CCA GTG TAG

10. The transformed animal cell according to Claim 6, wherein said recombinant vector has a construction shown in Figure 1.

11. The transformed animal cell according to Claim 6, wherein the host cell is an animal cell possessing dhfr characteristics.

12. The transformed animal cell according to Claim 6, wherein said host cell is CITO dhfr cell.

13. The transformed animal cell according to Claim 6, wherein said host cell is TRC-29R cell.

14. The transformed animal cell according to Claim 6, which is M1-del 14-2 (FERM BP-3522).

15. A process for the preparation of M-CSF active polypeptide comprising: transforming an animal cell by introducing thereinto a recombinant vector capable of expressing M-CSF active polypeptide, containing an M-CSF-delete DNA and a dhfr marker DNA constructed in the downstream of said M-CSF-delete DNA, wherein said M-CSF-delete DNA is a DNA containing a portion of a DNA encoding an amino acid sequence starting from the N-terminal to the 179 amino acid of a polypeptide which contains M1-CSF signal peptide and not containing a part or whole of the DNA encoding amino acids from the 180 amino acid to the C-terminal, and said M-CSF-delete DNA is introduced by substitution into a dhfr marker DNA site of an expression vector possessing a promoter and the dhfr marker DNA;; cultivating the transformed animal cell in a medium containing methotrexate to select a methotrexate resistant transformed animal cell possessing a capability of a high and stable expression of M-CSF active polypeptide; and cultivating said selected methotrexate resistant transformed animal cell in a medium.

16. The process according to Claim 15, wherein said promoter is SV40 early promoter.

17. The process according to Claim 15, wherein said M-CSF-delete DNA encodes the following amino acid sequence.

Met Thr Ala Pro Gly Ala Ala Gly Arg Cys Pro Pro Thr Thr Trp Leu Gly Ser Leu Leu Leu Leu Val Cys Leu Leu Ala Ser Arg Ser Ile Thr Glu Glu Val Ser Glu Tyr Cys Ser His Met Ile Gly Ser Gly His Leu Gln Ser Leu Gln Arg Leu Ile Asp Ser Gln Met Glu Thr Ser Cys Gln Ile Thr Phe Glu Phe Val Asp Gln Glu Gln Leu Lys Asp Pro Val Cys Tyr Leu Lys Lys Ala Phe Leu Leu Val Gln Tyr Ile Met Glu Asp Thr Met Arg Phe Arg Asp Asn Thr Pro Asn Ala Ile Ala Ile Val Gln Leu Gln Glu Leu Ser Leu Arg Leu Lys Ser Cys Phe Thr Lys Asp iyr Glu Glu His Asp Lys Ala Cys Val Arg Thr Phe Tyr Glu Thr Pro Leu Gln Leu Leu Glu Lys Val Lys Asn Val Phe Asn Glu Thr Lys Asn Leu Leu Asp Lys Asp Trp Asn Ile Phe Ser Lys Asn Cys Asn Asn Ser Phe Ala Glu Cys Ser Ser Gln Gly His Glu Arg Gln Ser Glu Gly Ser Leu Leu Phe Tyr Arg irp Arg Arg Arg Ser His Gln Glu Pro Gln Arg Ala Asp Ser Pro Leu Glu Gln Pro Glu Gly Ser Pro Leu Thr Gln Asp Asp Arg Gln Val Glu Leu Pro Val

18. The process according to Claim 15, wherein said M-CSF-delete DNA has the following base sequence.

ATG ACC GCG CCG GGC GCC GCC GGG CGC TGC CCT CCC ACG ACA TGG CTG GGC TCC CTG CTG TTG TTG GTC TGT CTC CTG GCG AGC AGG AGT ATC ACC GAG GAG GTG TCG GAG TAC TGT AGC CAC ATG ATT GGG AGT GGA CAC CTG CAG TCT CTG CAG CGG CTG ATT GAC AGT CAG ATG GAG ACC TCG TGC CAA ATT ACA TTT GAG TTT GTA GAC CAG GAA CAG TTG AAA GAT CCA GTG TGC TAC CTT AAG AAG GCA TTT CTC CTG GTA CAA TAC ATA ATG GAG GAC ACC ATG CGC TTC AGA GAT AAC ACC CCC AAT GCC ATC GCC ATT GTG CAG CTG CAG GAA CIC TCT TTG AGG CTG AAG AGC TGC TTC ACC AAG GAT TAT GAA GAG CAT GAC AAG GCC TGC GTC CGA ACT TTC TAT GAG ACA CCT CTC CAG TTG CTG GAG AAG GTC AAG AAT GTC TTT AAT GAA ACA AAG AAT CTC CTT GAC AAG GAC TGG AAT ATT TTC AGC AAG AAC TGC AAC AAC AGC TTT GCT GAA TGC TCC AGC CAA GGC CAT GAG AGG CAG TCC GAG GGA TCC CTC TTG TTC TAC AGG TGG AGG CGG CGG AGC CAT CAA GAG CCT CAG AGA GCG GAT TCT CCC TTG GAG CAA CCA GAG GGC AGC CCC CTG ACT CAG GAT GAC AGA CAG GTG GAA CTG CCA GTG TAG

19. The process according to Claim 15, wherein said recombinant vector has a construction shown in Figure 1.

20. The process according to Claim 15, wherein the host cell is an animal cell possessing dhfr characteristics.

21. The process according to Claim 15, wherein said host cell is CITO dhfr cell.

22. The process according to Claim 15, wherein said host cell is TRC-29R cell.

23. The process according to Claim 15, wherein said transformed animal cell is Ml-del 14-2 (FERM BP-3522).